Role of the Na+/H+ antiporter in rat proximal
tubule bicarbonate absorption.
P A Preisig, … , R J Alpern, F C Rector Jr
J Clin Invest.
1987;
80(4)
:970-978.
https://doi.org/10.1172/JCI113190
.
Amiloride and the more potent amiloride analog, 5-(N-t-butyl) amiloride (t-butylamiloride),
were used to examine the role of the Na+/H+ antiporter in bicarbonate absorption in the in
vivo microperfused rat proximal convoluted tubule. Bicarbonate absorption was inhibited 29,
46, and 47% by 0.9 mM or 4.3 mM amiloride, or 1 mM t-butylamiloride, respectively.
Sensitivity of the Na+/H+ antiporter to these compounds in vivo was examined using
fluorescent measurements of intracellular pH with (2',
7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein (BCECF). Amiloride and t-butylamiloride were shown to be as potent
against the antiporter in vivo as in brush border membrane vesicles. A model of proximal
tubule bicarbonate absorption was used to correct for changes in the luminal profiles for pH
and inhibitor concentration, and for changes in luminal flow rate in the various series. We
conclude that the majority of apical membrane proton secretion involved in transepithelial
bicarbonate absorption is mediated by the Na+-dependent, amiloride-sensitive Na+H+
antiporter. However, a second mechanism of proton secretion contributes significantly to
bicarbonate absorption. This mechanism is Na+-independent and amiloride-insensitive.
Research Article
Find the latest version:
Role of the
Na+/H+
Antiporter in Rat
Proximal Tubule Bicarbonate
Absorption
P.A. Preisig,*H.E.
Ives,*
E.J.Cragoe,Jr.,'
R. J.Alpem,t and F. C. Rector,Jr.tDepartmentsof*Physiological Nursing and $Medicine, and Cardiovascular Research Institute,
University of
California,
SanFrancisco,California 94143-0532;and
WMerck
SharpandDohmeResearch Laboratories, West Point,Pennsylvania
19486Abstract
Amiloride
and the more potentamiloride analog,
54N-t-butyl)
amiloride (t-butylamiloride),
were used toexamine
the role ofthe Na+/H+ antiporter
inbicarbonate absorption
in the in vivomicroperfused
ratproximal convoluted tubule. Bicarbonate
absorption
wasinhibited 29, 46, and
47% by0.9
mM or4.3
mM
amiloride,
or 1 mMt-butylamiloride, respectively.
Sensi-tivity of the Na+/H+ antiporter
to thesecompounds
in vivo wasexamined using fluorescent measurements of intracellular
pHwith
(2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein
(BCECF). Amiloride and
t-butylamiloride
wereshown
to be as potentagainst
the
antiporter
in vivo
asin brush border
mem-brane vesicles.
Amodel
of
proximal
tubule bicarbonate
ab-sorption
wasused
tocorrectfor changes in the luminal profiles
for
pH and inhibitor
concentration,
and
for
changes inluminal
flow
ratein the various series. We conclude that the majority
of apical membrane proton secretion involved in transepithelial
bicarbonate
absorption
is mediated by the
Na+-dependent,
amiloride-sensitive Na+/H+ antiporter.
However,
asecond
mechanism
of
protonsecretion
contributes
significantly
tobi-carbonate
absorption.
This mechanism is
Na+-independent
and
amiloride-insensitive.
Introduction
Amiloride-sensitive
Na+/H+
antiporter
activity has been
dem-onstrated in brush border membrane vesicles from rabbit and
ratrenal
cortex(1-10).
Inaddition,
anH+-ATPase has been
demonstrated
in
endosomes
derived
from renal
cortexand in
brush border membranes
(11-17).
The
contribution of
Na+/H+ exchange
toapical
membrane
protonsecretion
in the
intact
proximal
tubule has been
investigated
in several
micro-perfusion studies
by
examining
the effect of
removing
luminal
and
capillary
Na+
orinhibiting
the
Na+/K+
ATPaseonthe
rateof
bicarbonate
absorption.
Mostof
thesestudies found that
80-100% of bicarbonate absorption
is
dependent
onthe
pres-enceof
Na+ and Na+
absorption (18-23).
The
interpretation of
these studies is
complicated
by the
fact that the
major
basolateral membrane
H+/HCO-
transportmechanism
in the
proximal
tubule is
Na+-coupled
and
rheo-genic (carrying negative charge) (24-30).
Removal of Na+
from the luminal and capillary fluids inhibits basolateral
membrane bicarbonate exit,
whichalkalinizes
the cell(25, 26,
AddressreprintrequestsandcorrespondencetoPartriciaPreisig, De-partment of InternalMedicine, University ofTexasHealth Science CenteratDallas, 5323 Harry HinesBlvd., Dallas,TX75235-9030.
Receivedfor publication7August 1986 andin
revisedform
15April
1987.
31),
and
cansecondarily inhibit
any apicalmembrane proton
secretory
mechanism. Inhibition
of Na+ transport by
inhibi-tion of
the
Na+/K'
ATPase will
depolarize
the
cell,which
hasbeen shown (25)
todecrease the
rateof the basolateral exit
step,and lead
to acell alkalinization. Indeed,
Wang etal.
(32)have
confirmed
that inhibition of
theNa+/K'
ATPase
withouabain alkalinized
the
cell.
To more
directly examine
the roleof
theNa+/H+
anti-porterin
bicarbonate
absorption, both Chan and Giebisch
(21)
and
Howlin
etal.
(33) used luminal
amiloride,
and found
lessinhibition of bicarbonate absorption than
would bepredicted
if
Na+/H+
antiport
werethe sole apical membrane
mechanism
of
protonsecretion.
Determination of the contribution of
Na+/H+ exchange
tobicarbonate absorption
wascomplicated
in these studies by
anumber of problems
andconcerns:
(a) thepossibility
that
amiloride
was aless potent inhibitor ofNa+/H+
exchange in the in vivo tubule than in vesicles;
(b)
the question
of
amiloride
absorption
along the length
of
the
tubule; (c) the
possibility
that inhibition of
Na+/H'
exchange acidified the
cell and
secondarily
stimulated
apical
membrane
protonse-cretion; (d)
the
effect of
achange in the axial luminal
bicar-bonate
concentration
profile
when
bicarbonate
absorption
is
inhibited; and (e)
aninability
togethigher, more potent
con-centrations of amiloride
into
physiological
solutions,
sothat
alarger percent
of
bicarbonate
absorption
could be
manip-ulated. However, these studies raised the
possibility
that
aso-dium-independent mechanism
of
protonsecretion
existed in
this
epithelium.
