Stimulation of sodium transport in frog skin by 2-imidazolines (guanidinbenzimidazole and phentolamine)

Life Sciences Vol . 15, pp " 539-542 Printed in the U.S .A .

Pergamon Preee

STIMUIATION OF SODIUM TRANSP(ä2T IN FROß SSIN BY 2-IMIDAZOLINES ((iUANIDIIiHENZIlIIDA7ALS AND PAFaTPOLAI~t~) . Fsderico OarciaRamsu laboratoire de Phyaialogie G1lulaire,Facultd dsa Scisacea Parc Valroee,06 Nice . (Received is final form 27 Juna 1974) The stimulating effscta of 2-guanidinbenaimidazols and pheatolamine on sodium transport through the isolated akin of t)u frog Rang eeculeata are described. Theas eubatancee only act whoa added to the ep t~helial eide,snggesting that they affect the psrdsability of the external barrier of sodium co~spartmsnt . The role of the ieidasols group is the activation of sodium trsneport is discussed. