Sodium chloride absorption by the urinary
bladder of the winter flounder. A
thiazide-sensitive, electrically neutral transport system.
J B Stokes
J Clin Invest.
1984;
74(1)
:7-16.
https://doi.org/10.1172/JCI111420
.
The urinary bladder of the winter flounder absorbs NaCl by a process independent of the
transepithelial voltage. In contrast to most other epithelia which have a neutral
NaCl-absorptive system, the flounder bladder has a high transepithelial resistance. This feature
simplifies analysis of the cellular transport system because the rate of ion transfer through
the paracellular pathway is rather low. Experiments were designed to distinguish among
three possible mechanisms of neutral NaCl absorption: (a) Na/K/2Cl cotransport; (b) parallel
Na/H and Cl/OH exchange; (c) and simple NaCl cotransport. A clear interdependency of Na
and Cl for net absorption was demonstrated. NaCl absorption was not dependent on
mucosal K and was minimally sensitive to loop diuretics. Thus a Na/K/2Cl transport system
was unlikely. The mechanism was not parallel exchange as evidenced by insensitivity to
amiloride and to 4,4'-diisothiocyano-2,2'-disulfonic stilbene, an inhibitor of anion exchange.
In addition, inhibitors of carbonic anhydrase had no effect. Net absorption was almost
completely abolished by hydrochlorothiazide (0.1 mM). Its action was rapid, reversible, and
effective only from the mucosal surface. Metolazone, a structurally dissimilar diuretic in the
benzothiadiazide class had qualitatively similar actions. The mechanism of NaCl absorption
in this tissue appears to be a simple interdependent process. Its inhibition by thiazide
diuretics appears to be a unique feature. The flounder bladder may be a model for NaCl […]
Research Article
Sodium Chloride Absorption
by the Urinary Bladder
of the
Winter Flounder
A
Thiazide-sensitive,
Electrically
Neutral
Transport System
John B. Stokes,
with the technicalassistanceof
Ivan Lee and Melissa D'Amico
The Mount Desert Island Biological Laboratory, SalsburyCove,
Maine04672;Laboratory of Epithelial Transport andKidney
Physiology, Department of Internal Medicine, University ofIowa,
Iowa City, Iowa52240
Abstract. The
urinary
bladder of the winter
flounder
absorbs
NaCl by
a process
independent of
the
transepithelial
voltage.
In
contrast to most
other
epithelia
which
have a neutral
NaCl-absorptive
system, the flounder
bladder has
a
high
transepithelial
resistance. This feature
simplifies analysis
of the cellular transport system because
the rate
of ion transfer through
the
paracellular pathway
is
rather low.
Experiments
were
designed
to
distinguish
among three
possible
mechanisms of neutral
NaCl
ab-sorption: (a) Na/K/2Cl
cotransport;
(b) parallel Na/H
and
Cl/OH
exchange;
(c)
and
simple
NaCl cotransport.
A
clear
interdependency
of
Na
and
Cl
for
netabsorption
was
demonstrated. NaCl
absorption
was not
dependent
on mucosal
K
and
was
minimally
sensitive
to
loop
di-uretics.
Thus a
Na/K/2Cl
transport system
was
unlikely.
The
mechanism
was not
parallel
exchange
as
evidenced
by
insensitivity
to
amiloride
and to
4,4'-diisothiocyano-2,2'-disulfonic stilbene,
an
inhibitor
of anion
exchange.
In
addition, inhibitors of carbonic
anhydrase had no
effect.
Net
absorption
was
almost
completely abolished
by
hy-drochlorothiazide
(0.1 mM).
Its
action
was
rapid,
re-versible,
and
effective
only
from the mucosal
surface.
Metolazone,
a
structurally
dissimilar diuretic
in the
ben-zothiadiazide
class had
qualitatively
similar
actions. The
Dr. Stokes is an Established Investigator of the American Heart Asso-ciation.
Received for publication 11 October 1983 and in revised form 6 December 1983.
mechanism of NaCl absorption in this tissue appears to
be
a
simple interdependent
process. Its inhibition
by
thia-zide
diuretics
appears to be a
unique
feature. The flounder
bladder
may be a model for NaCl absorption in the distal
renal
tubule.
Introduction
Thereis now considerable evidence indicating that NaCl can
beabsorbed across the
apical
(luminal)
membrane of certain epitheliaby electrically
silent processes(1-3).
Such processes arecurrently believed to consist of three separate mechanisms forNaCl entry into the cell. Onewidely
studied mechanism is thatby which Na, K,
andCl
arecoupled.
Examples of
this
process can be found in Ehrlich ascites tumor cells (4), the thick ascending limb of Henle's loop (5-7), the intestine of the winter flounder(8), and the distal nephron of the amphiuma (9,10).
The
systemis
characterized
byits dependence of all three
ions toeffect net NaCl transport and its inhibitionby loop
diuretics such asfurosemide. The apparentstoichiometry
is 1Na/
1 K/ 2Cl.
Asecondmechanism for
neutral NaClabsorption is
parallel countertransport systems forNa/H
andCl/OH'
exchange.
When the systems areoperating
atthe same rate, netNaCl absorption
occurs
neutrally.
Thesedual exchangers
canbe experimentally separatedby
selective inhibitors; amiloride
inhigh
concentrations (1 mM)inhibits
Na/H exchange(1
1), andstilbenes (inhibitors of anion exchange processes) inhibit Cl/OH transport (12, 13).Epithelia where
these parallel systems operate usually containcarbonic
anhydrase and are sensitive to inhibitors of the enzyme. Examplesof this
system include the rat ( 13, 14) and rabbit ( 15) smallintestine,
thecortical thick ascending limb of Henle's loop1. Inthis article the anion exchange process will be designated asC1/
OH exchange because the system was nominally freeofCO2.However, itis possible that this putative exchanger might utilize HCO- rather thanOH-, especially in vivo where HCO- is more abundant.
7 NaCI TransportinFlounder Bladder
J.Clin. Invest.
©TheAmerican Society for Clinical Investigation, Inc.
ofthe mouse (16), and the rabbitproximal tubule(17-19).The
third mechanism by whichneutralNaCiabsorption occurs
in-volves "simple"
NaCl
cotransport. Although this mechanism has been postulated for many epithelia, clear evidence of its existence involves, at a minimum,demonstration that the othertwo mechanisms are not present. This system is not so well
characterized
as are the first two, and thus the extent to which it contributes to neutral NaCl transport is not clear. There is someevidence
that it may be present in the apical membrane of the Necturus gallbladder (20).The
urinary
bladderof
the winter founder is another example of anepithelium which possesses a neutral NaCltransport sys-tem.Renfro (21, 22) and Dawson and Andrew (23) have dem-onstrated that net Na andC1
absorption occur at approximately equal rates. Furthermore, net transport of either species is atleast partially dependent on the presence of the other (22). A
similar
interdependence of Na and C1 absorption has beende-scribed
in the trout urinary bladder (24). However, the flounder bladderdiffers
from many other epithelia that absorb NaCl neutrallyin
at least oneimportant
aspect. Whereas other epithelia have ahigh
transepithelial electrical conductance
(GT),2 typically4-20 mS/cm2,
the GT of the flounder bladder is much lower,typically 0.2-1.5 mS/cm2.
