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Paradoxical stimulation of sodium transport in toad skin by ethacrynic acid
Gen. Pharmac..
1977. |b/ 8. pp. fgg~to 196. Perqanum
Press Printed in Great Britain
PARADOXICAL STIMULATION OF SODIUM T R A N S P O R T IN T O A D SKIN BY E T H A C R Y N I C ACID FRANCISCO C . HERRI'RA AN[) MONTSERRAT ESIEVE*
Departamento de Fisiologia, Ccntro de Biofisica y Bioquimica, Instituto Vcnezolano dc lnvestigaciones Cientificas, IVIC, Apartado 1827, Caracas. Venezuela (Received 4 February 1977) Abstract 1. Ethacrynic acid, like anti-diuretic hormone, increases active sodium transport across the skin of BuJo marinus.
2. This is reflected in an increase in short-circuit current accompanied by a rise in potential difference and a marked fall in skin resistance, followed by a slow rise. 3. These effects result from an increased sodium uptake across the outer epithelial surface, where the sites of action of ethacrynic acid appear to be located. 4. These results suggest that the anti-diuretic effect of cthacrynic acid in nephrogcnic diabetes insipidus may bc related to an increased sodium rcabsorption at tubular sites where water reabsorption is obligatory.
INTROD u(TrlON Ethacrynic acid, 2,3-dichloro-4-(2 methylcncbutyryl)-phenoxy acetic acid, is a potent diuretic believed to act mainly as an inhibitor of sodium transport at the level of the ascending limb of Henle's loop (Goldberg et al., 1964; Seldin et al., 1966; Puse h e t t & Goldbcrg, 1968). However, in nephrogenic diabetes insipidus it causes a considerable decrcase in water intake and urine output (Steele et al., 1965; Brown et al.. 1966) which has been ascribed to an increased reabsorption of water and sodium at the level of the proximal tubule consequent to a contraction of the extracellular compartment. Dirks et al. (1966) obscrvcd an e n h a n c e m e n t of proximal tubule reabsorption of sodium following ethacrynic acid (EA) administration in micropuncture experiments. These authors also suggest that this increased rcabsorption would be consequent to acute depletion of extracellular volume, a response opposite to that produced by ,saline infusion. Nevertheless, a direct action of EA on tubular sodium reabsorption, although somewhat paradoxical, has not been ruled out. EA could facilitate sodium reabsorption at tubular sites where water rcabsorption is obligatory (Steele et al.. 1965). EA has been found to have an inhibitory effect on sodium transport across the epithelium of the toad urinary bladder (Hcrrera, 1975), but in frog skin this inhibitory effect is preceded by a one-hour long stimulation (Baba et al., 1966). In the present investigation, the effect of EA on ion transport across amphibian skin has been further explored. The results of Baba et al. have been confirmed and the site of the changes in the sodium transporting mechanism in toad skin epithelium responsible for the observed effects of EA on sodium transport has been located. METItODS
Specimens of the toad Bulo marinus captured in the environs of Valencia. north-central part of Venezuela. were * With the technical assistance of Yolanda Mandujano. GP
8'3--D
IS9
used in all cxperimcnts. The toads were double pithed: in some cases the ventral skin was removed and divided into symmetrical halves and the results of experiments on one half compared to thosc of the other which either served as control or on which the converse experiment was performed. Otherwise, each skin served as its own control: treatment with ethacrynic acid would be instituted following a control period of sutlicicnt duration to obtain stable values for the baseline bioclcctric parameters and fluxes. To minimize cdgc damage, the skins were mounted sandwiched between parafilm (Marathon Products, Ncenah, Wi, U.S.A.) gaskets lightly smeared with stopcock grease (Lubriseal, A. H. Thomas Co., Philadelphia, PA, tJ.S.A.I as a diaphragm separating two lucite chambers filled with the appropriate Ringer's solution. Two different Ringer's solutions were employed. In some cxpcrimcnts conventional frog Ringer's containing sodium chloride, 112 mM: potassium bicarbonate. 2.4 raM; calcium gluconatc, 0.5 mM. was used; this solution was termed chloride Ringer's. In experiments in which a high passive anionic conductance was undesirable, sodium chloride was replaced by 56mM sodium sulfate (Herrera. 1968. 1975): this solution was termed sulfate- Ringer's. The skins were short-circuited automatically and the current was interrupted every 8 min for 80 sec to record potential difference. LJnidircctional sulhltc and sodium fluxes were measured by adding ssSO] or sodium-22 to the solution bathing one of the surfaccs of the skin and sampling the solution at the opposite side every 20 min. The details of the methods have been published previously IHerrcra, 1966). The rate of sodium uptake across the outer surface of the epithelium was determined by a modification of the method used by Biber and Curran (1970). F'aired skin halves were stabilized for 1 hr in sodium sulfate Ringer's in chambers provided by an inlet port on the mucosal side connected by means of plastic tnbing to a three-way stopcock which in turn was attached to a 20ml syringe filled with Ringer's solution containing sodium-22 and tritiated mannitol. Once short-circuit current and potential difference were stabilized, 1 mM EA dissolved in Ringer's was added to the inner solution of the experimental skin half and the same volume of Ringer's added to the control. Sodium uptake was determined either at the time of maximum EA effect on short-circuit current or at the time of maximum time-rate of change of the current. In either case, at the appropriate instant, the same procedure was followed simultaneously in control and experimental skin
I~tl
I"RAN('IS('() ( ' . l J l R R t R A ,X\I) M ( I N I S l R R A I
JiSll\l 3000
./-/"
~"
SCC \
/ ImM Ethocryn~c ac,d outs,(Je
ImlVl Ethacrynic ocld reside
1o
I:ig I. Time course of short-circuit current 1%('(.'1. potential difference IPI)) and de. resi>tance iRI in skins bathed in sulfate Ringer's and treated with EA on the inner side (left-hand graph) and on the ot, tcr side (right-h:md graph). Arrow along the abscissa indicates the time-scale. halvcs. The outer solution ~ a s quickl.~ removed: the radioactive solution held in the syringe was rapidly injected into the outer chamber and duplicate I(R)itl samples wcrc immediately taken from the radioactive soh, tion. The latter was left in contact with the outer surface of the skin for exactly 35 sec; this period was terrain,clod bx removal of the outer .solution. removal of the skin ~mdv..ich and rapid blotting of both skin surfaces ~ith W h a t m a n No. 54 tilter papcr. The skin area exposed to the sohHlons x,.as cut out. and dricd overnight at 105 ('. Thc cxposcd skin area measured 3.14cm:. The dried skins xsere then extracted for at least 2 h r m 2nll (I.I N H N O ~ Ahquots o[ this extract were counted along with the salmples taken from the radioactive sx)lution and Iritiatcd mannitol and sodium-22 wcrc determined simultaneously on a beta counter by' the double-label counting rncthod of Okita et al. (1957). The mannitol space accessible from the ot,tcr skin st, rface was then determined. Sodium t, ptakc ~ a s estimated from the incorporation of radioactive sodium after corrcction for mannitol space. The data have been analyzed statistically by mcans of one-way and two-way analysis of ~ariancc and orthogonal comparisons have bccn performed on the rcsuhs [Sokal & Rohlf, 19691.
RESL?I,'I'S E~lbct oI" E A on shm't-circuit current acros.~ toad .skin
Paired s y m m e t r i c a l halves of thc ventral skin of the t o a d werc m o u n t e d in c o n v e n t i o n a l c h a m b e r s a n d
b a t h c d with sulfate R i n g e r ' s on b o t h sides. After p o t e n t i a l ditt'e,'encc a n d s h o r t - c i r c u i t c u r r e n t h a d stabilized, t h e baseline values of s h o r t - c i r c u i t current. p o t e n t i a l difference a n d d.c. resistance ( m e a s u r e d as the ratio of p o t e n t i a l difference to s h o r t - c i r c u i t current) were recorded d u r i n g a c o n t r o l period of 1 hr d u r a t i o n . T h e n 1 m M EA was a d d e d to the internal s o l u t i o n of o n e of t h e s k i n halves a n d to the e x t e r n a l s o l u t i o n of the other. F i g u r c I s h o w s t h e t i m e c o u r s e of p o t e n t i a l difference, s h o r t - c i r c u i t c u r r e n t a n d total skin resistance. In b o t h cases s h o r t - c i r c u i t c u r r e n t rises d r a m a t i c a l l y , potential difference rises r a t h e r m o r e slowly a n d relatively less t h a n s h o r t - c i , c u i t current b r i n g i n g a b o u t a m a r k e d fall in resistance. W h e n EA is a d d e d to the inner side s h o r t - c i r c u i t c u r r e n t rises to a peak value a n d t e n d s to fall off slowly a n d linearly with time. W h e n EA is a d d e d to the ot, ter side it cat, ses an a b r u p t rise in c u r r e n t after w h i c h tile curFent r e n l a i n s m o r c or Icss steady. T h e time rate o f c h a n g e of the electrical p a r a m e t e r s is q u i t e clearly m u c h g r e a t e r v,hcn EA is a d d e d to the e x t e r n a l solution t h a n w h e n it is a d d e d to the hlncr one. T a b l e I s u m m a r i z e s the rcsults of 7 experinaents. Short-circuit c u r r e n t ( S t ' ( ' ) rises to vahtes abot,t d o u b l e the c o n t r o l ones. the i n c r e a s e s being h i g h l y statistically signiticant bul they do n o t differ between s k i n s treated with EA on the o u t s i d c or on lhc inside. T h e potential difference also is i n c r e a s e d but p r o p o r t i o n a -
Table l. Changes in short-circuit current, potential difference and resistance caused b\ cthacrynic acid added either to the outside or the inside of skins bathed in sulfate Ringer's
Before [A
Maximum value after EA
A
t'~
/. animals {cllange)
t" m vs out
t,,,, ~ (mini
/ t,,.~
k animals t,..,
Short-circuit current (Hcqui', hr × 3.14 cm-'l I'A outside EA inside
2.07 3.11
4.89 5.84
2.83 2.73
61.2W 41.07 ~'
4.74" 5.64"
0.06
16 39
(,i.31 ~,
11.15 ~
Potential difference (mV) EA outside EA inside
98 106
132 123
34 17
57.70 h 33.2&
2.51 4.74"
10.52"
13 37
36.39t,
3.15
1884 1379
1125 g82
759 498
9t).31 h 52.10 h
24.95 ~' 20.49 ~'
16.53 t,
12 32
17.59"
2.72
Resistance 1~1 EA outside EA inside
a F is signilicant at or bclo\~ the 0.05 level but not at the 0.O1 level. ~' F is signilicant at or below the t).01 level.