In
the present
studies,
weexamined
the effect of
a more potentamiloride
analog,
5-(N-t-butyl)
amiloride
(t-butylami-loride),'
and
ahigher concentration of amiloride
onvolume,
glucose, and bicarbonate
absorption
in the in vivo
microper-fused
ratproximal
convoluted tubule.
Perfused
and collected
concentrations
of amiloride
andt-butylamiloride
weremea-sured.
Inaddition,
the effects of these
agents oncell
pH, and
their inhibitory
potency
onapical
membrane
Na+/H+
ex-change
activity
in
vivo
weredetermined
using
the fluorescent
measurement
of
cell pH. The results demonstrate the
most,but
notall, of
apical
membrane
protonsecretion is
mediated
by Na+/H+ exchange,
whereas the
remaining fraction
is
me-diated
by
aNa+-independent,
amiloride-insensitive
process.
Based
onthe
findings by
othersof
H+-ATPaseactivity in brush
border membrane vesicles and endosomes
derived
from the
renal
cortex(1 1-17),
wesuggestthat this second mechanism is
a H+-ATPase.Methods
General
methodsfor
microperfusion
studies. Male Wistarrats(CharlesRiver
Breeding
Laboratories,Wilmington, MA) weighing
200-300
g1. Abbreviations usedinthispaper:BCECF,
(2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein;
DCCD,dicyclohexylcarbodiimide;
SITS,
4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate;
t-butylamiloride,
54N-t-butyl)amiloride.
J. Clin.Invest.
©The AmericanSocietyforClinicalInvestigation,Inc.
0021-9738/87/10/0970/09 $2.00
wereprepared for in vivo microperfusion as previously described (34). Briefly, the rats were anesthetized with an intraperitoneal injection of Inactin (100 mg/kg bodywt with additional dosage if needed) and placed on a heated table that maintained body temperature at 36.5-370C. Theright femoral arterywascatheterized formonitoring blood pressure (transducer, Gould Inc., Oxnard, CA, and monitor, Stentor Co., KansasCity, MO)andobtaining blood samples.Theleft jugular veinwascatheterized for continuous infusion ofRingers' bi-carbonate (in mM: NaCi, 105; NaHCO3, 25; Na2HPO4, 4; KCI, 5; MgSO4, 1; CaCI2 1.8)at 1.5cc/h. The left kidney was exposed by a flank incision and immobilized inaLucite cup. Theureter was can-nulated(PE-50) to ensure the freedrainage ofurine. Proximal tubule transittime wasmeasured fromanintravenousinjection of 0.02 ml of 10%lissamine green dye. Only thosekidneyswith transit times of< 12 s wereacceptedforstudy.
In vivomicroperfusion techniqueforflux studies.Proximal convo-luted tubules wereperfusedwithathermally insulatedmicroperfusion
pump (Wolfgang Hampel, Berlin, FRG) as previously described (34).The perfusion pipettewasplaced inaproximalloop.Anoil block was placed proximal to the perfusion pipette, and a hole was left proximal to the block for glomerular ultrafiltrate to escape.A collec-tionpipettewasplaced inalateproximal loop,anoil blockinserted
distally,andatimedcollection made. Collectedsampleswerestored under Hepes-equilibrated paraffin oil (35). After the collection the
perfusedsegmentwasfilled withMicrofil(CantonBio-Medical Prod-ucts, Boulder, CO). Onasubsequent day the kidneywasincubated in 6 N hydrochloricacid at37°C for 70 min,allowingdissectionof the Microfil casts and measurement of the length of the perfused segment. In vivomicroperfusion technique for
fluorescence
measurements. Pipettes were placed using adissecting microscope (E. Leitz, Inc., Rockleigh, NJ). Peritubularcapillaries wereperfused aspreviouslydescribed (25) witha 12-14 ;m tip pipette designedto allowrapid
changes betweentwoperfusionfluids.
Thelumenofaproximalconvoluted tubulewasthenperfusedat 40 nl/min for 5-7 min usingathermally insulatedmicroperfusion
pump with a solution containingthe acetoxymethyl derivative of
(2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein (BCECF), as pre-viously described (36). After 5-7min, this pipettewasremovedanda
secondluminal pipettewasplaced in a more distal loop of the same nephron. Thissecond pipette, which was similar to that used in the
peritubularcapillary except that it had a smaller tip (7-9Mum),perfused theproximal tubular lumen inaretrograde fashionaspreviously de-scribed(36, 37). This method of lumen and capillaryperfusionhas beendemonstratedtogivecomplete control of luminal andcapillary
fluids (25, 36).
After placement of the pipettes, the dissecting microscopewas moved out of position, and an epifluorescence microscope (MPV Compact system, E.Leitz,Inc.)wasmovedinto position. Fluorescence intensity was measured as previously described (25) and used to calcu-late cell pH.
Generalmethodsforvesiclestudies. Theactivityoffive amiloride analogs against the antiporter was measured in rabbit renal brush border vesiclesusingtheacridine orange method (6). The brush border vesicles were prepared by
Mg2e
aggregation as previously described (8). Vesicles (400Mgof protein)inpH 6.0 buffer (50 mM Mes/Tris, 250 mMsucrose, 150mMK gluconate) were added to 2 ml of pH 7.5 assaysolution(50 mM Mes/Tris, 250 mM sucrose, 150 mM K glconate, 6
,MMacridine orange, 20 Migvalinomycin)andassayed forNa+/H+ ex-change in an SLM 8225 spectrofluorometer as previously de-scribed (8).
Amiloride hyrochloride dihydrate and its analogs
5-(N-t-butyl)-amiloride, 5-(N,N-dimethyl)amiloride,
54N-ethyl-N-propyl)amiloride
and5-(N-amino-N-methyl)amiloridewere prepared for this study by the procedures described previously (38). Using similar methods,
54N-methyl-N-methallyl)amiloride
wassynthesized. This compound has a melting point of 206-207°C.Perfusion
solutions
In vivo
microperfusion flux
studies: rates of volume, bicarbonate, andglucose absorption. Inthesestudies tubules were perfused with a plasma ultrafiltrate-like solution (TableI,luminal perfusion solution No. 1), except when 4.3 mM amiloridewasaddedto theperfusate.