Tissues with GT as low as this usually have arelatively
lowparacellular (passive)
transport rate. In thatthis
component represents only a fraction of the cellular(active)
transport, theanalysis of
cellulartransport
istechnically
easier.
Thus,this
epithelium
is well suited for a detailedeval-uation
of the
natureof NaCl
transport.The
resultsof the experiments demonstrate
that theflounder
urinary bladder has
acombination of properties
that have notbeen
previously
described.
NaClabsorption is
aninterdependent
process
operating independently of
mucosal K.Furthermore,
the
systemis selectively
inhibitedby thiazide diuretics.Methods
Winter flounder,
Pseudopleuronectes
americanus, were obtained (bytrawl) off thecoastof Mount DesertIsland, Maine,in June andJuly
andmaintained inflowingseawater tanks forupto10 d before death. After cervicaltransection,the
bladder3was
dissected free ofthe mesentary and thegonad, which surrounds the distalportion.
Themuscularlayerremained attachedtotheepithelium. Thebladderswereopened
lon-gitudinallyandpinned,mucosal side up,onasoft
plastic
sheet. A section2. Abbreviations usedin this
paper: 8-Br-cAMP,
8-bromoadenosine3',5'-cyclic monophosphate;
DIDS,
4,4'-diisothiocyano-2,2'-disulfonic
stilbene;gj,partialionicconductance;
GT, transepithelial
conductance; HCTZ,hydrochlorothiazide; I.e,
shortcircuit current;J;, unidirectional flux;ims,
mucosa-to-serosaflux;
J",
serosa-to-mucosaflux; VT, transepithelial
voltage.
3. Technicallyspeaking,this organ isabladder insofarasit actsas a
reservoir for urine. In contrast to
urinary
bladders ofamphibia,
theembryonic originof the flounder bladder is mesodermal and is formed
bythe fusion of the
mesonephric
ducts(21).
Therefore, itmaybeap-propriatetoconsider this tissueanextension of thekidney.
ofthe-bladder was then mounted in concentric plastic rings as described byDawson and Andrew (23). Depending on the size of the bladder, from one to four pieces of tissue (usually two) could be obtained. The rings, which exposed 1.25 cm2 of the bladder, were mounted in Ussing chambers and bathed with 10 ml of solution on both mucosal and serosal surfaces. The solution wasamodified Forster's solution containing (inmillimoles/liter) NaCl, 140; KC1, 2.5;CaCl2, 1.5; MgCl2, 1.0; N-2-hydroxyethylpiperazine-N'-2 ethane sulfonic acid (Hepes), 7.5; Na Hepes, 7.5;and glucose, 5. Osmolality was 305±3 and pH was 7.5. In solutions where Cl was removed, gluconate salts of Na, K, and Ca were substituted. The S04 salt was used for Mg. In solutions where Na was removed, choline Cl was substituted forNaCl and the buffer system was Tris-Hepes withapH = 7.5.
The bladders were mounted in the chambers and gassed (and stirred) using room air and allowed to equilibrate at room temperature. Electrical measurements were made using two electrodes connected to each side of the chamber via polyethylene tubing containing an appropriate elec-trolyte solution in agar. One electrode measured transepithelial voltage (VT) and the other passed current. The electrical conventions used for these studies were that VT was measured with the serosal (basolateral) surface being ground. A positive current represents a flow of positive charge from serosa to mucosa. In most cases, VT was clamped to0mV
by a voltage clamp made by the University of Iowa Bioengineering Department. GT was determined from the change in the transepithelial current after brief (0.5-1.0 s) pulses of 10 mV imposed every 100-200 s. The series resistance of the solutions and the bridges were compensated before inserting the tissue so that the current deflection reflected only tissue conductance.
Preliminary evaluation of this tissue indicated that GT was unstable with erratic behavior apparently corresponding to muscular contraction. Dawson and Andrew (23) had previously observed this phenomenon and reported that verapamil abolished the variations in GT without affecting the short circuit current (I,,) or the NaCI transport rate. Our experience was similar to theirs and consequently 50MMverapamil was constantly present in the serosal solution. In four experiments, verapamil had no effect on I, GT, or mucosa-to-serosa Na flux
(JN.,
ranging from 5.83±0.96 to 5.60±1.02AM/cm2'
h). With verapamil present,JN,
I4,
and GT were stable for up to 8 h.
Unidirectional
22Na
and/or -6C1fluxes were made by adding the appropriate isotope to one side of the chamber to reach an activity of 0.5-1.0MCi/ml.
Samples were made from the "hot" side every 20 min formucosal-to-serosal (absorptive) fluxes and every 30 min for serosal-to-mucosal fluxes. The"cold"side was sampled by removing 1 ml and replacing it with an equal volume of solution. At the midpoint of each collection a small aliquot was taken from thehotside and diluted 100-fold, and an aliquot was counted. Atalltimes the isotope activityonthe cold side was <1% of that ofthe hot side. In general,two flux measurements were made for eachperiodorexperimentalmaneuver.
The expression used to calculate the unidirectional flux
(JA)
is:JA
= (cpm)/(pA t), where cpm is the counts per minute of the tracer
appearing on the cold side during the fluxperiod,p is the
specific activity
of the tracer
(cpm/MM),
Ais thearea(1.25cm2),
andtis the time of collection. When22Na
and36CIfluxesweremeasuredtogether,separate aliquots were obtained for gamma counting and liquid scintillation counting. Standard corrections forcrossoverwereemployed.
Itsoon became apparentthattheNaCItransportratesvaried
widely,
even betweentwopieces ofthe samebladder. Thus,theuseof
paired
TableI. ComparisonofJ7aand J"¶after Ouabainand Papaverine with J" andJ"7
Flux
Mucosa-to-serosa after Serosa-to-mucosa ouabain andpapaverine
(n=9) (n=6) P
AfI/cm2*h UMI/cm2-h
PharmaceuticalCo.(Orange, NJ). Verapamilwaspreparedas a50 mM
aqueousstock.
Resultsarepresentedasmean±standarderror. Statisticalanalysis
wasby analysisofvariance,orpairedorunpairedttestasappropriate.
For paired or unpaired comparisons, a significant difference was
concluded when P < 0.05. For experiments where multiple paired comparisonswere made,asignificantdifference wasconcluded when P< 0.025.
