Spironolactone. An aldosterone agonist in the
stimulation of H+ secretion by turtle urinary
bladder.
A Mueller, P R Steinmetz
J Clin Invest. 1978;61(6):1666-1670. https://doi.org/10.1172/JCI109087.
In the isolated turtle bladder, spironolactone inhibits sodium transport in the presence of aldosterone or endogenous mineralocorticoid hormone. In contrast to this antagonism for the stimulation of sodium transport by aldosterone, the stimulation of hydrogen ion secretion by aldosterone is not inhibited by spironolactone. In hormone-depleted bladders,
spironolactone stimulates hydrogen ion secretion. The extent of stimulation is similar to that of aldosterone. Spironolactone functions as an agonist for aldosterone for the stimulation of urinary acidification.
Research Article
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Spironolactone
AN ALDOSTERONE
AGONIST
IN THESTIMULATION
OF H+SECRETION
BY TURTLE URINARY BLADDERALLAN MUELLER and PHILIP R. STEINMETZ, Department of Medicine,
University of Iowa College ofMedicine, Iowa City, Iowa 52242
A B S T R A C T Inthe isolated turtlebladder,
spirono-lactone inhibits sodium transport in the presence of
aldosteroneorendogenous mineralocorticoid hormone.
In contrast to this antagonism for the stimulation of sodium transport by aldosterone, the stimulation of
hydrogenion secretionby aldosteroneisnotinhibited
by spironolactone. In hormone-depleted bladders, spironolactone stimulates hydrogen ion secretion. The extent ofstimulation is similar to that of aldosterone.
Spironolactone functions as an agonist for aldosterone
for the stimulation of urinary acidification.
INTRODUCTION
The stimulation ofsodium transportinmanyepithelia by themineralocorticoid hormone, aldosterone,canbe
abolished by steroidal spirolactones (1-6). Crabbe
(1-3), Porter(4),andSakauye and Feldman(6)have shown
that spironolactone (in a concentration of -50-200 times that of aldosterone) antagonizestheaction of al-dosteroneinthetoad urinarybladder.This antagonism
has beenshown to occurbythebinding of
spironolac-tone tocytoplasmic and nuclearreceptorswith the
dis-placement of aldosterone(7-11).Thisinhibitory effect of the spirolactones on sodiumtransporthas alsobeen
showninthe distal nephron of the kidneyand has
be-comethebasis for theuseof spironolactoneas adiuretic in patients with statesofhyperaldosteronism andsodium retention (3, 5, 12).
In the kidney,
aldosterone
stimulates not only thereabsorption of sodium but
also
the secretion of hy-drogen ion and potassium (5). Little is known, how-ever, about the mechanisms by which potassium andhydrogensecretion are increased. The increasescould be caused bythe increased electrical potential difference
This study was presented atthe 61st Annual Meeting of the Federation ofAmericanSocietiesforExperimental Biology,
Chicago, Ill.,6April, 1977.
Receivedforpublication 25October1977 andin revised for-m1 February1978.
(lumennegative)generatedbyincreased sodium
trans-port. Alternatively, aldosterone might stimulate these secretory processes by separate mechanisms.
Recent studies have shown that aldosterone can
directly stimulate hydrogen secretion in two urinary
epithelia;inthe bladder of the Colombian toad (13) and thebladder of the freshwater turtle, Pseudemys scripta (14). The studiesinturtle urinary bladder have further shown that the stimulation of hydrogen secretion pre-cedes that of sodium transport and has different
sub-straterequirements(14). In this tissue, the stimulation of both transport systems, however, appeared to be abolished by actinomycin D.
In the present study, the differences between the stimulation of sodium and hydrogen ion transportwere
further explored in the turtle bladder by means of spi-ronolactone, SC 9420, which is arelativelypure antag-onistfor sodium transport in toad bladder
(6).
The study indicates that in turtle bladder, spironolactone has its usualinhibitoryeffect on sodium transport by antagonism to aldosterone, but that it functions as an agonist for aldosterone in the stimulation of urinary acidification.METHODS
Paired hemibladders ofthefreshwater turtle,P.scripta, were mountedinlucite chambersaspreviously described (15). Con-trol and experimental halves were obtained from the same bladder.H+secretion,JH1was measuredby pHstat titration at pH7.0andexpressedasnanomoles per minuteper 8cm2.Net sodiumtransport, JNa,wasobtained from the short-circuit cur-rent corrected for JH and was expressed in microamperes per8cm2.