Then it was necessaryto removesulfate and phosphate from the lu-minalperfusatetoincrease thesolubilityof amiloride(Table I,luminal
perfusionsolution No. 2). This substitution didnotaffect bicarbonate
absorptionwhencompared with tubulesperfused withoutthe
substi-TableL
Perfusion Solutions
Luminalsolutions Capillarysolutions
1 2 3 4 5 6 1 2 3
mM mM mM mM mM mM mM mM mM
NaCI 120 122 120 122 127 120 122 142
NaHCO3 25 25 25 25 25 25 25 5
KCI 5 5 5 5 5 5 5 5 5
MgSO4 1 - 1 1 - 0
CaCl2 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8
Na2HPO4 1 1 1 0
Glucose 5 5 5 5 5 5 5
Alanine 5 5 5 5 5 5 5
Urea 5 5 5 5 5 5 5 5 5
MgCI2
1 1 1 1 1 1Choline
C-
127CholineHCO3 - 25
SITS - 1
Amiloride ±0.9 ±4.3 ±0.9 ±3.6 ±3.6 ±3.6
t-Butylamiloride ±1 ±0.35 ±0.35 ±0.35
tutions(I25±5 [n=8] vs. 124±13 [n=4]pmol/mm-min [P> 0.5]). In thet-butylamiloride studies, it was necessary to dissolve this inhibi-tor indimethylsulfoxide (DMSO) before adding it to the perfusion solution. The final concentration of DMSO in the perfusate was 0.1%, which did not affect the rate ofbicarbonate absorption when compared tothat measured in the absence of DMSO (125±5 [n=8] vs. 127±8 [n
=3] pmol/mm * min [P>0.5]).
Todetermine the rate of glucose absorption, we added tracer amounts(0.0041 mmol) of
['4C]glucose
(New England Nuclear, Bos-ton,MA) to the aboveperfusates.Inaddition, the perfusates contained 0.05% FD and C green dye No. 3 (WarnerJenkinson, St. Louis, MO), to identify the perfused segment, and dialyzed [methoxy-3H]inulin(NewEngland Nuclear) as a volume marker. All solutions were bub-bledwith7%CO2 and 93%
02-In vivomicroperfusionfluorescence studies: effect ofinhibitoron cell pH. Inthesestudies both the lumen and capillaries were perfused with plasmaultrafiltrate-like solutions (Table I, capillary perfusate No. 1 with luminal perfusate No. 3, or capillary perfusate No.2with luminal
perfusateNo.4). The substitutions made in the secondsetof solutions were necessary to keep the higher concentrations of amiloride and
t-butylamiloridein solution. Allsolutionswerebubbledwith7%CO2 and93%
02-In vivomicroperfusionfluorescencestudies: in vivo rate ofNa+/H+ exchangeandamiloride andt-butylamiloride inhibition. Capillaries
were perfused with the same plasma ultrafiltrate-like solution de-scribed above, except that 20 mM bicarbonatewasreplaced with 20 mMchloride, and 1 mM SITSwasadded(Table I, capillary perfusate No. 3). Thesesubstitutions/additionswere madetoallowchanges in the luminalNa+/H+antiporterrate tohaveamaximumeffectoncell pH(36). Theluminal perfusatewaseitherNo. 5 or No.6.Again, all solutionswerebubbledwith7%CO2 and 93%02.
Analysis
Invivomicroperfusion fluxstudies: volume measurements. The total collected volumewas measured usingcalibrated constant-bore
pi-pettes. From this volumeanaliquotwasremoved formeasurementof total CO2and inhibitorconcentration. The remainingvolume was placed inavialcontaininga1:4mixture of aceticacid:Aquasol(New
England Nuclear) forliquid scintillation counting (Tri-Carb460C, Packard InstrumentCo., Inc., Downers Grove, IL). Because the solu-tionscontained both[3H]inulinand
['4C]glucose,
appropriate correc-tionsweremadeforcross-countingbetween theisotopewindows.TotalCO2concentration. The totalCO2concentration in both the
perfusate andcollected fluid was measured using microcalorim-etry(39).
Inhibitorconcentration inperfusateandcollectedfluid. The con-centration of amiloride or t-butylamiloride innanoliter samples of
perfusateand collected fluidwasmeasuredby fluorescenceintensityin an8225
spectrofluorometer
(SLMInstrumentsCo., Urbana, IL) using single photoncounting.Toincrease thesignal-to-noise ratio,allmea-surements weremade in ethanol where amiloride and
t-butylamiloride
exhibita 10-fold increase in fluorescenceintensity. Measurements were madeat
kX.
=360,X,4 =420toavoid the Ramanpeaksofwater (408 nm) and ethanol (400 nm) at X,, = 360. 12.5-nl samples ofperfusateorcollectedfluidwereaddedto500
Al
ethanol in2-mlplastictubestofacilitatemixing.Thesolutionwasthen transferredto500-,l
quartzcuvettes(UvonicInstruments,Plainview, NY).Excitationslits were16nm,emission 8nm.On eachday of measurements, standards wereperformedusing 12.5-nl samples of reference solutions ranging in concentration from 0.05 to 1 mM t-butylamiloride (1 mM t-butyl-amiloridestudies),0.04to0.9 mMamiloride (0.9 mM amiloride stud-ies), or 0.9 to 4.3 mM amiloride (4.3 mM amiloride studies). Over these ranges, fluorescencewas alinearfunction of concentration for bothcompounds (r value ranged from0.960 to0.999 forallstudies).
The limit of detection under these conditions is
10-'
M,orin the undiluted tubular fluid samples, 50X 10-6M.In vivo microperfusion
fluorescence
measurements. As described previously (25),fluorescencewasmeasuredalternatelyat500 and 450nmexcitation (emission 530 nm) using an epifluorescence microscope with interference filters (Corion Corp., Holliston, MA). All results were corrected by subtracting background. The fluorescence excitation ratio
(F500/F450)
was calculated as the mean of the two 500-nm excitation measurements divided by the 450-nm excitation measuremnt. Use of the fluorescence excitation ratio provides a measurement unaffected by changes in dye concentration.2 To convert fluorescent excitation ratios to an apparent cell pH value, we used results of our previously reported intracellular calibration (25).Calculations
Volume measurements. The perfusion rate,
Vp
nl/min,wascalculated asVp
=(Ij/1P)
(Vc),
whereIcandIP
are the collectedfluid and perfusate inulin concentrations, respectively, andVc
is the collectionrate.Volume flux per tubular length,Jo,nl/mm.min,wascalculated as
J,
=(Vp
-VJ)/L,
whereLis theperfused length.