Sodium flux and ethacrynic acid _No CI . ~
. . . . .
f \
~_,~-
R No2SO 4
+ .
:
-
:
~ ,
•
•
.
•
.
.
191
R NozSO44"ImM Elhocrynic a c i d reside . . . . . . . . . . . .
t
f r f ~ , . .
---.11 " ~ ~ / , ~ , , ~ / ~ ' , ~ ; ~ s c c
Fig. 2. Time course of short-circuit current (SCC). potential difference (PI)). d.c. resistance (RI and sodium influx (hatched area denoted by 4~i'I in skins pre-incubated in chloride Ringer's (R-NaCI) and then exposed to a control period in sulfate Ringer's followed by treatment with EA inside (R Na 2 SO4 + I mM EA inside). Arrow along the abscissa indicates the time-scale. tely less than short-circuit current, bringing about a statistically significant fall in resistance. The increase in potential difference is smaller in skins treated with EA on the inside and so is the decrease in resistance probably becausc the increase in SCC was relatively smaller since mean SCC before EA was higher in skins treated with EA inside than in those exposed to EA on the outside. Significant differences in response of the electrical parameters to EA a m o n g animals could bc detected. The time required to reach 90°~o of the maximum value of the response to EA of the three parameters, t,m.,, was much shorter when the diuretic was added to the outer side of the skin suggesting that the site of action of the drug was further removed from the inner skin surface than from the outer: no further effect was elicited when EA was added to the same side or to the side originally free from EA. These results suggest that EA increases short-circuit current through a decrease in resistance to the movement of ions through the skin. However, the ions whose transport is increased must be identified. Therefore, experiments were performed in which the mucosal to serosal sodium fluxes were measured.
Determination of mucosal to .serosal .sodium influx across toad skin under the influence of EA Experiments similar to those described above were performed on toad skins exposed to either chloride or sulfatc Ringer's. The skins were equilibrated and short-circuited. In order to determine transepithelial sodium movement approx 1/~Ci/ml of sodium-22 was added to the outer solutions of the skins and 500pl samples were taken from the inner solution at 20-min
intervals, replacing this volume with non-radioactive Ringer's. U p o n completion of a l-hr control period, I m M EA was added to the inner solution and the sampling of the internal solution was continued for another 4 hr. Short-circuit current, potential difference and total resistance were recorded throughout the experiment. A typical experiment is shown in Fig. 2 in which a skin exposed to sulfate Ringer's is treated with EA. The diuretic causes a rapid rise in potential difference and a fall in total resistance. Short-circuit current and inward sodium ftux increase almost simultaneously, short-circuit current being quite close to the sodium intlux. However, the increase in sodium influx sccms to lag slightly behind the increase in short-circuit current. Since, except for the lag in the increase in short-circuit current, the increase in short-circuit current and inward sodium flux agree quite closely in skins exposed to sulfate or chloride Ringer's, the increase in short-circuit current is mainly accounted for by an increase in the active inward transport of sodium. No other ion appears to be transported to any appreciable extent, chloride especially not being involved. Tables 2 and 3 show the orthogonal comparisons of inward sodium fluxes and short-circuit current during control and three subsequent l-hr periods (three flux determinations in each period), beginning 20 min after the addition of EA, in skins exposed to sulfate and chloride. Ringer's. Short-circuit current during each period was determined by graphical integration of the current. The control values of short-circuit current and sodium influx correspond to those obtained during the hour period immediately preceding EA addition
"lziblc 2. ()rthogonal comparisons of short-circuit current and sodium inward tlux between control period (('1 and ethacr)nic acid periods 4t) ~t) IT~ I. 100 140 1"1)) and 200 241) (/3) rain after addition of the drug to skins exposed to chloride Ringer's c
7~ 20 80
"r2 3r) 80 140 180--240 (rain after EA)
Sodium mucosal to serosal 11ux (l~equiv/hr x 3.14 cm 2) C vs (T~ + 7"2) 5.69 8.07 7.36 7"1 vs 7"2 8.07 7.36 (C + T~ + 7~) vs "F3 5.69 8.07 7.36 Short-circuit current (,uequiv/hr x 3.14 cm 2) C vs (Tt + 7"2) 5.29 8.6,~ 6.43 T~ vs 7) 8.6~ 6.43 (C + + 7)) vs "/~ 5.29 8.86 6.43
71
F
P
19.17
<0.01
1.79
n.s.
4.58
31.83
<0.01
4.19
15.26 11.33 22.87
<0.01 <0.01 <0.01
192
J R ' , \ ( I~,(ll (" JJlRRIR-,, .;\11 M ( ) N I S l R R ; I
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ctlrlclll
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conlro] period i ( l and clhacr',llic ;tcid pcliods 41) Sll I'/il. 10It 14()(/i,i cind 21Rl 2411 I'/~) nlin ~iftcr ;iddilion of the drug in skins cxpo,,¢d Io sti][':.ilc Rlilgcr's (
Soditiin nltlcosal
("
%s ('l~ '1"~ x s 7 ,
((' a 1; t 1-I ~s 7 I,
1
I'
1.9 =
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~,,x': .',S"
,x<,i "S 2 "~(
<;I).(I n.s.
i.U:--
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].';(,
24.71
< ). I
,:It.Ill
currcnl {/IL'qtlJ', hi" : 3.14 cnl 2)
(" xs I1~ -F 'I,) 'I m ~s 1", (('"I~ -
I. "I, ,<4(1 14() IX() 24U Imin aflcr I:\1
It) hc'l'l)sH] Ilu\ (/~¢Lll.ll\ ]11' :>~ "~14 CI]1")
- I.I
S[lorl-cJl'Ctlil
1"1 20 Xl)
I . I ~> Ii~
• 12
14.'
.iX2
19.49
.t S" .;S2
133
ll,b,,
2.12-
l 12 4.42
I I.(',H
<'-I).l)l
Iperiod ("). ARer EA, samples t~eie tnken e~ el') 20 rain and the samples takcn during the period bcl~cen 20 rain and ,'4(/min aftcr I{A were colleclcd into lilt first I{A period {period /~ I: ihosc taken het~cen ,xO and 140 rain ~ere collected into the second EA period (period 72i :.ind finall 3. the s,:lnlplcs l:.ikcn hctwcen 180 and 240 rain altcr tIA ~¢rc collected its the third I{A period (period 1,0. r h c short-cii-ctiii Cul-renl \~,:.lS similarly divided ;Mid lllCail short-circuh CUlrCll{ dtlring each period ~as calculated h\ graphical integration of the time cotlrsc of the CLIITCnt. ITA periods I alld 2 hrackcl lhc nlHxillltlnl rise in short-circuit Ctilieill as sllo~n ill Fig. 2: I{.:\ period 3 rcprcscnls the kisl three ltLIX periods ~iftcr I{A with their COrlespending short-circuil Ctll'I'Cllt. Tile (H'lhogt)ll;.iJ conlparisoils \%ere lnadc sticcessi,oditlnl hiilu\ ilccOtllltS ror pructicall 3 all of lhs increase m short-circuit ctirrcllt except flu a possihlc lag in flux wilh respecl Io the ctlrrenl dtlring lhe initial increase ill ctlrrclll and l'lti\. ('onlp.:uhl 7 Tables 2 and 3 it ma\ bc ohscixcd thut the mtlcosal Io scro~ll sodium l]tl\ aild shorl-circuit currcnl arc milch higher in skins L'kpo~cd lo clHoridc Ringer's. This appears it) be the rcsuh of ~1 decl-casc m sodium acli~c Iransporl in the skins c\poscd io
2tO
sLtll~tte ,;ince short-circuit CLIIrcn[ alld nlucoscli to scro~zl sodium flux agree closcl.,,. Morcovcr. il mal~ he obscr~ed in Table 2 that during lhe period I~ {20 ~40 mm after E.,\L .S('(' is higher than hlfltlx b ~, ;.ll-lout 0.611equi\.hr x 3.14cm 2, this figure being statisticall) significant below tile 0.05 level. During I2 (140 140ram clflcr I(/\1. S('(" is 0.9/lequi~ hr x 3.14 cm 2 lower (P < 0.051 than mucosal Io sciosal sodium flux as ~votild bc expected if short-circuit ctlrrClll is the difference belween inthix and outt]ux of sodium. In skins cxposcd to still'ale Rhlger's these diffcrcnccs xvcic not statistically signifi('illlt.
l'hc hchaxilu of tllc rcsistancc dill'ors in chloride and sulfate Ringer's. In the fol'nlel" C;.tSC contlol reslslancc is considerably lower than in the latter. After I(A addilJon resistance rises gradually duririg the whole of lhe E.,\ exposurc period. In lhc case of skins cxposed Io sulfate Ringer's. there is at marked fall in resistance immedialel~ after addition of t!A followed m some cases by a gradual rise. In chloride Ringer's thc ch;.in,,ese of resistance of the sodium pathway arc nl;.lsked b) tile high conducl~.lilce of the ailioni¢ pnihway. ~vhcl-eas m stllfalc Ringel"S the anionic Collductallce is quite low and mosl of tile ionic coilduclance is represenled by thai of tile soditnll transport path~;.iy. tile clltillges ill Collduciatlce of tilts ionic rotlle nol being masked b.,, lhe high anionic conductance (tlerrera, Ig75). ] h c lime Ct)tll%C t)[ Ihe resistance in chloride aild sulf:.itc Ringer's is shown in Fig. 3. The nlechani,sm underlying the increusc in in~aid sodiunl llux rcnlahlect to be determined. lllerefore. the soditlnl uptake across file otltcr Stllface of tile cpiihcliunl ~a> studied b~ Ihc method outlined by I*)iber ililtl ('tnra'li (1970). .~'lJdittm l//)/gl~t' ~i(l'lJ,,.,, lhc olllt'r
('pilh~.,liol
.\/ir/{/('t,
The ploccdtire outlined under .Methods was p¢i-[ormed on skins incubalcd ill sulfate Ringer's. In one group of experiments sodium uptake across lhe outer epithelial h o m e r was determined in control and I!A treated skin halxes at tile time of maximum EA effect on short-circuit CUl'ien[ (60 80 r a i n aftci- addition o1 [:% to tile internal solution]. In anotilcr group of experiments sodium uptake was delermmcd al the inSlalll e l nlaxinltlnl lime r;.itc o[ rise of shorl-circuil
Sodium flux and ethacrynic acid
2500-r
T
Sulf0te
193
Ringer's
I
f
0 . . . . . . . . . CONTROL-Jr ETHACRYNIC ACID E- PERIOD PEqlOO 1700~
'
I Chloride
8504
__.
"~
-~
Ringer's
-
j
J
t-
a >
0
lh
-/*'5/
"'"
.,-.o-~'''~ "°'c"
I
Fig. 3. Time course of d.c. resistance of skins exposed to sulfate Ringer's {upper graph) and chlorideRinger's (lower graph). Each experiment is identified by a different symbol. Note difference in scale of the ordinates. A larger scale has been used in the skins exposed to chloride- Ringer's in order to emphasize the slow rise in resistance after EA. The arrow below the abscissa mdicatcs the lime-scale. current. In the former experiments the procedure was as follows: short-circuit current was recorded in the skin to be treated with EA immediately before addition of the diuretic and at the .~tme instant in the control skin: l m M EA dissolved in Ringer's was added to the internal solution of the experimental skin and the same volume of Ringers was added to the control: as soon as short-circuit current reached a plateat, or maximum value in the EA-treated skin. short-circuit current was recorded in both skins and the sodium uptake procedure instituted in both skins. The same procedure was followed in those skins in which uptake across the outer epithelial border was determined at the instant of m a x i m u m time rate of rise of short-circuit current following EA except for the instant at which sodium uptake was determined. The mannitol space accessible from the outer side of the skin a m o u n t e d to 2.01 + 0.27/di3.14cm 2 in controis and 2.03 4- 0.31 ltl.3.14cm a in EA-treated skins in which sodium uptake was measured at the instant of maximum rise in short-circuit current caused by EA. In skins in which the sodium uptake was measured at the instant of maximum rate of rise of short-circuit current after EA. the mannitol spaces were similar to the above: 1.98 + 0.39/d..'3.14cm 2 in controls and 1.79 +0.211d/3.14cm-' in EA treated skins. These figurcs agree well with those reported by Biber et al. (1972) of 0.4301~1/cm 2 of frog skin. The results are summarized in Table 4. It may bc observed that the m a x i m u m increase in short-circuit current is 4.231,equiv/hr x 3.14cm-' whereas at the instant of the m a x i m u m rate of rise of current, this figure is considcrably Icss, 1.71/~cquiv,.'hr x 3.14cm 2. The increase in the uptake of sodium across the outer skin border caused by EA arc statistically significant. rising from 17.4Hcquiv/hr x 3.14cm 2 in control skins
to 26.6#equiw'hr x 3.14cm 2 in those skins in which maximum EA effect on short-circuit current was attained, and from 13.8,uequiv./hr x 3.14cm 2 in controls to 25.21~equiv.hr x 3.14cm 2 in those skins in which sodium uptake was determined at tile instant of the maximum time rate of rise of short-circuit current. Thus, at the instant of the maximum time rate of rise of short-circuit current, sodium uptake had already reached the value of that at the m a x i m u m EA effect on current. Therefore, the increase in sodium influx across the skin induced by EA appears to be consequent to the increase in sodium uptake across the outer epithelial border. This effect mimics the response of fl'og skin towards antidiu,'etic hormone (Curran et al.. 1963). E[l~'ct o[ EA on su!late fluxes across the skin In an effort to determine the effect of EA on the passive movements of a relatively impermeant anion. the inward flux of sulfate was studied on skins exposed to sulfate Ringer's during a I-hr control period and I hr following EA treatment, when the effect of EA was maximal. Inward sulfate flux rose from 0.042/2equiv hr x 3.14 cm" to 0.083/,equiv., hr x 3.14cm 2, the increase being highly statistically significant (P < ().011. Thus the sulfate inward flux is about 1/100 of the concomitant actively transported sodium flux. EA causes an increase in the permeability of the passive sulfate pathway somewhat similar to that seen for sodium in passive movements in toad bladder dterrera. 1975).
I)ISCI_,%.~ION The results of the experiments performed in the present investigation indicate that EA causes an in-
194
}'R,\N( IS( (I ('. I{IRRtRA A \ I ) ~,'|[INISI:RRA[ 1:~,11.\1 " l a b l e 4. S o d i u m u p t a k e acros'; the o u t e r ~,urface of the e p i t h e l i u m a n d COll,.2Olllilall( ~,hort-citcuit currcl'tt
('ontrol skin Sl',ort-circuit Sodiurn ctnrent uptake (Hcqui~ hr x 3.14 cm-')
t'A-trcated skin Short-circuit Sodium current
up[ak¢
~l~equi,. hr , 3.14 cm-'l
.~. S o d i u m
uptake
C u r r e n t and s o d i u m u p t a k e m e a s u r e d at time of m a x i m u m ilA effect on current
Control period Influx period AS(;(" t" {A St.'(') F (A Sodium uptake)
4.21 4.21
3.44 7.67 4.23
17.4
o.ol)
26.6
9.3
56.29~' 6.1t, a
Current and sodium t, ptake measured at time of maximum rate of rise of short-circuit current after EA Current period 4.14 3.19 lntlux period 3.79 13.8 4.89 25.2 AS('(" -0.35 1.71 I'" IASCC) 25.31 ~ t' IA Sodium uptake)
11.4 6.09"
" l' is signilicant at or below the 0.05 le',el bt, t not at the O.t)l level. t. F is significant at or belm~ the 0.01 level.
itial increase in short-circuit current which rctlects the concomitant increase in sodium influx across the skin. Thus. EA initially stimulates sodium transport across thc preparation. These results contirm those of Baba e t a / . (1966l. ttowever. EA can act from either sidc of the skin in the case of Bulb marim~s whereas it acts from the tuner side only in the case of the frog used by Baba et al. [1966j. Therefore. it would appear that species differences may' play a role in the accessibility of the sites of action of EA of the different skin epithelia. If a sufficiently hmg period. 3 hr. is allowed after EA treatment short-circuit current and sodium influx fall below control values. In toad bladder EA also causes a ['all in short-circuit current and sodium inttux: this decrease is not preceded by a rise in these parameters which begin to fall promptly after EA addition; however about two hours are required for them to reach a m i n i m u m value (Herrera. 1975). The close correspondence between short-circuit current and sodium influx in control and EA periods indicates that the diuretic does not stimulate transport of any other ion, on the one hand. and on the other that the passive sodium backflux is not appreciably increased. In this respect, toad skin responds quite differently to EA as compared to toad bladder: in the latter. EA caused a marked rise in sodium backflux (Herrera. 1975). Direct contirmation of this was sought in a group of experiments in which sodium outtlux was measured d u r m g control and EA periods. EA caused no consistent change in sodium outflux which a m o u n t e d to between ().It) a n d 0.23 imquiv,'hr x 3.14-cm 2, less than I0". of inllux. The tinding that short-circuit current is higher than sodium inttux during period 1~ could possibly bc due to a displacement of intracpithelial potassium towards the bathing solutions, presumably' across the inner, potassium permeable membrane, following increased intraepithelial sodmm accumulation due to the increase in permeability to sodium of the external epithelial barrier caused by' EA. In the remaining periods, sodium influx was generally about I0". higher than short-circuit current.
The increase m sodium transport caused by EA is in a way reminiscent of thc effect of antidiuretic h o r m o n e {ADH). C u r r a n et al. {19631 observed that this h o r m o n e causes a rise in sodium transport across flog skin through an increase in the sodium permeability of the ot, ter surface of the skin epithelium. EA likewise increases sodmm uptake across this barrier in toad skin leading to an increased delivery of sodmm to the pump. According to Biber and ( ' u r r a n (19701 the relationship between sodium concentration and sodium uptake across the outer barrier may be described as the sum of a saturable and a linear component. Only the saturable component would be involved in transepithelial sodium transport. The linear c o m p o n e n t would represent sodium entering a tissue space or spaces inaccessible to mannitol but not directly involved m sodium transport: these spaces are not detined at present and could be represented by the space accessiblc to sodium diffusing through the sl'mnt p a t h ~ a y between epithelial cells and the sodium space represented by gland cells and other epithelial components. In the present investigation these two c o m p o n e n t s have not been explicitly separated but it is clear from the results that EA can increase Na uptake across the outer barrier. This mcrease is more than enough to account for the increase in transepithelial s o d m m transport, represented by the short-circuit current, its may be seen m Table 4. In experiments performed on the skin of Rtma pipiens. Cruz (1976) found that sodium influx a m o u n t e d to 3.62 Hequi', hr x c m 2 and concomitant short-circt, il current was 1.76,uequiwhr x cm 2 when the outer surface of the skin was exposed to Ringer's containing 103.7mequiv 1 sodium. Using the linear coelticicnt calculated by Biber et al. (1972). it can be calculated from their rest, Its and those of Cruz {1976) that 1.46 Hequiv. h," × cm 2 of sodium intlux (about 40".) belongs to the non-saturable or linear component. The maximum increase in short-circuit current caused by EA. from 4.21 tlequiv..hr × 3.14cm 2 to 7.67 Hequix .hr x 3.14 cm 2, a m o u n t s to 3.46/mquiv
Sodium flux and cthacrynic acid hr x 3.14cm 2. The concomitant change in influx, from 17.4pcquiv/hr x 3.14cm z to 26.6l~equiv/ hr x 3.14cmZ, 9.2 #equiv/hr x 3.14cm 2, is more than double that required for the increase in short-circuit current. Should the saturable inttux represent sodium moving across the outer barrier involved in inward active sodium transport it cannot be less than shortcircuit current. Using the figures calculated from Biber et al. (1972), 40°. of the increase caused by EA might be tentatively ascribed to sodium entry into a non-transporting compartment but 60°~,, could be ascribed to the transport component: this would amount to 5.5#equiv/hr x 3.14cm 2, more than accounting for the 3.46 l~equiv..;hr x 3.14 cm 2 increase in short-circuit current caused by EA. In the case of A D H a 20-min lag period betwccn addition of the hormone and the initiation of its effect is observed. This lag period is indicative of the occurrence of a series of intracpithelial events initiated at the internal epithelial border (the hormone only acts from the inner side of the epitheliuml and culminating in an increased permeability of the outer surfaces towards sodium. In the case of EA, thcrc is practically no lag phase even when the agent is added to the inner side where the rate of increase of its effect is slower than on the outer side, but the onset is nevertheless immediate. This behavior of the skin towards EA suggests that this agent does not act through a series of relatively slow events as does ADH. The increase in sodium permeability of the outer border caused by EA takes place before the rate of increase of short-circuit current has reached its maximum value since the increase in sodium permeability does not show any further change when determined at maximum short-circuit current. Moreover, the fall in total resistance caused by EA in skins exposed to sulfate Ringer's is probably a consequcnce of the increased sodium conductance across the outer barrier of the epithelium. If the resistance of the active sodium transport pathway includes two series components, thc resistance of the outer barrier towards sodium and the resistance of the active pumping mechanism at the basal border of the cells, a decrease in the former will bring about a fall in the resistance of the whole of the active pathway, all other resistances of the active pathway remaining constant. Since increased delivcry of sodium to the pump increases the transport rate across it, increasing pump conductance, the decrease in the resistance in the outer barrier towards sodium will bring about a secondary fall in the resistance across the pump thus probably bringing about a larger decrease in the resistance of the active pathway than would be expected from the fall in the outer resistance alone. In chloride Ringer's this effect is masked by the low resistance of the skins because of the high parallel anionic conductance. therefore only a gradual rise in resistance is seen which corresponds to the increase of resistance of the active pathway during the late partial inhibiton of sodium transport which is seen to follow the marked early rise in current caused by EA. The increase in sulfate passive fluxes caused by EA indicates that its action is not limited to the active sodium transport path. However. the possible influence of the EA-induced increase of sulfate conductance on total skin conductance in skins exposed to
195
sulfate-Ringer's is limited since the sulfate fluxes are quite small compared to the active sodium flux. The passive sodium flux across the skin, however, does not appear to be appreciably modified by EA as it is in toad bladdcr (Herrera, 1975), its contribution to changes in total skin conductance being thereby negligible. The fact that EA increases sulfate passive fluxes but not the passive sodium backflux requires explanation. Sodium backflux is considerably larger than sulfate tluxes and possibly takes place through a transcellular pathway while sulfate could take an intercellular route. EA could increase the sulfate permeability of the intercellular pathway, presumably acting on the tight junctions between cpithelial cells, while the passive sodium transcellular route remains unmodified, since the main resistance to passive sodium movemcnt. the basal epithelial membrane, presumably is not affected by the drug. The late fall in sodium transport following the initial stimulation is possibly caused by an inhibitory action of the diuretic on the sodium pump itself. There is some evidence that EA can inhibit the sodium pump. Herrera (1975) has already shown that EA can block sodium transport in the toad urinary bladder. However, no early stimulatory effect of EA on sodium transport was observed in this organ. An early increase in transepithelial potential difference occurs simultaneously with the increase in sodium permeability: however, it is not possible at present to say whether it is the result of an increased sodium diffusion potential across the outer epithelial barrier or to increased electrogenic pumping of sodium at the inner epithelial border. EA may act from either side of the skin: however, its action is much faster when added to the outer solution. EA at 1 mM concentration results in a maximum effect: further addition either to the same side or to the opposite side of the skin does not cause further changes in the electrical parameters and sodium transport. This suggests that all sites of action are accessible to EA from either side of the skin. Moreover, the more rapid rate of onset of the effect of EA from the outer side as compared to its effect from the inner side suggests that the sites of action are more accessible from the outer side. possibly residing on the outer barrier of the epithclium itself. The action of EA on toad skin may be relevant to its pharmacological effects at the level of the kidney. The enhancement of tubular sodium reabsorption following EA administration (Dirks et al., 1966) and its anti-diuretic effect in nephrogenic diabetes insipidus, which have been ascribed to an enhanced proximal tubular reabsorption of sodium consequent to a contraction of the extracellular compartment (Brown et al.. 19661 may. at least in part, be explained on the basis of an increased sodium reabsorption at the proximal tubule due to a direct action of EA on tubular epithelial cells. Although Abramow (1974) has observed that EA antagonizes the hydrosmotic effect of ADH, it must be remembered that in certain epithelia. such as that of the toad bladder, the hydrosmotic and natriferic effects of A D H are independent, the hormonc acting possibly on two separate barriers in series, a dense diffusion barrier which retards all substances including water and a porous barrier. Vaso-
196
I-R AN{ IS('l I ( ". [ ] I RRI,R a AX;l) M t I:,, lSl RRA I ['.Sl I.'.. 1
pressin increases the p c r m e a b i l i t y to s o d i u m by' an effect o n the diffusion barrier a n d to w a t e r by' an effect o n the p o r o u s barrier {l,ichtcnstein & Leat: 1965, M e n d o z a et a/., 1967: P e t e r s e n & E d e l m a n , 1 t,~64). R I': l, I':R I':N (" I-:,%
AI~P,.aMow M. 11974) Etfccts of ethacrynic acid on the isolated collecting tubule..I, elm. Imest. 53, 796 804. B,',BA W. I.. SMmt A. J. & "Fo'.V',/SH~>,D M. M. (1966) A comparison of the effcct,~ of cthacrynic acid and a mercurial diuretic (Mersal',l) on sodium transport across the isolated frog skin. Br..I. Pharmac. 28, 238 245. BmI:R T. U. I... CRtZ 1.. J. & Ct RRA',/ P. F. 11972) Sodium influx at the outer surface of frog skin. Evaluation of different cxtraccllular markers. J. .%h,mhrane Biol. 7, 365 376. [:hm:R T. U. L. & (Tt:RuaY I). I". (19701 l)irect measurement of uptake of sodium ~,t the outer surface of fiog skin. J. #en. PIn'.siol. 59, g3 99.
BRow>, 1). hi.. Rl:','>:()ll)s J. W., MI('H,xEIS A. F. & Ut.sJroM R. A. 119661 The use and mode of acnon of ethacrynic acid in nephrogcnic diabetes insipldus. Pediatrics .t7, 447 455. CRUZ I.. J. (19761 Esludio dc la pcrmcabilidad tic sodio a tra~es dc la superticic extema de la piel aisklda de rana. Evaluation de los espacios extracelulares en el epifolio. M. 17). Thesis. Llniversidad Central de Veneztmla. CI:RRAN P. 1".. H'FRRIiRA ]'. (-'. & FI.ANI(;AN ~V. J. 11963) The effect of calcium and antidiuretic hormone on sodium transport across [rog skill..L ~tcn. Phv.siol. 46,
1011 1027. I)tR~s J. H.. ('lRkSl:X,,,, W. J. & Ih],~LIX~:R R. W. 11966) Micropuncture study of the effect of various diuretics on sodium reabsorption b). the proximal tubules of tile d o g .I. elm. hn'c.st. 45. 1S75 ISX5.
( i o l DmiR(; M., M t ' ( ' t RD',' I). K.. Irq~t.iz E. L. & t:lu I!MU
L. W., JR. Effects of cdlacrynic acid (a new ,~duretic agenO on renal diluting and concentrating mechanisnls: evidence for site of action in the loop of I-lenle. J. elm. Invest...13, 201 216. ['[I:RRt RA E. ('. 119661 Action of o u a b a m ill the toad urinar,, bladder. Am. J. l)hw'iol. 210. 9gO 9g6. IhRE~i.R,X I-'. C. (19681 Action of ouabain on bioelectric properties and ion content in toad urinary bladder. Am. ,I. Phvsi,l. 215, 183-189. th:Rm~Ra E. C. 11975)The role of tile active and passive sodium pathways~n the mechanism of action of ethacrvnic acid. (h'n. Pharmac 6 ")01-'~07 LWIlII:>gSTH>/ N. S. & LI ,'d: .,~. 119651 Effcct of amphotericm B on the permcabilit) of tile toad bladder. J. elm. hlrest. 4,1, 1328 1342. M I : N I ) o Z , ~ S. A.. II,'~Xl)l.liR J. S. & (.)RLOFF J. [1967) Effect of amphotericin B o n permeability and short-circuit c u r rent in toad bladder. 4m. ,I. Physiol. 213. 1263-1268. ()KItA (i. T., K.,~m~Ra, J. J.. RI('tI.,~RDSON E. & LERoY G. V. 119571 Assaying c o m p o u n d s containing H "~ and (,t4. Nucleonics 15, I I I 115. PIi'I'I-RSt!N M. J. & EI)I!I MAX: 1..~. (19641 Calcium inhibition of the action of',asopressin on the urinary bladder of the toad. ,I. elm. Inrest. 43, 583 594. Pt s('m!rT J. B. & GOLDm!R(i M. 119681 The acute effects of furosemide on acid and electrolyte excretion in man. J. Luh. elm. 3,h.,d. 71, 666 677. SI3.DP< 1). W.. EK,',OYAN G.. St:KI W. N. & RL('mR. JR. E. C. ( 1 9 6 6 ) Localization of diuretic action flom the p a t t e r n of water and electro h i e excretion..,hm. N. Y /h'ad. Sci. 13% 328 343. ,"kIK.ai. R. R. & ROHLt. I". J. 11969) Biometrv. W. II. Freenaan. San Francisco. ('A. StN:t ~ "F. H.. S m t p W. D. & WIN S. F'. 11965)The effect or ethacr)nic acid ill nephrogenic diabetes msipidus. (Ira. Rev 13, 314.