Themediacontained(expressedinmillimolesperliter) Na, 115; K, 3.5; Ca, 1.8; Cl, 121.5; HPO4, 0.3; anddextrose,5.The mucosal solution wasgassedwithCO2-freeairand the serosal solutionwasgassed with 1% CO2in air.
The bladders weredepletedofendogenousmineralocorticoid hormoneby soaking the turtles for2-3daysin 0.7% NaCl and by depletion overnight in the lucite chambers (Results).
D-1Abbreviations
used in this paper: JH, H+ secretion; JNa,net sodiumtransport.
Aldosterone (Sigma Chemical Co., St. Louis, Mo.)wasadded in 50,l of methanoltogivea finalconcentrationof 0.5 ,uM. This concentrationishalf theconcentration usedbyLeFevre (16) inthe turtle bladder and has been effectiveinthe stimula-tion ofJH andJNain ourprevious study (14). Spironolactone (Sigma Chemical Co.), 17-hydroxy-7a-mercapto-3-oxo-17a-pregn-4-ene-21-carboxylicacidy-lactone7-acetate,wasadded to50,ulofmethanoltogiveafinalconcentrationof50,uM.The same volume of methanol was added to the control halves. The addition of hormone or diluent was made only to the serosal solution.
RESULTS
Aldosterone action in turtle bladder: experimental
considerations. Threepractical aspects of these studies are emphasized before the presentation of the results
obtainedwith spironolactone. (a)It is virtually
impos-sible to obtain fresh turtle bladders thatare depleted ofendogenous mineralocorticoid hormone. In the ab-senceofasatisfactorymethod tomaintain adrenalecto-mized turtles, we have used the bladders of saline-soaked turtles which are only partially depleted of the hormone (pithing and bladder removal probably cause secretion of the hormone). To get near complete de-pletion of endogenous mineralocorticoid, the bladders were incubated for 20 h in Lucite chambers. After a 20-h period of depletion in vitro (this study and 17), the halves exposed to aldosterone showed increases in JH and JNacomparedtocontrolhalvesof the same bladders
inall 14bladdersstudied in our laboratory. The results wereconsiderably lesspredictable (17) when compari-sons were made during the first 6 h after mounting,
presumably because of the presence of endogenous hormone. (b) For an investigation of the interactions between aldosterone anditsantagonist, spironolactone,
itwould be ideal to compare four identical quarters of the samebladder: onecontrol, one exposed to aldoste-rone alone, one exposed to spironolactone alone, and one treatedwith spironolactone before exposure to
al-dosterone. Unfortunatelythe requirement to have four segments with comparable initial rates of JH
measur-ableby pH stat titration could not be met in any of the
eight bladders sostudied. To overcome thislimitation,
threesetsof separate comparisons were made between halves of bladders instead of the four-way comparisons in the quarters of bladders. First, exposure to aldoste-rone alone caused consistent stimulation ofJH and JNa comparedtocontrolhalves (14, 17). Second,
aldosterone-exposed halves were compared with and without pre-treatmentwithspironolactone.Finally,exposure to spi-ronolactone alone was compared to control halves. (c)
As in previous studies (14) the term "stimulation" re-fers tothe increased transport by experimental halves
overthe control halves of the same bladders. Most blad-ders have higher rates at the time of mounting than after 20 h in the chambers even when exogenous
al-dosterone and glucose are present. The initial rates
were measured to ensurethat both bladder halves had adequate and comparable transport rates for H+ andNa+.
Aspointed out above, the experimental bladder halves
exposed for 20 h to 0.5 ,uM aldosterone consistently
exhibitedhigher transport rates than the cointrol halves, although the rates were often less than the rates ob-served in the fresh state. The survival time of epithelial cell layers in vitro is -48h. Little is knowni about the mechanism of this decline in finction, it is likely that variousnutrient and poorly understood humoral factors areinvolved. For the pturpose of our study, however, it is important that the presence of aldosterone plays a major role in sustaining the transport rates in vitro.
Effects of pretreattment with spironolactonte oni the
action of aldosterone. Table I shows that JNa was
in-hibited in the bladder halves pretreated with spirono-lactone andsubsequently exposed for20htoexogenous aldosterone. In contrast, JH was unaffected by spirono-lactone. The transport rates at the time of mounting of the bladder halves before hormone addition were the same for the control and experimental halves. JNa was
183 +23
,uA
and JH wasinitially 12.1±0.9nmol/min, 20 h before the values of Table I were obtained.Fig. 1 shows in six other experiments how JH was uIn-affected by spironolactone pretreatment, whereas JNa was inhibited by 25±5%. In these experiments, both halves wereagain depleted of hormone in the chamber for 20 h. Bothhalves were exposedtoaldosterone for4h.