Total CO2 flux measurements. The rate of bicarbonate absorption,
Jacom pmol/mm. min, wascalculated as
Jmco2
=[(CpVp)
-(CcVJ)]/L,
whereCp
andCcare the totalCO2 concentrations of the perfusate and collected fluid, respectively, as determined by microcalorimetry.Glucose absorption. The rateof glucose absorption,
J&
pmol/ mm-min,was calculated asJgu
= 5 mM {Vp
-[V,
X(GcJGp)J}/L,
where
GC/Gp
is the ratio of counts per minute of['4C]in the collected fluid andperfusate,respectively, and 5 mM is the concentration ofcold glucose in the perfusate.Log mean inhibitor concentration. Log mean concentration of ei-ther t-butylamiloride or amiloride was calculated as: log mean con-centration=
(AP
-Aj)/
ln(AWAc),
whereAp
andAc
are the concentra-tion of inhibitor in theperfusateandthe meanconcentration ofinhibi-tor in the collectedfluid,respectively.Initial rate
of
cellpHchange. In some fluorescence studies, the initial rate of cell pH change was calculated, as described previously (25). During a fluid change, fluorescence was followed with 500 nm excitation on a chart recorder. The slope of a line drawn tangenttothe initial deflection[d(F50)/dt]
defined the initial rate of change of 500 nmfluorescence. We have previously demonstrated that fluorescence with 450 nm excitation is not measurably affected by cell pH, and thus can be considered constant (25). The rate of change in the fluorescence excitation ratio[(Fvo/F45o)/dt]
can then be calculated using the for-mula:d(F50/F45o)/dt
=[d(F50)/dt]/F450,
whereF43o
represents the calculated 450 nm excitation fluorescence corrected for background at the time of the fluid change (interpolated from the measurements before and after the fluid change).Statistics
The results are expressedasmeans±SE. In the flux studiesstatistical
significancewas assessedbytheStudent'sttestusingan analysis of
covariance.Inthe in vivo fluorescence studiesexaminingtheeffect of the inhibitorsoncell pH, statisticalsignificancewasassessedby the Student's t test forpaireddata.Inthefluorescence studiesdetermining
the potency of amiloride andt-butylamilorideinvivo,statistical
signif-icance was assessed by the Student's ttestforunpaireddata.
2. In these studies BCECF was excited alternately at 500 and 450 nm andemission was measured at 530 nm. Theseexcitation wavelengths are afair distance from the excitation wavelength of amiloride (360 nm), and thus it is unlikely that there is significant amiloride fluores-cence.Inaddition two observations were made in the in vivo studies. One is that the lumen
of
thetubules do notvisibly fluoresce when amiloride is added to the luminal perfusate, suggesting that little or no fluorescent light is being emitted from the amiloride. The second is that in thestudiesdone toexaminetheeffectof amilorideoncellpH, theaddition ofamilorideort-butylamiloridetotheperfusatedid not change the fluorescent ratioortheintensityof 500or450nm fluores-cence,again suggestingthat the inhibitorsare notemittingany sub-stantialamountof fluorescence.Table II.
Microperfusion Flux
DataControls 0.9 mM Amiloride 4.3 mMAmiloride 1mMt-Butylamilonde
(n = 15) (n =7) (n=7) (n= 10)
J,
(nl/mm
min)
2.13±0.22.02±0.4
1.16±0.2*
0.97±0.2§
Jco2
(pmol/mm
min)
125±490±140
68±8§ 67±10§JGI (pmol/mm min) 30.7±1.3 29.0±2.5 26.5±2.1
20.9±1.8§
Perfusionrate(nl/min) 16.7±0.2 16.6±0.5 16.8±0.2 16.0±0.4
Length (mm) 1.87±0.1 2.32±0.2 1.94±0.2 1.99±0.3
Collected tCO2
(mM)
12.5±0.8 15.6±1.6 17.1±1.4* 18.0± 1.1§*P<0.01 controlvs.inhibitor.
tP
<0.005 controlvs.inhibitor. §P<0.001 controlvs.inhibitor.Results
In vivo
microperfusion
flux studies. In the initialsetofstudiestubuleswere
perfused
invivo withaplasma ultrafiltrate-likesolution containing 25 mM
bicarbonate
(TableI,luminalper-fusate No. 1). Under these
conditions
J,
was 2.13±0.2nl/mm
min, Jtco
was 125±4 pmol/mmmin,
andJglu
was30.7±1.3 pmol/mm * min
(Table
II). When 0.9 mM amiloridewasaddedtothe perfusate
Jv,
andJtco
wereinhibited by 5.2%(P> 0.1) and 28.7% (P <0.005), respectively, and
Jglu
wasunaffected
(30.7±1.3
vs. 29.0±2.5 pmol/mm*minm P>0.5).This degree of inhibition is similartothat observedbyHowlin
etal.(33). Because of the small degree of inhibition of bicar-bonate
absorption
wenextexamined
the effect ofhigher (4.3mM)
amiloride concentrations
(TableI,luminal perfusateNo.2). When
compared
with control tubules,J,
was inhibited45.5% (P<
0.01)
and Jico2wasinhibited
45.9% (P<0.001)(Table II). Again,
Jglu
wasnotaffected(P>0.1).Thisdegree ofinhibition of
bicarbonate
absorption was less than thatpre-dicted by kinetics from vesicle studies assumingthat all of
apical membraneprotonsecretion is effected by
Na+/H+
anti-port.
Brushborder membrane vesicle studies: amiloride analogs. To find a more potent inhibitor of
Na+/H+
exchange, wescreeneda groupof analogues of amiloride in whichthe pro-tonsof the5-amino groupwerereplaced byone ortwoalkyl
groups.To facilitate the screeningprocess, wemeasured
activ-ity against
Na+/H+
exchange in isolated rabbit renal brushborder membrane vesicles. Inhibitory potency of amiloride
and its
5-(NN-dimethyl)-
(1), 5-(N-ethyl-N-propyl)-(2),5-N-methyl-N-methallyl)-
(3), 5-(N-amino-N-methyl)- (4), andFLUORESCENCEUNITS/s /mgprothin/[Noa]
Figure1.Inhibition of Na+/H+ exchange by t-butylamiloride.
Con-centration-response
curveforNa+(10-200 mM)wasobtainedat each of theindicated concentrationsof
t-bu-tylamiloride.Data
plot-ted in Eadie-Hofstee formasmean±SE
forn=3.
5-(N-t-butyl)-
(5)
derivatives
wereexamined.
Compounds 2, 3,
and
5all had
affinity
constantfor inhibitor
(K1)
=2-5
AM
against
the renal
Na+/H' antiporter,
whereas
compounds
1and
4 wereonly about twice
aspotent
asamiloride
(K1
=25
MM
[8]). Unfortunately, compounds
2 and 3 wereonlyslightly
soluble
in aqueous
solutions
and could
notbe used for in vivo
experiments
athigh concentrations. Thus,
amongthe
com-pounds
examined, only
t-butylamiloride
exhibited the
combi-nation of high aqueous
solubility
and potency
against
the
Na+/H'
antiporter.