1977. |b/ 8. pp. fgg~to 196. Perqanum
Press Printed in Great Britain
PARADOXICAL STIMULATION OF SODIUM T R A N S P O R T IN T O A D SKIN BY E T H A C R Y N I C ACID FRANCISCO C . HERRI'RA AN[) MONTSERRAT ESIEVE*
Departamento de Fisiologia, Ccntro de Biofisica y Bioquimica, Instituto Vcnezolano dc lnvestigaciones Cientificas, IVIC, Apartado 1827, Caracas. Venezuela (Received 4 February 1977) Abstract 1. Ethacrynic acid, like anti-diuretic hormone, increases active sodium transport across the skin of BuJo marinus.
2. This is reflected in an increase in short-circuit current accompanied by a rise in potential difference and a marked fall in skin resistance, followed by a slow rise. 3. These effects result from an increased sodium uptake across the outer epithelial surface, where the sites of action of ethacrynic acid appear to be located. 4. These results suggest that the anti-diuretic effect of cthacrynic acid in nephrogcnic diabetes insipidus may bc related to an increased sodium rcabsorption at tubular sites where water reabsorption is obligatory.
INTROD u(TrlON Ethacrynic acid, 2,3-dichloro-4-(2 methylcncbutyryl)-phenoxy acetic acid, is a potent diuretic believed to act mainly as an inhibitor of sodium transport at the level of the ascending limb of Henle's loop (Goldberg et al., 1964; Seldin et al., 1966; Puse h e t t & Goldbcrg, 1968). However, in nephrogenic diabetes insipidus it causes a considerable decrcase in water intake and urine output (Steele et al., 1965; Brown et al.. 1966) which has been ascribed to an increased reabsorption of water and sodium at the level of the proximal tubule consequent to a contraction of the extracellular compartment. Dirks et al. (1966) obscrvcd an e n h a n c e m e n t of proximal tubule reabsorption of sodium following ethacrynic acid (EA) administration in micropuncture experiments. These authors also suggest that this increased rcabsorption would be consequent to acute depletion of extracellular volume, a response opposite to that produced by ,saline infusion. Nevertheless, a direct action of EA on tubular sodium reabsorption, although somewhat paradoxical, has not been ruled out. EA could facilitate sodium reabsorption at tubular sites where water rcabsorption is obligatory (Steele et al.. 1965). EA has been found to have an inhibitory effect on sodium transport across the epithelium of the toad urinary bladder (Hcrrera, 1975), but in frog skin this inhibitory effect is preceded by a one-hour long stimulation (Baba et al., 1966). In the present investigation, the effect of EA on ion transport across amphibian skin has been further explored. The results of Baba et al. have been confirmed and the site of the changes in the sodium transporting mechanism in toad skin epithelium responsible for the observed effects of EA on sodium transport has been located. METItODS
Specimens of the toad Bulo marinus captured in the environs of Valencia. north-central part of Venezuela. were * With the technical assistance of Yolanda Mandujano. GP
8'3--D
IS9
used in all cxperimcnts. The toads were double pithed: in some cases the ventral skin was removed and divided into symmetrical halves and the results of experiments on one half compared to thosc of the other which either served as control or on which the converse experiment was performed. Otherwise, each skin served as its own control: treatment with ethacrynic acid would be instituted following a control period of sutlicicnt duration to obtain stable values for the baseline bioclcctric parameters and fluxes. To minimize cdgc damage, the skins were mounted sandwiched between parafilm (Marathon Products, Ncenah, Wi, U.S.A.) gaskets lightly smeared with stopcock grease (Lubriseal, A. H. Thomas Co., Philadelphia, PA, tJ.S.A.I as a diaphragm separating two lucite chambers filled with the appropriate Ringer's solution. Two different Ringer's solutions were employed. In some cxpcrimcnts conventional frog Ringer's containing sodium chloride, 112 mM: potassium bicarbonate. 2.4 raM; calcium gluconatc, 0.5 mM. was used; this solution was termed chloride Ringer's. In experiments in which a high passive anionic conductance was undesirable, sodium chloride was replaced by 56mM sodium sulfate (Herrera. 1968. 1975): this solution was termed sulfate- Ringer's. The skins were short-circuited automatically and the current was interrupted every 8 min for 80 sec to record potential difference. LJnidircctional sulhltc and sodium fluxes were measured by adding ssSO] or sodium-22 to the solution bathing one of the surfaccs of the skin and sampling the solution at the opposite side every 20 min. The details of the methods have been published previously IHerrcra, 1966). The rate of sodium uptake across the outer surface of the epithelium was determined by a modification of the method used by Biber and Curran (1970). F'aired skin halves were stabilized for 1 hr in sodium sulfate Ringer's in chambers provided by an inlet port on the mucosal side connected by means of plastic tnbing to a three-way stopcock which in turn was attached to a 20ml syringe filled with Ringer's solution containing sodium-22 and tritiated mannitol. Once short-circuit current and potential difference were stabilized, 1 mM EA dissolved in Ringer's was added to the inner solution of the experimental skin half and the same volume of Ringer's added to the control. Sodium uptake was determined either at the time of maximum EA effect on short-circuit current or at the time of maximum time-rate of change of the current. In either case, at the appropriate instant, the same procedure was followed simultaneously in control and experimental skin
I~tl
I"RAN('IS('() ( ' . l J l R R t R A ,X\I) M ( I N I S l R R A I
JiSll\l 3000
./-/"
~"
SCC \
/ ImM Ethocryn~c ac,d outs,(Je
ImlVl Ethacrynic ocld reside
1o
I:ig I. Time course of short-circuit current 1%('(.'1. potential difference IPI)) and de. resi>tance iRI in skins bathed in sulfate Ringer's and treated with EA on the inner side (left-hand graph) and on the ot, tcr side (right-h:md graph). Arrow along the abscissa indicates the time-scale. halvcs. The outer solution ~ a s quickl.~ removed: the radioactive solution held in the syringe was rapidly injected into the outer chamber and duplicate I(R)itl samples wcrc immediately taken from the radioactive soh, tion. The latter was left in contact with the outer surface of the skin for exactly 35 sec; this period was terrain,clod bx removal of the outer .solution. removal of the skin ~mdv..ich and rapid blotting of both skin surfaces ~ith W h a t m a n No. 54 tilter papcr. The skin area exposed to the sohHlons x,.as cut out. and dricd overnight at 105 ('. Thc cxposcd skin area measured 3.14cm:. The dried skins xsere then extracted for at least 2 h r m 2nll (I.I N H N O ~ Ahquots o[ this extract were counted along with the salmples taken from the radioactive sx)lution and Iritiatcd mannitol and sodium-22 wcrc determined simultaneously on a beta counter by' the double-label counting rncthod of Okita et al. (1957). The mannitol space accessible from the ot,tcr skin st, rface was then determined. Sodium t, ptakc ~ a s estimated from the incorporation of radioactive sodium after corrcction for mannitol space. The data have been analyzed statistically by mcans of one-way and two-way analysis of ~ariancc and orthogonal comparisons have bccn performed on the rcsuhs [Sokal & Rohlf, 19691.
RESL?I,'I'S E~lbct oI" E A on shm't-circuit current acros.~ toad .skin
Paired s y m m e t r i c a l halves of thc ventral skin of the t o a d werc m o u n t e d in c o n v e n t i o n a l c h a m b e r s a n d
b a t h c d with sulfate R i n g e r ' s on b o t h sides. After p o t e n t i a l ditt'e,'encc a n d s h o r t - c i r c u i t c u r r e n t h a d stabilized, t h e baseline values of s h o r t - c i r c u i t current. p o t e n t i a l difference a n d d.c. resistance ( m e a s u r e d as the ratio of p o t e n t i a l difference to s h o r t - c i r c u i t current) were recorded d u r i n g a c o n t r o l period of 1 hr d u r a t i o n . T h e n 1 m M EA was a d d e d to the internal s o l u t i o n of o n e of t h e s k i n halves a n d to the e x t e r n a l s o l u t i o n of the other. F i g u r c I s h o w s t h e t i m e c o u r s e of p o t e n t i a l difference, s h o r t - c i r c u i t c u r r e n t a n d total skin resistance. In b o t h cases s h o r t - c i r c u i t c u r r e n t rises d r a m a t i c a l l y , potential difference rises r a t h e r m o r e slowly a n d relatively less t h a n s h o r t - c i , c u i t current b r i n g i n g a b o u t a m a r k e d fall in resistance. W h e n EA is a d d e d to the inner side s h o r t - c i r c u i t c u r r e n t rises to a peak value a n d t e n d s to fall off slowly a n d linearly with time. W h e n EA is a d d e d to the ot, ter side it cat, ses an a b r u p t rise in c u r r e n t after w h i c h tile curFent r e n l a i n s m o r c or Icss steady. T h e time rate o f c h a n g e of the electrical p a r a m e t e r s is q u i t e clearly m u c h g r e a t e r v,hcn EA is a d d e d to the e x t e r n a l solution t h a n w h e n it is a d d e d to the hlncr one. T a b l e I s u m m a r i z e s the rcsults of 7 experinaents. Short-circuit c u r r e n t ( S t ' ( ' ) rises to vahtes abot,t d o u b l e the c o n t r o l ones. the i n c r e a s e s being h i g h l y statistically signiticant bul they do n o t differ between s k i n s treated with EA on the o u t s i d c or on lhc inside. T h e potential difference also is i n c r e a s e d but p r o p o r t i o n a -
Table l. Changes in short-circuit current, potential difference and resistance caused b\ cthacrynic acid added either to the outside or the inside of skins bathed in sulfate Ringer's
Before [A
Maximum value after EA
A
t'~
/. animals {cllange)
t" m vs out
t,,,, ~ (mini
/ t,,.~
k animals t,..,
Short-circuit current (Hcqui', hr × 3.14 cm-'l I'A outside EA inside
2.07 3.11
4.89 5.84
2.83 2.73
61.2W 41.07 ~'
4.74" 5.64"
0.06
16 39
(,i.31 ~,
11.15 ~
Potential difference (mV) EA outside EA inside
98 106
132 123
34 17
57.70 h 33.2&
2.51 4.74"
10.52"
13 37
36.39t,
3.15
1884 1379
1125 g82
759 498
9t).31 h 52.10 h
24.95 ~' 20.49 ~'
16.53 t,
12 32
17.59"
2.72
Resistance 1~1 EA outside EA inside
a F is signilicant at or bclo\~ the 0.05 level but not at the 0.O1 level. ~' F is signilicant at or below the t).01 level.