JNa
Jcl
0.51 ±0.05 1.41 ±0.13
0.58
±0.11
1.71 ±0.18
Concentrationof papaverine in the mucosal solution, 0.5 mM;
con-centration of ouabain in theserosalsolution,0.1 mM.Na and Cl fluxesweredeterminedsimultaneously. Pvaluescompareserosato mucosa and mucosato serosafluxes of thesameion (by unpaired
analysis).
(23) had previously shown that either papaverine or ouabain could inhibitnet Na andCl transport. In ourpreliminary experiments, we noted thatouabain didnot alwaysabolish the
I,
andneverdidsoasquicklyaspapaverine.Wethus electedto usetheseagentsin combination (papaverine 0.5mM to mucosaandouabain 0.1mM toserosa)toinhibit activetransport.With this methodweobtainedanestimate of the passive flux foreachtissue.Thecomparison oftheinhibitedmucosa-to-serosa
fluxandthe serosa-to-mucosa flux forNa andCl is depicted in Table
I.ForeachexperimentNa andC1weredeterminedsimultaneously.The tissueswereeither exposedtoouabain and papaverine for 20 min(for
mucosa-to-serosa flux) or untreated (for serosa-to-mucosa flux). The mean valuesfor theNafluxesarenotdifferent by paired analysis. The
sameistruefor the Cl fluxes. Thus,J"' followingouabain and papaverine is representative for Jsm for that tissue.
Inexperiments where Na, K, orCl concentrations wereincreased incrementally,they were added to the mucosalsolutionas aconcentrated solution.Attheendof the appropriate flux period,asmallaliquotwas
taken for measurement of the actualconcentration. Na and K were
determined by flame photometry (Instrumentation Laboratory, Inc.,
Lexington, MA) andClbytitration (Cotlove).
Unlessstatedotherwise, eachagent wasadded in itspowderform directly into the chamber. Manyofthe agents were tested in
concen-trationsthat approached their solubility:hydrochlorothiazide (HCTZ),
metolazone, furosemide,andbumetanide areexamples.
The following reagents were obtained from Sigma Chemical Co.,
St. Louis, MO:4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS),
acetazolamide, 8-bromoadenosine 3',5'-cyclic monophosphate
(8-Br-cAMP),ouabain, papverine,HCTZ,andHepes (both acid and Na salt).
DIDSwaspreparedas a 10 mM stock solution in ethanol and 100Ml
was added to themucosalsolution to make a finalconcentrationof0.1
mM. In controlexperimentsthis amount of ethanol had no effect of I.,
GT,
orJma.
Ouabain was prepared as a 10 mM aqueous stocksolutionand 8-Br-cAMP was prepared as a 100 mM aqueous stock
solution. Amiloride was obtained from Merck Sharp & Dohme (West
Point,PA),furosemidefromAmericanHoechst Corp.(Somerville,NJ),
bumetanide from Hoffmann La RocheInc, (Nutley, NJ),metolazone
from Pennwalt Corp. (Philadelphia, PA), and verapamil from Knoll
Results
To ascertain
that,
under thepresent
experimental
conditions, Na and Cl transport wereinterdependent,
experiments wereconducted where the mucosal solution was
nominally
Na orCl-free
initially
andtheappropriate
tracerfluxwasmeasured.Fig.
1 demonstrates thatby raising
Cl concentration from 0.5 to 6 and then to 20mM, Jma
increased from 0.59±0.13 to 1.61±0.33 to 2.29±0.45MM/cm2
-h. Subsequent addition of ouabain andpapaverine
reducedJma
to 0.65±0.09 AM/cm2*h,indicating
that the increase observedby
the addition of Clwasowing
toactivetransport.The periodsaredifferent (byanalysis
of
variance)
and eachperiod
isdifferent from the previousperiod
by paired analysis. Changes
inI,,
andGT
were variable and werenotsignificant.
Fig. 2 demonstrates theeffect ofraising [Na] from 2 to8
mM and then to 20 mM to
Jd.
These maneuvers causedJd'
toincrease from 1. 12±0.22to5.50±1.58to7.38±1.95MM/
cm2-h. Addition of ouabain and papaverine causedJm'
to fallto 2.69±0.50
MM/cm2.
h. Aswastruefor theprevious
exper-iment,
each valuewassignificantly different from theprevious
one.Thus, thereappearstobe codependence onNa and Cl to effectnet transport.
Na/K/2CIcotransport. Inanumber of epithelia knownto
have interdependent Na and Cltransport,therehas beenrecent
3
2
E
O
z-1
0.5 6 20 OUABAIN
[CI]m(mM) PAPAVERINE
Figure 1.Effect ofmucosalCl concentration on J". Cl wasadded (as NaCl)to mucosal solution which contained anominally Cl-free (gluconate) solution. Clconcentrationon abscissarepresents
mea-sured values at theend of the flux period.Bars represent SEM.Each
value is significantly different from the previously measuredvalue. Ouabain and papaverinevalue represents passivecomponentofthe unidirectionalflux(n= 8).
8
' 6
E
4
0)
2
E<, 2
2 8 20 OUABAIN
+
PAPAVERINE
[Nalm
(mM)Figure 2. Effect of mucosal Na concentration on
Jr,'.
Na was added (as NaCl) to mucosal solution which contained a nominally Na-free(choline)solution. Bars represent SEM. Each value issignificantly
dif-ferent fromthepreviouslymeasured one (n=4).
evidence indicating
thatmucosal
Kis
alsorequired. The
Kdependency of
NaCl transportin
flounder bladder
wastestedin
amannersimilar
to thatdescribed for
theprevious
experi-ments. Several
precautions
were taken to insure that all K was removedfrom
the mucosalsolution.
Themucosalsurface
was washed with K-free solution five times and, 2.5 mMBaCl2
was added to the mucosalsolution
toinhibit
Ksecretion.
Dawsonand
Andrew(25) have reported that when
aISC
is
present,it
represents K
secretion and
can beblocked completely with
mu-cosal Ba. The presentresults confirmed that the
spontaneous VT was lumenpositive and
thatthe
spontaneousIsc
was alsopositive. Furthermore, mucosal BaCl2 reduced the
Isc
to0.
Inorder
todetermine
whether
BaCQ2
affected NaCl
absorption,
5 mMBaCl2
wasadded
tothe mucosal solution of four tissues
and
JN.a
wasmeasured. BaCl2
had noeffect
onJN
(decrease
from 5.60±1.02
to5.13±0.72 MM/cm2.
h). Thus, 2.5 mM BaCl2
was
continuously
present inthe mucosal solution of
theex-periments that
examined
the Kdependency.
Table
IIdisplays
theeffect of
increasing
mucosal
Kconcentration
onJma and
Jma$
(determined in
separate
tissues).
There was nosignificant
effect
oneither flux.
Ouabain
andpapaverine
inhibited'thesefluxes
indicating
the
componentof
active
transport.Because the
loop diuretics inhibit
theNa/K/2Cl
transport process(4, 6, 8, 9, 26), the effect of
1.0 mM(mucosal)
furosemide
on Na and
Cl
transportwasexamined.