Before
perfusing
tubules with
t-butylamiloride,
weexam-ined its potency
against
the
Na+/H'
antiporter
in
moredetail
(Fig. 1).
Theseexperiments
showed that
t-butylamiloride
is
amixed inhibitor
of
Na+/H'
exchange
asdescribed for
amilor-ide (8) with
aKi
=2.5
MM.
To showthat
t-butylamiloride
could inhibit
Na+/H'
exchange completely
under
physiologi-cal
conditions,
wemeasured
Na+/H'
exchange
with 200
mMNa'
and 200
MM
t-butylamiloride.
Na+/H'
exchange
wasin-hibited
at96±3%
(n
=4) under these conditions.
In vivo
microperfusion flux studies:
I mMt-butylamiloride.
Next
weexamined
theeffect of
t-butylamiloride
on bicarbon-ateabsorption.
When
I mMof
inhibitor
wasadded
tothe
perfusate (Table
I,
luminal
perfusate
No.1), J,
wasinhibited
54.5% (P
<0.001) and
Jico2
wasinhibited 46.9%
(P
<0.001)
(Table
II).
Inthese
studies,
however,Jgju
wasinhibited
signifi-cantly when compared with control
values,3
(30.7±1.3
vs.20.9±1.8
pmol/mm -min,
P<0.001). Once
again,
Jtco2
wasinhibited less
thanwould
beexpected if
theNa+/H+ antiporter
mediated all of apical membrane proton secretion. Possible
causesfor
this
discrepancy in all series include loss
of
inhibitor
from the
luminal
fluid, resistance
of
the
antiporter
tothe
in-hibitors in vivo, and secondary effects of the inin-hibitors
onluminal
andcell pH.
Presence
of
inhibitor along
the perfused
segment. To ensurethat the inhibitor
waspresent along the entire
perfused
seg-ment, theconcentrations
of
amiloride
and
t-butylamiloride
in
theperfusate
andcollected
fluid were measuredby
fluores-cenceintensity (see Methods).
Ascanbeseenin TableIII,
both3. Aneffectonglucosetransportwasonly observed in the
t-butylami-loride studies.Wefeel itlikely that this isadirect effectonthe Na/glu-cosetransporter,asamiloride analogueshavebeenshowntohavea
higher affinity forthe transporter than doesamiloride(40).However,if
t-butylamilorideinhibitedtheNa+/K+-ATPase,thiswouldfurther in-hibit bicarbonate absorption (18-23) and overestimate the role of Na+/H+ antiporter.
20
co
E
I-z I1J
cn
z
3
u.
0
U-Table III. Concentration
of
Inhibitor in Perfusate and Collected FluidMeasuredconcentration
Log mean Inhibitor Perfusate Collectedfluid Mean collected concentration* permeability
mm mM mM mm cm/s
0.9 mMamiloride 0.8±0.0 0.4-0.8 0.5±0.1 0.7 11.0
4.3 mMamiloride 4.0±0.1 2.9-3.6 3.2±0.1 3.6 6.1
1mMt-butylamiloride 0.99±0.0 0.02-0.18 0.08±0.04 0.35 43.5
* Calculated from theequation: logmeanconcentration=
(Ap
-A,)/ln
(AW/AJ).
amiloride and
t-butylamiloride
wereabsorbed
from the
lu-minal
fluid,
t-butylamiloride
to amuch
greaterextent, consis-tentwith its
morehydrophobic
nature.Loss of
inhibitor could
possibly explain
someof the difference between the observed
inhibition of
Jgco
and that
predicted from
vesicle
studies if
the
Na+/H+
antiporter
mediated all of
apical
membrane
protonsecretion.
We
corrected
for
the loss of
inhibitor
by
using
amodel
of
proximal
bicarbonate
absorption.
In
vivo
rate
ofNa+/H+
exchange activity
and
inhibition by
amiloride and
t-butylamiloride.
Previous studies had left open
the
possibilities
that
in
vivo the
ratepithelium
wasless
sensi-tive
toamiloride
than in the vesicle studies
orthat
amiloride
was notgaining
access tothe
antiporter,
and
thereafter that
useof
these
compounds underestimates
the role of
Na+/H+
ex-change
in
bicarbonate
absorption (33).
To rule
outthese
possi-bilities,
weexamined the
rateof Na+/H+ exchange
activity
in
the
presenceand absence
of the
inhibitors in vivo. The
proto-col
for
these
studies
wassimilar
tothat
previously
described
by
us
(36).
The
peritubular capillaries
wereperfused
with
asolu-tion
containing
5 mM
bicarbonate
and
1 mMSITS
(Table I,
capillary
perfusate
No.
3).
Wehave
previously
shown that
with
this
capillary perfusate
the
basolateral
Na+/(HCOj)0,>
trans-porter
is
inhibited,
but that cell
pH
isnotalkalinized
enough
tosuppress
the
luminal
membrane
Na+/H+
antiporter
(36).
This
maneuver,then,
allows
changes
in the
rateof
the
luminal
membrane
Na+/H+
antiporter
tohave
the
greatesteffect
oncell
pH
(36).
The
luminal
perfusate
waschanged
between
onecontain-ing
152 mMNa+
andone with zeroNa+ (Table I,
luminalperfusates
No.5
and No.
6). Mean luminal inhibitor
concen-trations from the flux
studies
wereused.
Inthe
controlcondi-tion when the luminal Na+ concentracondi-tion
wasdecreased
from
152
to0 mM, cell pH decreased
at a rateof
2.51±0.35 pHunits/min
(Table IV). When
luminal Na+
was returned to 152mM,
cell
pH
increased
at a rateof 1.78±0.34 pHunits/min.
When
3.6 mM
amiloride
wasadded to theluminal
perfusate,
the
rateof change in cell pH,
uponluminal
Na+ removaland
replacement,
wasreduced 87
and
91%,respectively
(TableIV). Insimilar studies with t-butylamiloride
(luminalperfusates No.5 and
No.
6, andcapillary
perfusate No. 3) (TableI),
0.35mM
inhibitor reduced
the rate of cellpH change by 86 and89%, respectively (Table
IV). A typicaltracing is shown in Fig.2.
Ingeneral, these
studies
demonstrate that the Na+/H+ anti-porteris
verysensitive
toamiloride
and its analogue in vivo.Effect of amiloride
and
t-butylamiloride
on intracellularpH.
Aneffect of the inhibitors
on cell pH would affect theinterpretation
of the
observedinhibition
of bicarbonateab-sorption.