Sodium flux and ethacrynic acid _No CI . ~
. . . . .
f \
~_,~-
R No2SO 4
+ .
:
-
:
~ ,
•
•
.
•
.
.
191
R NozSO44"ImM Elhocrynic a c i d reside . . . . . . . . . . . .
t
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---.11 " ~ ~ / , ~ , , ~ / ~ ' , ~ ; ~ s c c
Fig. 2. Time course of short-circuit current (SCC). potential difference (PI)). d.c. resistance (RI and sodium influx (hatched area denoted by 4~i'I in skins pre-incubated in chloride Ringer's (R-NaCI) and then exposed to a control period in sulfate Ringer's followed by treatment with EA inside (R Na 2 SO4 + I mM EA inside). Arrow along the abscissa indicates the time-scale. tely less than short-circuit current, bringing about a statistically significant fall in resistance. The increase in potential difference is smaller in skins treated with EA on the inside and so is the decrease in resistance probably becausc the increase in SCC was relatively smaller since mean SCC before EA was higher in skins treated with EA inside than in those exposed to EA on the outside. Significant differences in response of the electrical parameters to EA a m o n g animals could bc detected. The time required to reach 90°~o of the maximum value of the response to EA of the three parameters, t,m.,, was much shorter when the diuretic was added to the outer side of the skin suggesting that the site of action of the drug was further removed from the inner skin surface than from the outer: no further effect was elicited when EA was added to the same side or to the side originally free from EA. These results suggest that EA increases short-circuit current through a decrease in resistance to the movement of ions through the skin. However, the ions whose transport is increased must be identified. Therefore, experiments were performed in which the mucosal to serosal sodium fluxes were measured.
Determination of mucosal to .serosal .sodium influx across toad skin under the influence of EA Experiments similar to those described above were performed on toad skins exposed to either chloride or sulfatc Ringer's. The skins were equilibrated and short-circuited. In order to determine transepithelial sodium movement approx 1/~Ci/ml of sodium-22 was added to the outer solutions of the skins and 500pl samples were taken from the inner solution at 20-min
intervals, replacing this volume with non-radioactive Ringer's. U p o n completion of a l-hr control period, I m M EA was added to the inner solution and the sampling of the internal solution was continued for another 4 hr. Short-circuit current, potential difference and total resistance were recorded throughout the experiment. A typical experiment is shown in Fig. 2 in which a skin exposed to sulfate Ringer's is treated with EA. The diuretic causes a rapid rise in potential difference and a fall in total resistance. Short-circuit current and inward sodium ftux increase almost simultaneously, short-circuit current being quite close to the sodium intlux. However, the increase in sodium influx sccms to lag slightly behind the increase in short-circuit current. Since, except for the lag in the increase in short-circuit current, the increase in short-circuit current and inward sodium flux agree quite closely in skins exposed to sulfate or chloride Ringer's, the increase in short-circuit current is mainly accounted for by an increase in the active inward transport of sodium. No other ion appears to be transported to any appreciable extent, chloride especially not being involved. Tables 2 and 3 show the orthogonal comparisons of inward sodium fluxes and short-circuit current during control and three subsequent l-hr periods (three flux determinations in each period), beginning 20 min after the addition of EA, in skins exposed to sulfate and chloride. Ringer's. Short-circuit current during each period was determined by graphical integration of the current. The control values of short-circuit current and sodium influx correspond to those obtained during the hour period immediately preceding EA addition
"lziblc 2. ()rthogonal comparisons of short-circuit current and sodium inward tlux between control period (('1 and ethacr)nic acid periods 4t) ~t) IT~ I. 100 140 1"1)) and 200 241) (/3) rain after addition of the drug to skins exposed to chloride Ringer's c
7~ 20 80
"r2 3r) 80 140 180--240 (rain after EA)
Sodium mucosal to serosal 11ux (l~equiv/hr x 3.14 cm 2) C vs (T~ + 7"2) 5.69 8.07 7.36 7"1 vs 7"2 8.07 7.36 (C + T~ + 7~) vs "F3 5.69 8.07 7.36 Short-circuit current (,uequiv/hr x 3.14 cm 2) C vs (Tt + 7"2) 5.29 8.6,~ 6.43 T~ vs 7) 8.6~ 6.43 (C + + 7)) vs "/~ 5.29 8.86 6.43
71
F
P
19.17
<0.01
1.79
n.s.
4.58
31.83
<0.01
4.19
15.26 11.33 22.87
<0.01 <0.01 <0.01
192
J R ' , \ ( I~,(ll (" JJlRRIR-,, .;\11 M ( ) N I S l R R ; I
[:ib]c 3. ( ) r l h o g o l i a l
ctH)lp;.lll,~on,, o[ ,,[)orI-circtiil
ctlrlclll
I',11\1
anti ~,t~t.]itllii
ill\~,Hld t'iLl\ [)CDACCII
conlro] period i ( l and clhacr',llic ;tcid pcliods 41) Sll I'/il. 10It 14()(/i,i cind 21Rl 2411 I'/~) nlin ~iftcr ;iddilion of the drug in skins cxpo,,¢d Io sti][':.ilc Rlilgcr's (
Soditiin nltlcosal
("
%s ('l~ '1"~ x s 7 ,
((' a 1; t 1-I ~s 7 I,
1
I'
1.9 =
3 53 .~'.5"~
~,,x': .',S"
,x<,i "S 2 "~(
<;I).(I n.s.
i.U:--
"~
].';(,
24.71
< ). I
,:It.Ill
currcnl {/IL'qtlJ', hi" : 3.14 cnl 2)
(" xs I1~ -F 'I,) 'I m ~s 1", (('"I~ -
I. "I, ,<4(1 14() IX() 24U Imin aflcr I:\1
It) hc'l'l)sH] Ilu\ (/~¢Lll.ll\ ]11' :>~ "~14 CI]1")
- I.I
S[lorl-cJl'Ctlil
1"1 20 Xl)
I . I ~> Ii~
• 12
14.'
.iX2
19.49
.t S" .;S2
133
ll,b,,
2.12-
l 12 4.42
I I.(',H
<'-I).l)l
Iperiod ("). ARer EA, samples t~eie tnken e~ el') 20 rain and the samples takcn during the period bcl~cen 20 rain and ,'4(/min aftcr I{A were colleclcd into lilt first I{A period {period /~ I: ihosc taken het~cen ,xO and 140 rain ~ere collected into the second EA period (period 72i :.ind finall 3. the s,:lnlplcs l:.ikcn hctwcen 180 and 240 rain altcr tIA ~¢rc collected its the third I{A period (period 1,0. r h c short-cii-ctiii Cul-renl \~,:.lS similarly divided ;Mid lllCail short-circuh CUlrCll{ dtlring each period ~as calculated h\ graphical integration of the time cotlrsc of the CLIITCnt. ITA periods I alld 2 hrackcl lhc nlHxillltlnl rise in short-circuit Ctilieill as sllo~n ill Fig. 2: I{.:\ period 3 rcprcscnls the kisl three ltLIX periods ~iftcr I{A with their COrlespending short-circuil Ctll'I'Cllt. Tile (H'lhogt)ll;.iJ conlparisoils \%ere lnadc sticcessi,
2tO
sLtll~tte ,;ince short-circuit CLIIrcn[ alld nlucoscli to scro~zl sodium flux agree closcl.,,. Morcovcr. il mal~ he obscr~ed in Table 2 that during lhe period I~ {20 ~40 mm after E.,\L .S('(' is higher than hlfltlx b ~, ;.ll-lout 0.611equi\.hr x 3.14cm 2, this figure being statisticall) significant below tile 0.05 level. During I2 (140 140ram clflcr I(/\1. S('(" is 0.9/lequi~ hr x 3.14 cm 2 lower (P < 0.051 than mucosal Io sciosal sodium flux as ~votild bc expected if short-circuit ctlrrClll is the difference belween inthix and outt]ux of sodium. In skins cxposcd to still'ale Rhlger's these diffcrcnccs xvcic not statistically signifi('illlt.