Figs.
3and
4display
the results of individual
experiments
forJN.
andJd.
Thesefigures illustrate three
important
features:(a)
therewas alarge
variability in thecontrol
ratesof transport;(b) only
inbladders
transporting
athigh
rates wasthere any effect offurosemide;
and (c)
in nobladder did furosemide
inhibit the transportrateas
completely
asdid
ouabain
andpapaverine.
In 12 of the blad-dersdisplayed
inFigs.
3 and4,
the fluxes of Na and Clweredetermined
simultaneously
and themean values for theseex-periments
arepresented
inTable III. The inhibitionwasgreater
onJdS
(1.33±0.3 jUM/cm2
-h)
than onJN1
(0.70±0.28
WMM
cm2
-h) by
paired analysis.
The
effect
ofbumetanide
(0.1
mM in the mucosalsolution),
amore potentinhibitor
thanfurosemide
insometissues,
wasTableII. Effectof Increasing Mucosal K
Concentrationon
Jcls
andJN7J
Mucosal[KI(mM)
Ouabain
0.02 0.07 0.56 1.05 +papaverine
JN,
AM/cm2h
4.05 4.18 4.35 4.24 0.58*(n=7) ±0.41 ±0.36 ±0.39 ±0.32 ±0.11
J&-,
p.M/cm2.h
3.61
3.803.77
3.85 1.71* (n=6) ±0.47 ±0.49 ±0.51 ±0.66 ±0.18Mucosal [K] was increased sequentially and wasmeasuredatthe end of each period. Short circuit current was 0 in all periods (because of the additionof mucosal Ba+). GT was 0.569±0.094
mS/cm2
in the tissues whereJN.
was measured and was0.378±0.074 mS/cm2
in thosewhereJaf
wasmeasured.These valuesdid not change signifi-cantlyafteraddition of K.Quabain+papaverine valuesrepresent es-timatesofthebackfluxes(JV
andJO!)
in these tissues.* Value different from previously measured values.
examined in four
bladders. In these bladders control JF1a was low(1.32±0.19
MM/cm2.
h) and bumetanide had no effect(1.31±0.18
MM/cm2.
h).Thus, it appears that bumetanide does not actdifferently
than
furosemid, in this tissue.Parallel
Na/H
and
Cl/OH
exchange. Coupled Na and
C1
transport can occur bysynchronous
operation of parallelNa/H
andCl/OH
exchangers.
To examine whether these pro-cessescould explain NaCl absorption
inthis tissue, specific
in-hibitors
were used.Amiloride
has been shown to inhibit Na/H exchangeand
is particularly effective
when Na concentrationis
low(1
1). Table IV displays the results of experiments where 1 mM amiloride was added to the mucosal solution containing 15 mM Na(choline
replacement). There was no effect onI,,,
GT,
orJN..
Subsequent
addition
of ouabain
andpapaverine
produced
their
usual effect. Table V displays the effect of 100 MM DIDS onIS, GT,
andJd5.
Inthese experiments, mucosal
andserosal
solutions
werethemodified
Forster's solution.
DIDS had noeffect
on anyof
the measuredparameters.
Subsequent8
6
E
=L 4 \ \ ,Figure 3. Effect of 1.0 mM
Ez - \ \ furosemide onJ". Each
z z \lnrers
ntsteman
~~~~~~~~line
representstne
meanof
_________________ wtwo20-min flux
periods
ina
single
tissue under theCONROL OUABAIN thr conditions indicated
10
8
b
-a Figure4. Effect of 1.0 mM 2
~~~~~~~~~mental
protocol as in Fig.F
3. Some ofthe values
dis-o playedinthisfigurewere
CONTROL FUROSEMIDE OUABAIN determined simultaneously
PAPAVERINE with somein Fig. 3.
addition
of ouabain
andpapaverine
demonstrated the usual inhibition.Although the lack of effect of amiloride and DIDS indicated that NaCl
absorption
wasprobably
notviaparallel exchangers,
one
additional experimental
maneuver wasattempted.
Mostepithelia
that utilize this system contain carbonic anhydrase, theactivity
ofwhich iscritical
for fulloperation
ofthe cotransport system.Acetazolamide, acommonly used inhibitorof carbonicanhydrase,wasaddedtoboth mucosal and serosal solutionsto a
concentration of
1.0 mM. There was noeffect
onI,GT,
orJNa
in fourtissues whereasouabain
andpapaverine
had their usualeffect.
Thus, these resultsareconsistent
with the notion that NaClabsorption does notoccurby
parallel exchangers.Effects
of thiazide diuretics.
Theeffects
of two members of thebenzothiadiazide
classof
drugs wereexamined.
The agents chosen were HCTZ and metolazone. Theeffect of
0.1 mM HCTZapplied to the mucosalsurface onIC
andGT is depictedin
Fig. 5. HCTZ reducedIC
(when present) to near 0. The response beganin 5-30 s and was usually complete within 2-3 min.Washing
outHCTZ produced a prompt return ofI.
Tissue conductance showed a similar time course. However,
GT
consistently
increased from a meanvalueof
0.541±0.091TableIII.
Effect
of Furosemide on Simultaneously DeterminedJ7a
andJc!j
Furosemide Ouabain
Control (ImM) + papaverine
I.a MM/cm2h 3.52 2.82* 0.63* ±0.56 ±0.30 +0.06
JMsM/cm2
h 4.49 3.16* 1.38*±0.70 ±0.34 +0.08
*Significantlydifferent from previous period (n= 12).
Table IV. Effect ofAmiloride onIs, GT, and JNa
Amiloride Ouabain Control (ImM) +papaverine
sc,
AI/cM2
6.5 6.5 0.1*±1.8 ±1.9 ±0.1
GT,
mS/cm2 0.786 0.718 0.543±0.088 ±0.127 ±0.120
JM,
MM/cm2h 3.30 3.28 0.42*±0.88 ±0.90 ±0.15
Naconcentration of mucosal solutionwas 15 mM.Amiloridewas
addedtomucosalsolution.
*Valuesignificantly different from control and amiloridevalues
(n=4).
mS/cm2 to 0.791±0.128 mS/cm2. The time course for change in GTwasthe same as that for
Isc
andwasreadily reversible.In these same tissues, Jms was measured and the results are
displayedinFig. 6. HCTZ inhibitedtheabsorptiveflux tovalues
notdifferent from thosesubsequentlymeasuredduring ouabain and papaverine exposure. Thus, it appears that HCTZ
com-pletely inhibitsnetNaabsorption.
Theeffect of 0.1 mM HCTZ appliedtothe serosalsurface
onlywasevaluated in fourtissues. .JP wasunchanged (2.06±0.72
to 2.08±0.72
'4M/cm2.
h) as were GT and I. Thus, HCTZ produces its characteristic effects only from the mucosalside.The dose-response relationship for
kc
and GT is displayed inFig. 7. Tissues were used only if they displayed a spontaneousI.