Inthe presence of onlyendogenousmineralocorticoid hormone, spironolactone appeared to inerease JH in eight freshbladders,but the increasewasof borderline significance (Fig. 2). JNa, however,wasagain inhibited by spironolactone.
These results show that spironolactone does not act
TABLE I
Effect of Spironolactoneon
JIN,a
andJHin Bladders Subsequently ExposedtoAldosteroneiNa JH
Control SPIRO AJN, Control SPIRO AJH
+ALDO* +ALDO (Sp-c) +ALDO +ALDO (Sp-c)
,uA nmol/min
114 53 -61t 9.5 9.6 +0.1
+35 ±19 +23 ±3.0 ±2.2 ±1.9
*ALDO, aldosterone; Sp-C, spironolactone-control; SPIRO,
spironolactone.
ISignificant by paired analysis (P<0.05). If the ratio JNa/JNaO were compared, the P value was <0.005. SPIRO caused no significant change in either JH/JHO or AJH. Mean values+SEM are given for six paired hemibladders. Bladders wereexposedfor 20 h to 0.5
A.M
ALDO. Experimental halves werepretreated with 50 ,uM SPIRO 1 h before the addition ofALDO. The serosal solution contained 5 mMglucoseand1%CO2 inair.
lactone and aldlosteronie caused significant increases
ill JH.
DISCUSSION
These results inidicate that spironolactone acts as ani an-tagoniistforaldosterone inthe stinmulatioin ofsodiumil
tranisport in the tturtle bladder as in other uriniary
epi-thelia. This aintagoInisiml wasobservedwheineindogenlouIs
minieralocorticoid hormiione or aldosterone were pres-ent.Intheabsenice of aldosteroneinbladders depleted of endogenous hormiionefor 20 h in thechamber,the ef fects ofspironolactonie wererathervariable. In 2 of the
11bladderis(Table II), sodium transport was inhibited, in the others it was stimulated. It is possible that the two bladders had residual mineralocorticoid activity
and that the others were so depleted that spironolac-tone servedas apartialagonist. Sakauye and Feldmani
(6)reported that spironolactone hadnoagonistic prop-ertiesby itself in toadbladder afterashorter period (6 h)
0 1 2 3 4
TimeAfterAldosterone(h)
FIGURE 1 The effect of spironolactone (SPIRO) onthe
re-sponses ofJH and JNa to aldosterone (ALDO) in
hormone-depleted bladders. Ratios are graphed for six paired
hemi-bladders. Experimental halves werepretreated with 50,uM
spironolactone. Attimezero,0.5AMaldosterone wasadded
toSPIRO andcontrol halves. The serosal solution contained
5 mMglucose and1%CO2inair.*Significant by paired
analy-sis (P<0.05).
as an antagonist for the aldosterone stimulation of JH
and suggest that spironolactone mayact as an agonist
for aldosterone in the stimulation ofJH.
Effects of spironolactone aloneonJNa andJ, in hor-mone-depleted bladders. To explore this possibility
the effects ofspironolactonealonewereexaminedin 11
bladdersdepletedfor20 hofendogenous mineralocor-ticoid hormone and comparedwithcontrol halves ofthe
same bladders. As shown in Table II, spironolactone
causedno inhibition ofJNa inthese hormone-depleted bladders,aresultconsistent with theinterpretationthat
endogenous mineralocorticoid is required for the in-hibition by spironolactone. On the contrary, therewas anincrease inthemeanJNainthespironolactone-treated
halves. The increase, however, was of borderline
sig-nificance because ofvariability of the results.JH,onthe
otherhand,wasconsistentlyandsignificantlygreater in
spironolactone-treatedhalves thaninthe control halves. The difference of8.1+2.2 umol/min was comparable
to that observedpreviously in experimetnts with
aldo-steronealone(14) asshown inTable III.Both
spirono-JH
JHO
JNa
JNaO
SPIRO
CONTROL
0.9
I I
1.1 - ## I %.,. .4 I
*#
0.7
I-T
t CONTROL
-1
*
SPIRO
I I I I
0 1 2 3 4
Time After Spironolactone(h)
FIGURE 2 The effect of SPIROonJH andJNa infreshturtle
bladders. Ratiosare graphed foreight paired hemibladders.