l'hc hchaxilu of tllc rcsistancc dill'ors in chloride and sulfate Ringer's. In the fol'nlel" C;.tSC contlol reslslancc is considerably lower than in the latter. After I(A addilJon resistance rises gradually duririg the whole of lhe E.,\ exposurc period. In lhc case of skins cxposed Io sulfate Ringer's. there is at marked fall in resistance immedialel~ after addition of t!A followed m some cases by a gradual rise. In chloride Ringer's thc ch;.in,,ese of resistance of the sodium pathway arc nl;.lsked b) tile high conducl~.lilce of the ailioni¢ pnihway. ~vhcl-eas m stllfalc Ringel"S the anionic Collductallce is quite low and mosl of tile ionic coilduclance is represenled by thai of tile soditnll transport path~;.iy. tile clltillges ill Collduciatlce of tilts ionic rotlle nol being masked b.,, lhe high anionic conductance (tlerrera, Ig75). ] h c lime Ct)tll%C t)[ Ihe resistance in chloride aild sulf:.itc Ringer's is shown in Fig. 3. The nlechani,sm underlying the increusc in in~aid sodiunl llux rcnlahlect to be determined. lllerefore. the soditlnl uptake across file otltcr Stllface of tile cpiihcliunl ~a> studied b~ Ihc method outlined by I*)iber ililtl ('tnra'li (1970). .~'lJdittm l//)/gl~t' ~i(l'lJ,,.,, lhc olllt'r
('pilh~.,liol
.\/ir/{/('t,
The ploccdtire outlined under .Methods was p¢i-[ormed on skins incubalcd ill sulfate Ringer's. In one group of experiments sodium uptake across lhe outer epithelial h o m e r was determined in control and I!A treated skin halxes at tile time of maximum EA effect on short-circuit CUl'ien[ (60 80 r a i n aftci- addition o1 [:% to tile internal solution]. In anotilcr group of experiments sodium uptake was delermmcd al the inSlalll e l nlaxinltlnl lime r;.itc o[ rise of shorl-circuil
Sodium flux and ethacrynic acid
2500-r
T
Sulf0te
193
Ringer's
I
f
0 . . . . . . . . . CONTROL-Jr ETHACRYNIC ACID E- PERIOD PEqlOO 1700~
'
I Chloride
8504
__.
"~
-~
Ringer's
-
j
J
t-
a >
0
lh
-/*'5/
"'"
.,-.o-~'''~ "°'c"
I
Fig. 3. Time course of d.c. resistance of skins exposed to sulfate Ringer's {upper graph) and chlorideRinger's (lower graph). Each experiment is identified by a different symbol. Note difference in scale of the ordinates. A larger scale has been used in the skins exposed to chloride- Ringer's in order to emphasize the slow rise in resistance after EA. The arrow below the abscissa mdicatcs the lime-scale. current. In the former experiments the procedure was as follows: short-circuit current was recorded in the skin to be treated with EA immediately before addition of the diuretic and at the .~tme instant in the control skin: l m M EA dissolved in Ringer's was added to the internal solution of the experimental skin and the same volume of Ringers was added to the control: as soon as short-circuit current reached a plateat, or maximum value in the EA-treated skin. short-circuit current was recorded in both skins and the sodium uptake procedure instituted in both skins. The same procedure was followed in those skins in which uptake across the outer epithelial border was determined at the instant of m a x i m u m time rate of rise of short-circuit current following EA except for the instant at which sodium uptake was determined. The mannitol space accessible from the outer side of the skin a m o u n t e d to 2.01 + 0.27/di3.14cm 2 in controis and 2.03 4- 0.31 ltl.3.14cm a in EA-treated skins in which sodium uptake was measured at the instant of maximum rise in short-circuit current caused by EA. In skins in which the sodium uptake was measured at the instant of maximum rate of rise of short-circuit current after EA. the mannitol spaces were similar to the above: 1.98 + 0.39/d..'3.14cm 2 in controls and 1.79 +0.211d/3.14cm-' in EA treated skins. These figurcs agree well with those reported by Biber et al. (1972) of 0.4301~1/cm 2 of frog skin. The results are summarized in Table 4. It may bc observed that the m a x i m u m increase in short-circuit current is 4.231,equiv/hr x 3.14cm-' whereas at the instant of the m a x i m u m rate of rise of current, this figure is considcrably Icss, 1.71/~cquiv,.'hr x 3.14cm 2. The increase in the uptake of sodium across the outer skin border caused by EA arc statistically significant. rising from 17.4Hcquiv/hr x 3.14cm 2 in control skins
to 26.6#equiw'hr x 3.14cm 2 in those skins in which maximum EA effect on short-circuit current was attained, and from 13.8,uequiv./hr x 3.14cm 2 in controls to 25.21~equiv.hr x 3.14cm 2 in those skins in which sodium uptake was determined at tile instant of the maximum time rate of rise of short-circuit current. Thus, at the instant of the maximum time rate of rise of short-circuit current, sodium uptake had already reached the value of that at the m a x i m u m EA effect on current. Therefore, the increase in sodium influx across the skin induced by EA appears to be consequent to the increase in sodium uptake across the outer epithelial border. This effect mimics the response of fl'og skin towards antidiu,'etic hormone (Curran et al.. 1963). E[l~'ct o[ EA on su!late fluxes across the skin In an effort to determine the effect of EA on the passive movements of a relatively impermeant anion. the inward flux of sulfate was studied on skins exposed to sulfate Ringer's during a I-hr control period and I hr following EA treatment, when the effect of EA was maximal. Inward sulfate flux rose from 0.042/2equiv hr x 3.14 cm" to 0.083/,equiv., hr x 3.14cm 2, the increase being highly statistically significant (P < ().011. Thus the sulfate inward flux is about 1/100 of the concomitant actively transported sodium flux. EA causes an increase in the permeability of the passive sulfate pathway somewhat similar to that seen for sodium in passive movements in toad bladder dterrera. 1975).
I)ISCI_,%.~ION The results of the experiments performed in the present investigation indicate that EA causes an in-
194
}'R,\N( IS( (I ('. I{IRRtRA A \ I ) ~,'|[INISI:RRA[ 1:~,11.\1 " l a b l e 4. S o d i u m u p t a k e acros'; the o u t e r ~,urface of the e p i t h e l i u m a n d COll,.2Olllilall( ~,hort-citcuit currcl'tt
('ontrol skin Sl',ort-circuit Sodiurn ctnrent uptake (Hcqui~ hr x 3.14 cm-')
t'A-trcated skin Short-circuit Sodium current
up[ak¢
~l~equi,. hr , 3.14 cm-'l
.~. S o d i u m
uptake
C u r r e n t and s o d i u m u p t a k e m e a s u r e d at time of m a x i m u m ilA effect on current
Control period Influx period AS(;(" t" {A St.'(') F (A Sodium uptake)
4.21 4.21
3.44 7.67 4.23
17.4
o.ol)
26.6
9.3
56.29~' 6.1t, a
Current and sodium t, ptake measured at time of maximum rate of rise of short-circuit current after EA Current period 4.14 3.19 lntlux period 3.79 13.8 4.89 25.2 AS('(" -0.35 1.71 I'" IASCC) 25.31 ~ t' IA Sodium uptake)
11.4 6.09"
" l' is signilicant at or below the 0.05 le',el bt, t not at the O.t)l level. t. F is significant at or belm~ the 0.01 level.
itial increase in short-circuit current which rctlects the concomitant increase in sodium influx across the skin. Thus. EA initially stimulates sodium transport across thc preparation. These results contirm those of Baba e t a / . (1966l. ttowever. EA can act from either sidc of the skin in the case of Bulb marim~s whereas it acts from the tuner side only in the case of the frog used by Baba et al. [1966j. Therefore. it would appear that species differences may' play a role in the accessibility of the sites of action of EA of the different skin epithelia. If a sufficiently hmg period. 3 hr. is allowed after EA treatment short-circuit current and sodium influx fall below control values. In toad bladder EA also causes a ['all in short-circuit current and sodium inttux: this decrease is not preceded by a rise in these parameters which begin to fall promptly after EA addition; however about two hours are required for them to reach a m i n i m u m value (Herrera. 1975). The close correspondence between short-circuit current and sodium influx in control and EA periods indicates that the diuretic does not stimulate transport of any other ion, on the one hand. and on the other that the passive sodium backflux is not appreciably increased. In this respect, toad skin responds quite differently to EA as compared to toad bladder: in the latter. EA caused a marked rise in sodium backflux (Herrera. 1975). Direct contirmation of this was sought in a group of experiments in which sodium outtlux was measured d u r m g control and EA periods. EA caused no consistent change in sodium outflux which a m o u n t e d to between ().It) a n d 0.23 imquiv,'hr x 3.14-cm 2, less than I0". of inllux. The tinding that short-circuit current is higher than sodium inttux during period 1~ could possibly bc due to a displacement of intracpithelial potassium towards the bathing solutions, presumably' across the inner, potassium permeable membrane, following increased intraepithelial sodmm accumulation due to the increase in permeability to sodium of the external epithelial barrier caused by' EA. In the remaining periods, sodium influx was generally about I0". higher than short-circuit current.