To
rule
outthis possibility,
theeffect
ofamiloride
and
t-butylamiloride
oncell
pH wasdetermined in vivo.Peri-tubular
capillaries
wereperfused
with a plasmaultrafiltrate-like solution (Table I, capillary perfusate
No. 1 or No. 2).Thetubular
lumens
wereperfused with
aplasmaultrafiltrate-like
solution
with
sulfate
and phosphate
removed toincrease
inhib-itor
solubility
(Table I,
luminal
perfusates
No.3 or No. 4). Theinhibitor
wasadded
tothe luminal
fluid
only, atamiloride
concentrations
of 0.9
or3.6
mM,
or at-butylamiloride
con-centration of 0.35 mM.
As canbe
seenin Table
V,cell
pHwas notaffected
significantly
in
anyof the studies, suggesting
thatTable
IV. Potencyof
Inhibitors
In VivoRateof change Percent Luminal[Na]changes of cellpH inhibition*
pH units/min
Control (n=8)
Na'152to0 2.51±0.35
Na'0to 152 1.78±0.34
3.6mMamiloride(n=7)
Na' 152to0 0.33±0.06 87
Na+
0 to 152 0.16±0.07 910.35mMt-butylamiloride (n=6)
Na+ 152to0 0.36±0.05 86
NaO0to 152 0.19±0.03 89
* Calculatedas
inhibited/control
rateofchange
in cellpH.
LUMINAL
[No+],meq/liter
11521
0 | 1521 ----I~~~~~~~~~~~~~~~~~
Fluorescence Intensity )tex a500nm
Xem'530nm
Control
11521
0 1152f
1~~
t-butylomiloride
Figure 2. Inhibition ofNa+/H+
exchange
by
t-butylamiloride
in vivo:typicaltracing.Fluorescence
intensity
with 500nmexcitation(pH-sensitivewavelength)as afunction of time
during
luminalNa+
re-moval and readdition.Table
V.Effect of
Inhibitor on CellpH
Cell pH
-inhibitor* +inhibitor P value
0.9mMamiloride
(n
=7)
7.28±0.02 7.28±0.02 >0.53.6mM amiloride (n=7) 7.28±0.06 7.28±0.06 >0.2 0.35 mMt-butylamiloride
(n=7) 7.23±0.04 7.21±0.04 >0.2
* These valuesarethemeansof thepre-andpost-controlvalues.
the inhibition
ofbicarbonateabsorption
thatwasobserved was notsecondarily
alteredby
changes
in cellpH. (In
these studies there was aprogressive
cell alkalinization that has beenob-served by
uspreviously
(36).
To determine the effect of theinhibitors
on cellpH,
we meaned the pre- and post-controlvalues
for
comparison with the
experimental period
during
which the
inhibitor
waspresent.)
Thisfinding
is somewhatexpected
as we havepreviously
shown that the basolateralmembrane
Na+/(HCO)>1
transporter
in themajor
determi-nantof cell pH under
physiological
conditions (36).
Role
of
NaI/H+
exchange
inbicarbonateabsorption.
In themicroperfusion
flux
studies,
4.3 mMamiloride
and 1 mMt-butylamiloride only
inhibitedJ&co2
46 and47%,
respectively.
However, inhibition
of theNa+/H'
antiporter
did increase the pHand
bicarbonate concentration
ofthe collected fluid(Table
II).
This increase in the luminal bicarbonate axial concentra-tionprofile
would beexpected
to stimulate bicarbonateab-sorption (35).
TocorrecttheratesofNa+/H+ exchange
activity
for the
varying
bicarbonate
concentrationprofiles,
weusedamodification
of
ourpreviously published
model
of
bicarbon-ateabsorption
intheproximal
tubule(41).
The details of themodel
calculations
arepresented
inthe
Appendix.
In addition tocorrecting for changes
in the
bicarbonate
concentrationprofile,
the model also
adjusted
the
magnitude
of bicarbonate
inhibiton
for the
varying
inhibitor concentration
profile.
Theresults
of the model
arepresented
inTable
VI.The observed
inhibition
in column
oneis the data from reference 33
andTable
VI.Contribution of
Na+/H+
Exchange to
Jtco2
Conribution Observed Predicted of Na+/H+ inhibition of inhibition exchangeto
Atco, ofNa+/H+*
JtCO2*
(%) (%) (%)
0.9 mM amiloride
21.40-28.711
31.3-33.2 68.4-86.44.3mMamiloride 45.9 68.1 67.4
1.0 mMt-butylamiloride 46.9 73.2 64.1
*Calculated from model presented inAppendix. Corrects for
differ-encein axial luminal bicarbonate concentrationprofilebetweenthe
studies,andfor the varying inhibitor concentrationalong the per-fusedsegment.
tCalculatedasthe observedinhibition/predictedinhibition.See text forfurther description.
§Data arefrom reference 33. 11DataarefromTableII.
Table
II.The
predicted
inhibition of bicarbonate
absorption
(second column)
is calculated from themodel
determined
in-hibition of
J1co assuming
that
all ofJLco2
is mediated by the
antiporter.
This rate ofJhco2
has
beenadjusted
for
bothchanges
inthe axialbicarbonate
concentrationprofile
and for the loss ofinhibitor along
theperfused
segment. Thecontribu-tion
ofNa+/H+ exchange
toJco2
canbe determined from the
ratio of these
twovalues
(third column).
Asshown,
64.1-86.4%
ofbicarbonate absorption
wasmediated by the
antiporter.
The 65%Na+/H+ antiporter contribution
calcu-lated with 4.3
mMamiloride
and 1 mM t-butylamiloride are themoreaccurateestimates.Discussion
To
examinethe
role of theapical
membraneNa+/H+
anti-porter inbicarbonate absorption,
weperfused proximal
con-voluted
tubules
in vivo with 4.3 mM amiloride and amorepotent
amiloride analogue, t-butylamiloride.
In the search foran
amiloride analogue
that could be used to achieve greaterinhibition of Na+/H+ exchange
invivo,
we had twomajor
problems. First,
because the more potent amilorideanalogues
have hydrophobic
groups on the5-amino
group, theyhave
greaterlipid/water partition
coefficients anddecreased
aqueoussolubility
when compared withamiloride. Second,
although t-butylamiloride
was more soluble than someana-logues,
itexhibited significant
uptake into tissues and left theluminal
compartment.However,
even atthe measured meanluminal
concentration,Na+/H+
exchange activity was inhib-ited aspredicted.