It is clear that there aredose-dependent
changes
in both parameters. Theinhibition ofIc
in some tissues showedatran-siently
more negative value thandid
the steady-statevalue 5-10 min later. Although the reason(s) for this transient is (are) not clear, theeffect of the drug was recorded at the peak response.Table V.Effect ofDIDS on
I,,,
GT, andJ"O
DIDS Ouabain Control (100MM) +papaverine
c,p.A/cm2 2.7 1.6 0.1* +1.4
±1.2
±0.1GT, mS/cm2
0.429 0.479 0.404+0.075
±0.034
0.110JMS,
AM/cm2
h 5.67 5.74 1.88*+0.98 ±1.07 ±0.42
DIDS added to mucosal solution from stock solution in ethanol. Ethanol alone had no effect on these parameters.
*Value significantly different from control and DIDS values (n = 5).
2.0
.5
E
E CD
1.0
0.5
0
CONTROL HCTZ
14 12 10
E
-A/ 4
A2-0'
-2
-4
CONTROL HCTZ
.25
I I
21.00
El
TN~
T
\
0.75t-O
C-0.50
C 0-7 gQ-6 10-5 0-4 C iO-7 -6 iO0-5 0-4
CONCENTRATION (MOLES/LITER)
Figure 7. Dose-response relationshipbetween HCTZ concentration
(mucosal only)and peakresponseofICand GT (n =7). Curves
drawn byinspection. Figure5. Effect of0.1 mM (mucosal) hydrochlorothiazideon
I,,
andGT. Valuesarefor individualbladders. Mean changesarestatistically
significantbypairedanalysis. (4)indicatesfour bladders in which
I,,
= 0anddidnotchange.Thus,theslightly negativemeanvaluefor
IC
at0.1 mMreflects this transient. The fractional inhibition ofI,,
at 106,10-',
and10-4
M was0.06±0.03,
0.38±0.10, and (by definition) 1.00.Thefractional increase in GTattheserespective concentrations
was0.05±0.03, 0.34±0.09, and0.63±0.13. Although the data are not sufficient to conduct detailed kinetic analysis on the
inhibitory effect, it isapparentthat half-maximal inhibition of
IkC
andhalf-maximal
increase ofGT
occurs atapproximately
2-5 X l0-sM.
Theeffectof 0.1mMHCTZonsimultaneously determined Naand Cltracerfluxesis displayed in Table VI. Both
JNma
andJc',s
werereduced but the reduction inJc',S
wasconsistently (andsignificantly)greater than
JNa.
MucosalHCTZalsoreducedthe3.0
2.0 E
EZ .0
0
CONTROL HCTZ OUABAIN+
Figure6. Effectof 0.1 mM(mucosal)hydrochlorothiazide on
J".
Bladdersarethesame asdepictedinFig. 5. Reductionfollowing HCTZexposure issignificant; subsequent exposuretoouabain and papaverinehadnoeffect.
backflux(serosal-to-mucosal) of bothNa andCl; this effectwas
also greater on
Jc'm
than on JA. The net differences betweentheunidirectional fluxes,
JNet
andJdt,
werenotdifferentin thecontrol tissue, suggestinga 1:1 interdependence. AfterHCTZ,
Jfltwas slightly largerthan
JNe'
but it isnotclear whetheror notthis differencerepresentsatruedifferential effectonnetNaandC1 fluxesorratheravariable effectof HCTZonthebackflux
components.
Thecellularmechanism of action of the thiazide diuretics is not known. One possibility is that they inhibit
phosphodi-esteraseactivity(27, 28). To evaluate the possiblerateof cyclic AMP, six bladders were exposed to 1 mM 8-Br-cAMP (both
serosal and mucosal solutions). The results are displayed in
TableVII.Therewere nochanges in
Jma
orinIC
but GT increasedmodestly.
Metolazone isadiuretic which is structurally dissimilarto
the classic thiazidediuretics but which is usually included in this classofdrugsbecause of itsapparentlysimilarmechanism
ofaction (29). The time course of its effecton
Ik,
and GT in twosections of thesame bladderaredisplayed in Fig. 8. Thisfigure illustrates several featuresregarding the effect of
meto-lazone. First,insometissues metolazone hadapurelyinhibitory
effecton
Ik,
whereasinothers therewas atransientstimulatory effect. Second, the increaseinGTwasseenin most tissuesex-amined.Third, whentherewas an
Ik,
present, itwasnot com-pletely reduced to0 (as wasthe case forHCTZ). And fourth,the onset of actionwassubstantially longerformetolazonethan
itwasfor HCTZ. TableVIIIdisplaysthe effectson
Isk.
GTandJ~m foreighttissues. Both
Ik,
andJ11wereinhibitedbutIk,
wasfurther inhibitedbyouabain andpapaverine. Inan additional
fourtissues, theeffects of0.1 mM metolazoneapplied to the
serosalsolutionwereexamined.Therewas nosignificanteffect
on
Ik,
GT, orJNal.
Thus,theeffects of metolazone werequal-itatively similarto the effects of HCTZ and both acted only fromthe mucosal surface. Themajordifference relates tothe
longer timefor metolazonetocompleteitsactions,the occasional
transient stimulation of the
Ik,,
andquantitativedifferences inthemagnitude ofchange in
I,
Jza, andGT.5
N
E
U)
0
5
0
Table VI. Effect of HCTZonSimultaneously DeterminedNaandCl Tracer Fluxes
Flux
Mucosa-to-serosa (n = 8) Serosa-to-mucosa (n = 7) Net
GT JN7 ACT JN. JO
mS/cnm MM/cm2.h mS/cm2 M/cm2.h M/cm2.h
Control 0.669 1.70 2.45 0.346 0.52 1.34 1.18 1.11 ±0.147 ±0.25 ±0.30 ±0.035 ±0.07 ±0.12
HCTZ 0.951 0.40 0.69 0.665 0.23 0.29 0.17 0.40 (0.1 mM) ±0.186 ±0.05 ±0.24 ±0.115 ±0.02 ±0.04
P <0.01 <0.005 <0.005 <0.02 <0.002 <0.001
Pvalue representssignificance betweencontrol andHCTZ-treatedgroupbypaired analysis.Net fluxesarethedifferences between themean
unidirectional fluxes.
Discussion
The present results demonstrate some
characteristics of
elec-trically
neutralNaCl
absorption
across ahigh resistance
epi-thelium. Net NaCl transport involves aninterdependence
of Na andCl
but does notrequire
mucosal K. The processap-parently
does notinvolvesynchronous parallel exchange
ofNa:H andCL:OH.Animportant feature which characterizes this system isitssensitivity
tothiazide diuretics.Theseresults are
generally consistent with results
reportedby
others who have studied thisepithelium. There is
general agreement that the overall net ratesof
NaandCl
absorption areequal(22, 23,
and TableVI) and that thereis
codependency(22,
andFigs.