The increase m sodium transport caused by EA is in a way reminiscent of thc effect of antidiuretic h o r m o n e {ADH). C u r r a n et al. {19631 observed that this h o r m o n e causes a rise in sodium transport across flog skin through an increase in the sodium permeability of the ot, ter surface of the skin epithelium. EA likewise increases sodmm uptake across this barrier in toad skin leading to an increased delivery of sodmm to the pump. According to Biber and ( ' u r r a n (19701 the relationship between sodium concentration and sodium uptake across the outer barrier may be described as the sum of a saturable and a linear component. Only the saturable component would be involved in transepithelial sodium transport. The linear c o m p o n e n t would represent sodium entering a tissue space or spaces inaccessible to mannitol but not directly involved m sodium transport: these spaces are not detined at present and could be represented by the space accessiblc to sodium diffusing through the sl'mnt p a t h ~ a y between epithelial cells and the sodium space represented by gland cells and other epithelial components. In the present investigation these two c o m p o n e n t s have not been explicitly separated but it is clear from the results that EA can increase Na uptake across the outer barrier. This mcrease is more than enough to account for the increase in transepithelial s o d m m transport, represented by the short-circuit current, its may be seen m Table 4. In experiments performed on the skin of Rtma pipiens. Cruz (1976) found that sodium influx a m o u n t e d to 3.62 Hequi', hr x c m 2 and concomitant short-circt, il current was 1.76,uequiwhr x cm 2 when the outer surface of the skin was exposed to Ringer's containing 103.7mequiv 1 sodium. Using the linear coelticicnt calculated by Biber et al. (1972). it can be calculated from their rest, Its and those of Cruz {1976) that 1.46 Hequiv. h," × cm 2 of sodium intlux (about 40".) belongs to the non-saturable or linear component. The maximum increase in short-circuit current caused by EA. from 4.21 tlequiv..hr × 3.14cm 2 to 7.67 Hequix .hr x 3.14 cm 2, a m o u n t s to 3.46/mquiv
Sodium flux and cthacrynic acid hr x 3.14cm 2. The concomitant change in influx, from 17.4pcquiv/hr x 3.14cm z to 26.6l~equiv/ hr x 3.14cmZ, 9.2 #equiv/hr x 3.14cm 2, is more than double that required for the increase in short-circuit current. Should the saturable inttux represent sodium moving across the outer barrier involved in inward active sodium transport it cannot be less than shortcircuit current. Using the figures calculated from Biber et al. (1972), 40°. of the increase caused by EA might be tentatively ascribed to sodium entry into a non-transporting compartment but 60°~,, could be ascribed to the transport component: this would amount to 5.5#equiv/hr x 3.14cm 2, more than accounting for the 3.46 l~equiv..;hr x 3.14 cm 2 increase in short-circuit current caused by EA. In the case of A D H a 20-min lag period betwccn addition of the hormone and the initiation of its effect is observed. This lag period is indicative of the occurrence of a series of intracpithelial events initiated at the internal epithelial border (the hormone only acts from the inner side of the epitheliuml and culminating in an increased permeability of the outer surfaces towards sodium. In the case of EA, thcrc is practically no lag phase even when the agent is added to the inner side where the rate of increase of its effect is slower than on the outer side, but the onset is nevertheless immediate. This behavior of the skin towards EA suggests that this agent does not act through a series of relatively slow events as does ADH. The increase in sodium permeability of the outer border caused by EA takes place before the rate of increase of short-circuit current has reached its maximum value since the increase in sodium permeability does not show any further change when determined at maximum short-circuit current. Moreover, the fall in total resistance caused by EA in skins exposed to sulfate Ringer's is probably a consequcnce of the increased sodium conductance across the outer barrier of the epithelium. If the resistance of the active sodium transport pathway includes two series components, thc resistance of the outer barrier towards sodium and the resistance of the active pumping mechanism at the basal border of the cells, a decrease in the former will bring about a fall in the resistance of the whole of the active pathway, all other resistances of the active pathway remaining constant. Since increased delivcry of sodium to the pump increases the transport rate across it, increasing pump conductance, the decrease in the resistance in the outer barrier towards sodium will bring about a secondary fall in the resistance across the pump thus probably bringing about a larger decrease in the resistance of the active pathway than would be expected from the fall in the outer resistance alone. In chloride Ringer's this effect is masked by the low resistance of the skins because of the high parallel anionic conductance. therefore only a gradual rise in resistance is seen which corresponds to the increase of resistance of the active pathway during the late partial inhibiton of sodium transport which is seen to follow the marked early rise in current caused by EA. The increase in sulfate passive fluxes caused by EA indicates that its action is not limited to the active sodium transport path. However. the possible influence of the EA-induced increase of sulfate conductance on total skin conductance in skins exposed to
195
sulfate-Ringer's is limited since the sulfate fluxes are quite small compared to the active sodium flux. The passive sodium flux across the skin, however, does not appear to be appreciably modified by EA as it is in toad bladdcr (Herrera, 1975), its contribution to changes in total skin conductance being thereby negligible. The fact that EA increases sulfate passive fluxes but not the passive sodium backflux requires explanation. Sodium backflux is considerably larger than sulfate tluxes and possibly takes place through a transcellular pathway while sulfate could take an intercellular route. EA could increase the sulfate permeability of the intercellular pathway, presumably acting on the tight junctions between cpithelial cells, while the passive sodium transcellular route remains unmodified, since the main resistance to passive sodium movemcnt. the basal epithelial membrane, presumably is not affected by the drug. The late fall in sodium transport following the initial stimulation is possibly caused by an inhibitory action of the diuretic on the sodium pump itself. There is some evidence that EA can inhibit the sodium pump. Herrera (1975) has already shown that EA can block sodium transport in the toad urinary bladder. However, no early stimulatory effect of EA on sodium transport was observed in this organ. An early increase in transepithelial potential difference occurs simultaneously with the increase in sodium permeability: however, it is not possible at present to say whether it is the result of an increased sodium diffusion potential across the outer epithelial barrier or to increased electrogenic pumping of sodium at the inner epithelial border. EA may act from either side of the skin: however, its action is much faster when added to the outer solution. EA at 1 mM concentration results in a maximum effect: further addition either to the same side or to the opposite side of the skin does not cause further changes in the electrical parameters and sodium transport. This suggests that all sites of action are accessible to EA from either side of the skin. Moreover, the more rapid rate of onset of the effect of EA from the outer side as compared to its effect from the inner side suggests that the sites of action are more accessible from the outer side. possibly residing on the outer barrier of the epithclium itself. The action of EA on toad skin may be relevant to its pharmacological effects at the level of the kidney. The enhancement of tubular sodium reabsorption following EA administration (Dirks et al., 1966) and its anti-diuretic effect in nephrogenic diabetes insipidus, which have been ascribed to an enhanced proximal tubular reabsorption of sodium consequent to a contraction of the extracellular compartment (Brown et al.. 19661 may. at least in part, be explained on the basis of an increased sodium reabsorption at the proximal tubule due to a direct action of EA on tubular epithelial cells. Although Abramow (1974) has observed that EA antagonizes the hydrosmotic effect of ADH, it must be remembered that in certain epithelia. such as that of the toad bladder, the hydrosmotic and natriferic effects of A D H are independent, the hormonc acting possibly on two separate barriers in series, a dense diffusion barrier which retards all substances including water and a porous barrier. Vaso-
196
I-R AN{ IS('l I ( ". [ ] I RRI,R a AX;l) M t I:,, lSl RRA I ['.Sl I.'.. 1
pressin increases the p c r m e a b i l i t y to s o d i u m by' an effect o n the diffusion barrier a n d to w a t e r by' an effect o n the p o r o u s barrier {l,ichtcnstein & Leat: 1965, M e n d o z a et a/., 1967: P e t e r s e n & E d e l m a n , 1 t,~64). R I': l, I':R I':N (" I-:,%
AI~P,.aMow M. 11974) Etfccts of ethacrynic acid on the isolated collecting tubule..I, elm. Imest. 53, 796 804. B,',BA W. I.. SMmt A. J. & "Fo'.V',/SH~>,D M. M. (1966) A comparison of the effcct,~ of cthacrynic acid and a mercurial diuretic (Mersal',l) on sodium transport across the isolated frog skin. Br..I. Pharmac. 28, 238 245. BmI:R T. U. I... CRtZ 1.. J. & Ct RRA',/ P. F. 11972) Sodium influx at the outer surface of frog skin. Evaluation of different cxtraccllular markers. J. .%h,mhrane Biol. 7, 365 376. [:hm:R T. U. L. & (Tt:RuaY I). I". (19701 l)irect measurement of uptake of sodium ~,t the outer surface of fiog skin. J. #en. PIn'.siol. 59, g3 99.
BRow>, 1). hi.. Rl:','>:()ll)s J. W., MI('H,xEIS A. F. & Ut.sJroM R. A. 119661 The use and mode of acnon of ethacrynic acid in nephrogcnic diabetes insipldus. Pediatrics .t7, 447 455. CRUZ I.. J. (19761 Esludio dc la pcrmcabilidad tic sodio a tra~es dc la superticic extema de la piel aisklda de rana. Evaluation de los espacios extracelulares en el epifolio. M. 17). Thesis. Llniversidad Central de Veneztmla. CI:RRAN P. 1".. H'FRRIiRA ]'. (-'. & FI.ANI(;AN ~V. J. 11963) The effect of calcium and antidiuretic hormone on sodium transport across [rog skill..L ~tcn. Phv.siol. 46,
1011 1027. I)tR~s J. H.. ('lRkSl:X,,,, W. J. & Ih],~LIX~:R R. W. 11966) Micropuncture study of the effect of various diuretics on sodium reabsorption b). the proximal tubules of tile d o g .I. elm. hn'c.st. 45. 1S75 ISX5.
( i o l DmiR(; M., M t ' ( ' t RD',' I). K.. Irq~t.iz E. L. & t:lu I!MU
L. W., JR. Effects of cdlacrynic acid (a new ,~duretic agenO on renal diluting and concentrating mechanisnls: evidence for site of action in the loop of I-lenle. J. elm. Invest...13, 201 216. ['[I:RRt RA E. ('. 119661 Action of o u a b a m ill the toad urinar,, bladder. Am. J. l)hw'iol. 210. 9gO 9g6. IhRE~i.R,X I-'. C. (19681 Action of ouabain on bioelectric properties and ion content in toad urinary bladder. Am. ,I. Phvsi,l. 215, 183-189. th:Rm~Ra E. C. 11975)The role of tile active and passive sodium pathways~n the mechanism of action of ethacrvnic acid. (h'n. Pharmac 6 ")01-'~07 LWIlII:>gSTH>/ N. S. & LI ,'d: .,~. 119651 Effcct of amphotericm B on the permcabilit) of tile toad bladder. J. elm. hlrest. 4,1, 1328 1342. M I : N I ) o Z , ~ S. A.. II,'~Xl)l.liR J. S. & (.)RLOFF J. [1967) Effect of amphotericin B o n permeability and short-circuit c u r rent in toad bladder. 4m. ,I. Physiol. 213. 1263-1268. ()KItA (i. T., K.,~m~Ra, J. J.. RI('tI.,~RDSON E. & LERoY G. V. 119571 Assaying c o m p o u n d s containing H "~ and (,t4. Nucleonics 15, I I I 115. PIi'I'I-RSt!N M. J. & EI)I!I MAX: 1..~. (19641 Calcium inhibition of the action of',asopressin on the urinary bladder of the toad. ,I. elm. Inrest. 43, 583 594. Pt s('m!rT J. B. & GOLDm!R(i M. 119681 The acute effects of furosemide on acid and electrolyte excretion in man. J. Luh. elm. 3,h.,d. 71, 666 677. SI3.DP< 1). W.. EK,',OYAN G.. St:KI W. N. & RL('mR. JR. E. C. ( 1 9 6 6 ) Localization of diuretic action flom the p a t t e r n of water and electro h i e excretion..,hm. N. Y /h'ad. Sci. 13% 328 343. ,"kIK.ai. R. R. & ROHLt. I". J. 11969) Biometrv. W. II. Freenaan. San Francisco. ('A. StN:t ~ "F. H.. S m t p W. D. & WIN S. F'. 11965)The effect or ethacr)nic acid ill nephrogenic diabetes msipidus. (Ira. Rev 13, 314.