While it isconceivable that bulky
hydro-philic
groupsonthe 5-aminonitrogen
atomcouldovercomethese
difficulties,
the compound of this class which we have examined(5-[N-amino-N-methyl]amiloride)
is not much more potentthanamiloride
against theNa+/H+
exchanger.As shown in Table II, 0.9 mM
amiloride,
4.3 mM amilor-ide, and 1 mMt-butylamiloride
added to the luminalfluid
inhibited
Jtco
by 28.7, 45.9, and 46.9%,respectively. Before
utilizing these data todetermine
thecontribution of
Na+/H+ exchange to bicarbonateabsorption,
weaddressed
four poten-tial complications:(a)
the possibility that theinhibitors leave
the luminalcompartment and therefore
are not present along the entire length of the perfusedsegment, (b) the
possibility that the inhibitors are not as potent against theantiporter
in vivo as in vesiclepreparations,
(c)thepossibility
that the pres-enceof theinhibitors changed
cell pH,which secondarily af-fectedbicarbonate
absorption, and (d) the effect ofchanges
in theaxialluminal bicarbonate concentration
profile whenvary-ing
amountsof bicarbonate absorption
have beeninhibited.
Loss of inhibitor along the perfused segment. As shown in
Table III, theproximal tubule is permeable
toboth
amiloride andt-butylamiloride,
more so tot-butylamiloride
as isex-pected
because of its morehydrophobic
nature. Tocalculate aare
required
todefine the mechanisms of inhibitor efflux
invivo.
Potency ofinhibitors in vivo. The potency of amiloride and
t-butylamiloride
wasexamined by comparing
the ability oftheinhibitors
toreduce antiporter activity in vivo
to that observedin
vesicle
studies. These studies examined the effect of
luminal Na+removal
oncell
pH. As shownin
Table IV, the rate ofchange in cell pH
wasreduced by 89% when 3.6
mMamiloride
wasadded
totheluminal fluid,
and by 87.5% when 0.35 mMt-butylamiloride
wasadded.
For
amiloride,
ratvesicle
data havefound
a KNa of 8.3 mM(43)
and
aKi
of
48
gm
(44) for the
Na+/H'
anitporter.
Underthe
conditions
of
ouramiloride studies,
(Na'
concentration
of152 mM and amiloride
concentration of
3.6
mM),it
can becalculated that 80% of antiporter activity should
beinhibited.
In our
studies, the addition of
3.6 mMamiloride
to thelu-minal perfusate
reduced theeffect of changing
luminal Na+concentration
by 89%,
avalue similar
to80%. For
t-butylami-loride
the
KN,
in vesicles is
15 mM(rabbit brush
border) andthe
Ki
is 2.5 ,M. Under the conditions of the in vivo studies
(Na+
concentration of
152
mMand
aninhibitor concentration
of
0.35
mM),
it is predicted
that
92.6%
of
antiporter
activity
would
beinhibited. The
samecalculation using
aKNa of
8.3 mM(rat brush border
vesicle
studies
[43]),
would
estimate that
87.9% of Na+/H+
activity
would be inhibited. These numbers
aresimilar
to ourresults in vivo, in which 87.5% of antiporter
activity
wasinhibited.
These
studies
suggestthat
the failure
toobtain
morecom-plete inhibition
of bicarbonate absorption
in the
flux
studies
cannotbe
attributed
todecreased
sensitivity
of the
epithelium
to
the
inhibitors,
or to aninability of
the
inhibitors
togain
access to
the
antiporter.
These studies also demonstrate that in
this
setting
allof
the
effect of luminal Na+
oncell
pH
is due
toamiloride-sensitive Na+/H+ antiporter activity. This study
rules
outthe
presenceof amiloride-insensitive Na+/H+
anti-portin
this
preparation.
If
amiloride-insensitive Na+/H+
anti-porteractivity
werepresent,then the
effect of
aluminal
Na+
concentration change
oncell
pH
would
nothave been
blocked
aspredicted
in the
presenceof the inhibitors. Nakhoul and
Boron(45) and
Siebens
and Boron
(46)
have
recently
reported
an
important
role for
amiloride-insensitive Na+/acetate
and
Na+/lactate
cotransportin cell
pH
regulation
in the
proximal
tubule. However, neither
acetate norlactate
werepresentin
oursolutions.
Effect
of
inhibitors
oncell
and luminal pH.
Asshown in
Table
V,
inhibition
of the
Na+/H+
antiporter by amiloride
ort-butylamiloride did
notaffect cell
pH
in
situations where the
basolateral
mechanism for
bicarbonate exit is
operational.
Asmentioned, this
finding
is somewhat
expected
aswehave
pre-viously shown that the basolateral membrane
Na+/(HCOj),>,
transporter
is the
major determinant
of
cellpH
(36).
As
described in
theAppendix,
weaccounted for
changes
inthe axial luminal bicarbonate concentration
profile
by
modify-ing
aprevious
model. The
results,
aspresented
inTable
VI,
show that
-65%
ofbicarbonate
absorption
is
mediated
by
the
amiloride-sensitive Na+/H+
antiporter.
Comparison
with
previous
studies.
Asmentioned
above,
previous
in
vivo and in vitro
microperfusion
studies
havecon-sistently
found
that80-100% of bicarbonate
absorption
is
in-hibited by luminal
andperitubular
Na+ removal
(18-23).
While
these studies
wereinitially
interpreted
toimply
that80-100%
of
apical
membrane proton secretion is mediated
by
Na+/H+ exchange, this interpretation has recently been
com-plicated
bythedemonstration
of aNa+-coupled
basolateral membranebicarbonate exit
step(24-30). Based on the present results,the observed
80-100%inhibition
ofJ.co,
in previousstudies
canbe explained by
a 65% inhibition of apicalmem-brane
protonsecretion (Na+/H+ antiporter)
and > 90%inhibi-tion
of
basolateral membrane bicarbonate
exit(Na+/(HCOj)>1
symporter).
Microperfusion studies
havealso addressed the role of theapical
membrane Na+/H+ antiporter
byexamining the effectof Na+/K+-ATPase inhibition
onJ.co
The observedinhibi-tion, which has varied from
0 to 100%(18-23,
47), also cannotbe
specifically attributed
toeffects
on anapical
membrane Na+/H+antiporter.
If
aluminal membrane Na+-independent,
amiloride-in-sensitive
mechanism of
protonsecretion exists,
assuggested bythe
presentstudies,
then
onelikely possibility is
an H+-ATP-ase.Evidence for
such
an ATPase has been found in brushborder membrane
vesicles
and
endosomes derived from therenal
cortex(1 1-17).
Alsoin
supportof
an apical membrane H+-ATPasein the
ratis
thefinding
byFromter and Gessner of anacetazolamide-inhibitable
lumen-positive potential
differ-ence(37).
Alumen-positive
potential difference,
however, has not beenobserved in the rabbit
proximal tubule (48-50).Schwartz and
Al-Awqati
(51) have found further evidence
for
anapical
membrane
H+-ATPase in the in vitro perfused
proximal straight tubule.