1 and 2). The codependencyportion
of
the trans-portappeared to be reasonably completebecause
at the lowestconcentrations of
Na orCl examined,
thecoion flux
was equal toor(in
the caseof
Cl) less thanthe
backflux
componentes-timated
bysubsequent
treatmentwith
ouabain andpapaverine(Figs.
1 and 2).Renfro
(22)has reported an incomplete reduction in net Cl absorption after removal of mucosal Na as well asTable VII.
Effect
of I mM 8-Br-cAMPI.C CT Jm
JA/mcm2mS/cm2 MM/cm2.h
Control 4.6 0.480 1.82
+2.1 ±0.094 ±0.41 8-Br-cAMP 5.5 0.540 1.86
+2.4 ±0.102 ±0.51
P NS <0.05 NS 8-Br-cAMP was added to both mucosal and serosal solutions.
incomplete reduction in net Na absorption after removal of mucosal Cl. It is not clear whether the residual fluxesweredue
toincomplete elimination ofthe coion (at the apical membrane) orwhether there wasasecond transport mechanism(s) operating in parallel. One piece of evidence to favor the latter explanation is theobservation that in those studies removal ofC1 and ami-loride had additive effects on Na transport. In the present studies, amiloride hadnoeffect(Table IV).
Asillustrated in Fig. 3, this tissuetransports NaClover a
widerangeofrates.The controltransport rates appear toindicate thedegree to which furosemidewillinhibit transport. This partial (albeit modest) inhibition by high concentrations of furosemide
METOLAZONE
20-t A
_E101
I~0I
1.00-E ...
u0.75
E0.50
*( 0
--et201
At 101-0I
1.05-E0.80 -.
°0.55
E
O,0.30 ...
0_
B
0 20 40 60 80 100 120 TIME (min)
Figure 8. Effect of metolazone on
I4,
and GT on two sections of thesamebladder.
Table VIII.Effect ofMetolazoneonIc GT, and
JNa
Metolazone Ouabain Control (0.1mM) +papaverine
ISc, p.A/cm2 6.6 2.6* 0.1*
+1.6 ±1.3 ±0.1
GT,
mS/cm2
0.490 0.714 0.500+0.090 ±0.085 ±0.094
JAN,
1M/cm2.h
2.02 0.54* 0.39+0.36 ±0.06 ±0.09 Metolazone added to mucosal solution (n =8).
*Significantlydifferent than previously measured value.
is consistent with
Renfro's observation (21). The explanationfor this partial inhibition is
notcertain
but oneof
twopossibilities
seems
likely:
(a)furosemide
is a weak inhibitor of the system; or(b)
bladderswith high
ratesof
transport possess parallel sys-temsonly
someof which
areinhibited by furosemide.
Thebladders examined in the
presentexperiments behaved
asthough
they possessed
asingle
mechanism of NaCl absorption. Efforts
to
reveal
characteristics of
parallel Na/H andCl/OH
exchange were completely negative. High concentrations ofamiloride
had noeffect
onJP"
despite
thefact
that the Naconcentration
inthe mucosal solution
wassufficiently
low so that aneffect
shouldhave
beendetected (1
1). Thecomplete absenceof
aneffect of
DIDS (Table V) and
acetazolamide
supportthis conclusion.
Our
understanding
of the
natureof NaCl
cotransportis
evolving
rapidly.
It wasonly recently believed
thatfurosemide
inhibited
a(1:1)
NaCl entrymechanism in
avariety of epithelia
(2). There
is increasing evidence
thatfurosemide-sensitive NaCl
cotransport
also requires
Ksothat theNa:K:Cl
coupling ratio
is 1:1:2 (4). Such
amechanism likely exists in the thick ascending
limb of Henle's loop (5-7)
as wellasthe shark rectal
gland
(26)
and
theintestine of
thewinter flounder
(8).
The lackof
effect
of
increasing
mucosal Kconcentration
(Table
II)
on NaandCl
absorptive
fluxes
virtually
eliminates
thepossibility
that this entrymechanism
waspresent in these flounder bladders.The
sensitivity of this
absorptive
mechanism
to thiazidediuretics
(especially HCTZ)
further characterizes
itsunique
fea-tures. Theseagents have been inclinical
use for many years butastonishingly
little is
known about their cellular mechanismof
action. There is
general
agreement that themajor
renal tubular segment on whichthey
actis thedistal
convoluted tubule(30-33).
Thereis
also aneffect
on themedullary collecting
duct (34).Thiazides
andfurosemide
actsimilarly
on distal tubule Na andCl
transport but itis
notclear that separatetransport
systemsareinvolved. Both
furosemide
and chlorothiazidehy-perpolarize the basolateral
membrane(32),
but chlorothiazide appearsmoreeffective
ininhibiting
NaClabsorption
(33).
The
effects of thiazides
have also been examined in salientianurinary
bladders. Pendleton et al.(35)
found thatbendroflu-methiazide had a variety of effects in the toad bladder. They
found
thataddition
to the serosal solution caused a fall inI,,
andmucosa-to-serosa Na flux. In contrast, addition to the mu-cosalsolution
caused a modest stimulation of ISC. Addition to both solutions caused a biphasicresponse.
Marumo et al. (36) examined the effect of HCTZ in the frog bladder and found partialinhibition
ofISC after mucosal addition and no changein
electrical properties after serosal addition. Thus, effects ofthiazides
onepithelia
which have electrogenic Na absorption arenotclearly understood.The
effects of
HCTZ andmetolazone uncover some notablecharacteristics of
the passive transport and electrical propertiesof
theflounder
bladder. The sum of the partial ionic conduc-tancesof Na and C1, if they occur only through the junctional complexesvia
simple
diffusion, should approximate GT if the latter is also primarily a paracellular conductance. Under the conditionsof
noelectrochemicalgradients across the epithelium (i.e., identical bathing solutions and no transepithelial voltage) thepartial ionic
conductance(g1,
mS/cm2)
is calculated by thefollowing expression
(37):gj
=J1(z2F2)/(RT),
whereJi
is
theunidirectional flux,
z the valence ofthe ion, F Faraday's constant, Rthe
gas constant, and T the absolute temperature.Conve-niently,
underthe present experimental conditionsgi
= 1.04J1
whereJi
is the serosal-to-mucosal tracer flux (inMM/cm2
* h). Thus, the sum of the partial ionicconductances
can becompared
readily with GT measured in the same tissue. From Table VI
it is
clear thatin
the control periodthe sum of the partial ionic conductances of Na andCl
weregreater than GT, whereas thepost-thiazide
data show that(gNa
+gcl)
were not differentthan GT. Althoughit is
not clear thatin the
thiazide-treatedtissues
these
values are equal because theyshare the same conductivepathways, it is
clear that in the untreated tissues some of the tracerflux
occursthrough
pathways,which
areelectrically
silent. Thereduction of
JA
andJaP
by mucosal exposure to HCTZ strongly suggests that these neutral pathways are through the cell.Furthermore, the
mechanism
whereby HCTZinhibits
theabsorptive
flux
maybe the
same asthat
causing
theinhibition
of
thebackflux.