Experimentalhalves received 50,uM spironolactoneattime
zero (-4 h after mounting). The serosal solution contained
5 mMglucoseand1%CO2in air.*Significant by paired analy-sis(P<0.05).
1668 A. Mueller andP. R. Steinmetz
1.1
0.9'
1.1
1.0
JNa
JNao
0.9
0.8
0.7
SPIRO +ALDO
I
I
I %%
I %%
I
ofhormone depletion. Severalotherspirolactones, how-ever, had agonistic properties for sodium transport in
the absence of' aldosterone.
Ourresults suggest that the stimulation ofJH is not
inhibitedby spironolactone at any level of'endogenous mineralocorticoid activity orinthe presence of
aldoste-rone.After20hof' hormonedepletion,JH wasstimulated
in all bladders and the magnitude of stimulation was
comparable to that observed with aldosterone alone.
Spironolactone, therefore, funLetionied as a physiological agonist for aldosterone without any antagonistic
prop-erties.
How should the diff'erent eff'ects exerted by spirono-lactone On JHand Na+ absorption beinterpreted? Pre-viousstudies(18-20)haveshown that thetwotransport systems are notcoupled directly. They affect each other only indirectly via the electrical potential difference and the availability of
CO,
forJH (20). In the presenitstudy these indirect forms of' coupling vere excluded since all experiments were carried out in the
short-circuited stateand in the presence ofexogenous CO.
Severaldescriptive explanations forourresults must
be considered. First, the different behavior of spirono-lactone in the stimulation of the two transport systems is in accord with thepreviously reported differencesin
the substrate requirements and time-course between
the stimulation of'H and Na+ transport (14). Itis
pos-TABLE II
Effectof SpironolactoneonINaand JHinl Hormone-Depleted Bladders
JNa JH
AJN. AJH
Control SPIRO* (Sp-C) Control SPIRO (Sp-c)
AA tlmollmitl
100 206 +106 13.4 21.4 +8.1t
±24 ±40 ±48 ±1.8 ±2.7 ±2.2
(0.05<P (P<0.005) <0.10)
* Sp-C, spironolactone-control; SPIRO, spironolactone.
4 Significantby paired analysis. The P values were the same ifinstead ofA values, the ratios ofJNa/JNa0 were compared. Mean values+SEM are given for 11 paired hemibladders. The experimental halves were exposed for 20 h to 50 ,uM SPIRO. The transport rates in the control and experimental halves were the same at the time of mounting, 20 h before the results werepresented. JNao was 447+26,uAand JHO was
24.9±+1.6 nmol/min. These initial values were about twice the initial values of the experiments of Table I (see text). The experiments of this table were done in September and October, whereas those of Table I were done in March and April. The serosal solutions contained 5 mM glucose and
1%CO2 in air.
TABLE III
Comparisonof the Effects of Spironolactone and Aldosterone
on JH inHormone-Depleted Bladders JH
AJH
Control Steroid (St-c)
nmol/min
SPIRO* 13.4 21.4 +8.1t n = 11
±1.8 ±2.7 +2.2
ALDO 10.1 16.0 +6.01 n =6
±1.5 +3.2 +2.2
*
SPIRO,
spironolactone; St-C, steroid-control.Significant by paired analysis (P <0.05). Mean values +SEM are given for two different groups ofpaired hemi-bladders. The experimental halves wereexposedfor 20 h to either 50,M SPIROor 0.5
l.M
ALDO.The serosal solution contained 5 mM glucoseand 1%CO2inair.sible that both spironolactone and aldosterone stimulate JH by some direct mechanism altering the lipid
com-position of the cell membrane (21) and thereby the conductance in the active transport pathway for urinary acidification. This possibility, however,is notsupported by the observation that actinomycin D abolished both the stimulation ofHIand Na+ transportbyaldosterone (14); neither can it be considered excluded. The second set of'explanations invokes separate pathways for the stimulation of the two transport systems. The
separa-tion could take place at the level of the cytoplasmic hormone receptors or at subsequent sites in the path-ways leading to the synthesis of effector proteins. Our physiologic studies provide no information on whether there are separate hormone receptors, separate gene sitesthatresponddifferently to the same receptor
com-plexesorwhether the stimulatable transport processes
for Na+ and HI are contained in differentcell types.
ACKNOWLEDGMENT
This research was supported by grant AM 17568 from the National Institutes of Health.
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