Intheir
studies,CO2
stimulated
fu-sion
of
H+-ATPase-containing
intracellular vesicles with
theluminal membrane, suggesting the possibility of
aregulatory stepfor
participation of
these
pumpsin
bicarbonate
absorp-tion
in
the
proximal
tubule.
Arole
for these
protonpumpsin
transepithelial
transportis
supported by the
finding
that
dicy-clohexylcarbodiimide (DCCD) inhibits bicarbonate
absorp-tion (52); however, the
nonspecific
effects of DCCD
areof
concern.Insummary,
the
addition of high concentrations of
ami-loride
or morepotentamiloride analogues
tothe
luminal
per-fusate
inhibited
bicarbonate
absorption by
only 46% in the in
vivo
microperfused
ratproximal convoluted tubule. After
rul-ing
outsecondary effects
oncell and
luminal pH, the
possibil-ity
that the
epithelium
is insensitive
tothe
inhibitors
in
vivo,
and
the
effects
of
varying inhibitor concentration profiles,
weconclude that
twomechanisms of
protonsecretion exist in the
apical
membrane and
contribute
totransepithelial
bicarbonate
absorption.
The
amiloride-sensitive Na+/H+
antiporter
is
probably
the
major
mechanism.
Anadditional
mechanism is
Na+-independent
and
amiloride-insensitive.
Based
onother
studies
(11-17, 51, 52)
the
mostlikely
mechanism for
this
proton
secretion is
aH+-ATPase.
Appendix
The model usedtocalculate therate ofbicarbonate absorption per millimeterperfusedtubuleisamodification ofourpreviously
pub-lished model(41). Briefly,in the model theratesof bicarbonate
trans-portwere integrated alongthelength of the
perfused segment using
Euler's method. Theproximaltubulewasconsideredtobeacylinder
of volumeabsorption.Therateof transcellular proton secretion was
assumedtobetotally mediated by theapical membraneNa+/H+ anti-porter. After calculating the antiporterrate
(p),
as afunction of the abovedeterminants, it wasmodified fortheeffect of the inhibitorusing thefollowing equation:jINH
=[[Na+]x
+KNet
]*J([Ni.a+jx +KNa+
-4[
+([I]h/Ki)]}]P
(Al1)
where [Na+]x is the luminal Na+concentration,[I],
is the inhibitorconcentrationenteringtheintegration interval, KNais theaffinityof theantiporter for Na (8.3 mM), andKiis theaffinity for the inhibitor
(48AM
for amiloride and 2.5AM
fort-butylamiloride).4Theuseofthisequationtodetermine therateof the
Na+/H'
anitporterassumesthat thereis onlycompetitiveinhibition of theantiporter byeither inhibi-tor. Ifnoncompetitive inhibition of the antiporteris also involved,then the model will underestimate thepredictedinhibition ofJtco2and therefore overestimate the role of the
Na+/H'
antiporterinmediating transepithelialbicarbonateabsorption.The initialboundarycondition(s) for flowrate wastheperfusion
rate,and for luminal[HCO5],
[Na'],
and[inhibitor]weretheperfusate concentrationsof these substances. Peritubular HCO- wasthemea-suredplasmabicarbonate concentration. After calculation of therate
of transcellular protonsecretion,therateofnetbicarbonateabsorption
wascalculated bycorrecting for paracellularHCO- leakaspreviously
described (4 1).
Becausewehaveshown that bothamiloride andt-butylamiloride
aretransportedoutof the luminal compartment, theratesofNa+/H+
exchangewerecorrected for the concentrationprofileof the inhibitor. To dothis,weassumed that the inhibitorswereabsorbedfrom the tubule byafirst-order process described by theequation:5
JINH=
PINH
[I](A2)
whereJINH is the rate ofinhibitor lossfrom the luminal compartment and
PINH
is the inhibitorpermeability.Pm
wascalculatedby an itera-tive methodusingthe measuredperfusedandcollectedfluid inhibitor concentrations. The calculatedpermeability was then used in the model along with Eq. A2todetermine therateofinhibitorefflux.The concentrationofinhibitor along the tubulewasthendetermined using themassbalanceequation:Vh+,U=
VX(I] -JMI&X
(A3)where
[IJh+^,
is the inhibitor concentration leaving the integrationinterval, [I],, is the inhibitor concentration entering the integration
interval, Axis the size of the integration interval, V.is theluminal flow
4. Tojustify the use of the kinetic constants defined from vesicle studieswetestedtheinhibitor potencies in vivo (see Table IV). While these invivo studieswerenotsufficienttodefine a uniqueKNaand
Ki,
they did confirm the abilities of thekinetic constants to predict themagnitudeofinhibitionattheconcentration of Na and inhibitor used, which weresimilar to those used in the flux studies.
5. Inthesestudiesweassumedfirst-order kinetics for inhibitorefflux.
The worstpossiblesituation would be that the inhibitors left the lu-minal compartment at the beginning of the perfused segment and that the collectedconcentratioinwaspresent along the entire perfused seg-ment.Calculation of the predicted inhibition of the Na+/H+ antiporter under theseconditions doesnotchange the conclusion. In the 4.3 mM amiloridestudies,theNa+/H+ antiporter would be inhibited 81.7% in the presence of 4.0mMamiloride (measured perfusate concentration) and78.1% in the presence of 3.2mMamiloride (mean collected
con-centration). Inthe 1 mMt-butylamiloridestudies, the Na+/H+ anti-porter would be inhibited 95.5% inthepresence of 0.99mM t-butyl-amiloride (measured perfusate concentration) and 63.1% in the pres-ence of 0.08 mM t-butylamiloride (mean collected concentration). Both 78.1 and63%inhibitionarestill greater than the46%inhibition of
Jco2
thatwasobserved inthesetwoseries.rateenteringtheintegrationinterval,and Vx+, is the luminal flowrate leavingtheintegrationinterval.
Acknowledgments
The authorsgratefullyacknowledgetheexcellent secretarial assistance of Gracie Bernacki.
These studiesweresupportedbygrantsAM-27045 and AM-34127 from the National Institute ofArthritis, Diabetes,andDigestiveand
Kidney Diseases, bygrantDK-38875-01 from the National Institute of
Diabetes, Digestive, and Kidney Diseases, and byagrantfrom the Academic Senate CommitteeonResearchattheUniversityof
Califor-nia,SanFrancisco.Dr.Alpernis therecipientofaClinicalInvestigator
Award(AM-0 1229)from theNational Institute of
Arthritis, Diabetes,
andDigestiveandKidneyDiseases.
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