Astraightforward analysis of this
dataleadsmetosuspect that
HCTZ inhibits
aNaCl cotransporterlocated
onthe
apical
membranethrough which active
absorption
occursand
through which
aportion
of
the Na and Clbackflux
also occurs. Inthis
modelthe cotransporter wouldeffect
some Na and Clexchange diffusion
undernormal
operating
circum-stances.Insupport
of
thenotion
thataportion
of
theCl backflux
occursviaan
apical
NaCl cotransporter is the observation that in the absenceof
mucosal NaJdS
wassignificantly
smaller thanJflS
in the same tissuesexposed
toouabain
andpapaverine
with Napresent in the mucosalsolution
(Fig. 2).
The
mechanism(s)
underlying
theeffects
of HCTZon theelectrical
parameters is(are)
notimmediately
obvious.
Thecombination
ofreductions
inIc
andactive
transporttogether
with an increase inGT
isunusual.Areasonableexplanation
for
these results
might
beas follows. TheIS,
isowing entirely
toex-ample, by addition of bariumtothe mucosalsolution), the
I,,
is dependentonNa(Cl) absorptionsothat Kcanenterthe cell via the Na-K pump. Elimination of Naabsorption will thus stop Ksecretion and the
I,.
The increase inGT maybeowingto asecondary effectto increasethe(conductive) K permeability oftheapical membrane. Althoughthe reason for this(secondary) effect is unknown, there is precedent for increased apical K permeabilityafter inhibition of Naentryacross theapical
mem-brane of other transporting epithelia (38, 39). This scenario
might explain the transient increasein
I,,
seenaftermetolazone. Ifthe reductionin Naabsorptionwas notsufficiently
complete toreduceKentryviathe pumpbelow thesecretory rate,thenthe
(secondary)
increaseinapical
Kpermeability might
beex-pected to result in at least a transient rise inI. Whether or
notthisexplanation fortheseobservations is correct willrequire additional experimentation.
This transport system
probably
exists in the mammaliankidney. The clinical efficacyof thiazide diuretics supportsthe
notion that this systemoccurs in man. It isprobablyconfined tothedistalconvoluted tubule (30-33) (and perhapsthe
med-ullary collecting tubule, reference 34)
sincerecentevidencein-dicates that
there
is nothiazide-sensitive
NaClabsorption
inthecortical thick
ascending
limb ofHenle'sloop
wherefuro-semide
completely inhibits
transport(40). The
conclusionthatfurosemide
and thiazides inhibittwodifferentNa(Cl)
transport systemsin man issupported by the reportsdemonstrating
ad-ditive effects of these classes of diuretics (41, 42).
In summary,
the
urinary bladder of
thewinter flounder
hasa
NaCl
absorption
system which operates in theabsence
oftransepithelial
electricalactivity.
The mechanism of thisab-sorptive
processdoes not appear toinvolve
Na/K/Cl cotransportnor
parallel
Na/H andCl/OH exchange. Rather,
it appears that themechanism involves
asimple NaCl
cotransport processmostlikely
located on theapical
(luminal)
membrane.This
system isinhibited rapidly
andreversibly byhydrochlorothiazide
whichacts only
from
the mucosalsurface.
Metolazonebehaves
in aqualitatively
similar
fashion but with a longer time course. Theflounder
bladder may be auseful
modelfor
NaCl absorptionby
thedistal renal tubule.Iappreciate the encouragement and advice from Dr. David Dawson and David Andrew, and the critical review of the manuscript by Dr. Russell Husted and Dr. Michael Welsh. Ms. Syd Harned provided secretarial assistance.
This study was supported in part by National Institutes of Health grantAM-25231.
References
1.Warnock, D. G., and J. Eveloff. 1982. NaCl entry mechanisms in the luminal membrane of the renal tubule. Am. J.
Physio.
242(Renal Fluid Electrolyte Physiol. 11):F561-F574.2.Frizzell,R.A., M. Field, and S. G. Schultz. 1979. Sodium-coupled
chloride transportby epithelialtissues. Am. J. Physic!. 236(Renal Fluid andElectrolyte Physiol. 5):F1-F8.
3.
Kirschner,
L. B. 1983. Sodium chloride absorption across thebody surface: frog skins and other epithelia. Am. J.
Physio.
244(Regulatory Integrative Comp Physiol. 13):R429-R443.
4.Geck,P.,C.Pietrzyk,B. C.Burckhardt,B.Pfeiffer,andE. Heinz. 1980.Electricallysilent cotransport ofNa+,K+,andCl- in Ehrlich cells. Biochim. Biophys.Acta. 600:432-447.
5. Greger, R., and E. Schlatter. 1981. Presence of luminal K+, a
prerequisite for active NaCI transport in the cortical thick ascending
limb of Henle'sloopof rabbit kidney.
Pflugers
Arch. Eur. J. Physio.392:92-94.
6. Greger, R., E. Schlatter, and F. Lang. 1983. Evidence for elec-troneutral sodium chloride cotransport in the cortical thickascending
limb of Henle's loop of rabbit kidney. Pflugers Arch. Eur. J. Physic!. 396:308-314.
7.Greger, R.,and E. Schlatter. 1983.Properties of thebasolateral membrane of thecortical thick ascending limb of Henle's loop of rabbit
kidney:amodel for secondary active chloride transport.
Pflugers
Arch. Eur. J. Physic!. 396:325-334.8. Musch,M.W., S. A.Orellana, L. S.Kimberg,M.Field,D.R. Halm, E. J. Krasny, Jr., and R. A. Frizzell. 1982.Na+-K+-C1 cotransport inthe intestine of a marine teleost. Nature (Lond.). 300:351-353.
9. Oberleithner, H., W. Guggino, and G. Giebisch. 1983. The effect offurosemide on luminal sodium, chloride and potassium transport in the early distal tubule of Amphiuma kidney. Effects of potassium ad-aptation.PJlugersArch. Eur. J. Physic!. 396:27-33.
10.Oberleithner, H., F. Lang, R. Greger, W. Wang, and G. Giebisch. 1983. Effect of luminal potassium on cellular sodium activity in the early distal tubule ofAmphiuma kidney.
Pflugers
Arch. Eur. J. Physio. 396:34-40.11.Kinsella, J. L., and P. S. Aronson. 1981. Amiloride inhibition of the Na+-H+ exchanger in renal microvillus membrane vesicles. Am. J.Physiol. 10:F374-F379.
12.Cabantchik, Z. I., and A. Rothstein. 1972. The nature of the membranesites controlling anion permeabilityofhuman red blood cells asdetermined by studies with disulfonic stilbene derivatives. J. Membr. Biol. 10:311-330.
13.Liedtke, C. M., and U. Hopfer. 1982. Mechanism of C1 trans-location across small intestinal brush border membrane. II. Demon-stration of Cl--OH- exchange and C1- conductance. Am. J. Physiol. 242(Gastrointest. Liver Physiol. 5):G272-G280.
14.Liedtke, C. M., and U. Hopfer. 1982. Mechanism of C1- trans-location across small intestinal brush border membrane. I. Absence of Na+-Cl- cotransport. Am. J. Physio. 242(Gastrointest. Liver Physiol. 5):G263-G27 1.
15.Fan,C., R. G. Faust, and D. W. Powell. 1983. Coupled sodium-chloride transport by rabbit ileal brush-border membrane vesicles. Am. J. Physio. 244(Gastrointest. Liver Physiol. 7):G375-G385.
16.Friedman, P. A., and T. E. Andreoli. 1982. C02-stimulated NaCI
absorption in the mouse renal cortical thick ascending limb of Henle: evidence for synchronous Na+/H+ andCl-/HCO- exchange in apical plasma membranes. J. Gen. Physio. 80:683-711.
17.Kinsella, J. L., and P. S. Aronson. 1980. Properties of the Na+-H+ exchanger in renal microvillus membrane vesicles. Am. J.Physio.
238(Renal Fluid Electrolyte Physiol. 7):F46 1-F469.
18. Warnock, D. G., and V. J. Yee. 1981. Chloride uptake by brush border membrane vesicles isolated from rabbit renal cortex: coupling
toprotongradients and K+ diffusion potentials. J.Clin.Invest.
67:103-115.
19. Warnock, D. G., W. W. Reenstra, and V. J. Yee. 1982. Na+/
H' antiporter of brush border vesicles: studies with acridine orange uptake. Am. J.Physiol. 242(Renal Fluid Electrolyte Physiol. 1 l):F733-F739.
20. Ericson, A., and K. R. Spring. 1982. Coupled NaCi entry into Necturus gallbladder epithelial cells. Am. J. Physiol. 243(Cell Physiol.
12):C140-C145.
21. Renfro, J. L. 1975. Water and ion transport by the urinary bladder of the teleostPseudopleuronectesamericanus. Am.J. Physiol.
228:52-61.
22.Renfro,J.L.1978.Interdependence of activeNa'andCl- trans-portby the isolatedurinarybladder of theteleost, Pseudopleuronectus
americanus. J. Exp. Zool. 199:383-390.
23. Dawson, D. C., and D. Andrew. 1979. Differential inhibition of NaCl absorption and short-circuit current in the urinary bladder of the winter flounder,Pseudopleuronectesamericanus. Bull. Mt. DesertIsA.
Biol.Lab. 19:46-49.
24. Fossat, B., and B. Lahlou. 1979. The mechanism of coupled transportofsodium andchlorideinisolated urinary bladder ofthe trout. J.Physiol. (Lond.). 294:211-222.
25. Dawson, D. C., and D. Andrew. 1980. Activepotassiumsecretion
byflounderurinarybladder: role ofabasolateral Na-K pump andapical potassiumchannel.Bull. Mt. DesertIsL.Biol. Lab. 20:89-92.
26. Hannafin, J., E. Kinne-Saffran, D.Friedman, and R. Kinne. 1983. Ion fluxes and furosemidebindingin rectalglandplasma membrane vesicles; evidence for the presence of a Na, K,Clcotransport system.
KidneyInt. 23:257.(Abstr.)
27.Moore, P. F. 1968. The effects ofdiazoxideandbenzothiadiazine
diuretics uponphosphodiesterase.Ann. NYAcad. Sci. 150:256-260. 28.Vulliemoz,Y., M.Verosky,and L. Triner. 1980. Effectof
ben-zothiadiazine derivativesoncyclicnucleotidephosphodiesteraseandon
thetension of the aorticstrip. BloodVessels. 17:91-103.
29.Gilman, A. G.,L.S.Goodman, and A. Gilman, editors. 1980. ThePharmacologicalBasis ofTherapeutics.MacMillanPublishingCo., New York. 899-903.
30. Kunau, R. T., Jr., D.R. Weller,andH. L.Webb. 1975.
Clar-ification of the site of action of chlorothiazide in the rat nephron. J.
Clin.Invest. 56:401-407.
31.Costanzo, L. S., and E. E. Windhager. 1978. Calcium and sodium transport by the distal convoluted tubule of the rat. Am. J. Physiol.
235(RenalFluid Electrolyte Physiol. 4):F492-F506.
32. Hansen, L. L., A. R. Schilling, and M. Wiederholt. 1981. Effect of calcium, furosemide, and chlorothiazide on net volume reabsorption andbasolateral membrane potential of the distal tubule. Pflugers Arch. Eur. J.Physio. 389:121-126.
33.Velazquez, H., and F. S. Wright. 1983. Renal distal tubule path-ways forsodium, chloride, and potassium transport assessed by diuretics. Kidney Int. 23:269. (Abstr.)
34.Wilson, D. R., U. Honrath, and H. Sonnenberg. 1983. Thiazide diureticeffect on medullary collecting duct function in the rat. Kidney Int. 23:711-716.
35. Pendleton, R. G., L. P. Sullivan, J. M. Tucker, and T. E. Ste-phenson III. 1968. The effect of a benzothiadiazide on the isolated toad bladder. J. Pharmacol. Exp. Ther. 164:348-361.
36. Marumo, F., T. Mishina, and H. Shimada. 1982. Effects of diazoxide andhydrochlorothiazide on waterpermeabilityandsodium transport in thefrog bladder.Pharmacology. 24:175-180.
37.Schultz, S. G. 1980. Basic Principlesof Membrane Transport. Cambridge University Press, New York. 119.
38. Nagel, W., andW.Hirschmann. 1980. K+-permeabilityof the outerborder of thefrogskin (R.temporaria).J.Membr.Biol.
52:107-113.
39. Halm, D., E. Krasny, andR.Frizzell. 1981.Apicalmembrane
potassium conductance in flounder intestine: relationtochloride ab-sorption. Bull. Mt. DesertBio. Lab. 21:88-93.
40.Schlatter, E., R.Greger,andC.Weidtke. 1983. Effect of'high
ceiling'diureticsonactive salt transport in the cortical thickascending
limb of Henle's loop of rabbitkidney: correlationofchemicalstructure
andinhibitory potency.
Pflugers
Arch.Eur.J.Physio. 396:210-217.41. Ghose, R. R., and S. K. Gupta. 1981. Synergistic action of metolazone with 'loop' diuretics.Br. Med. J. 282:1432-1433.
42. Wollam, G. L., R.C. Tarazi,E. L. Bravo, andH. P. Dustan.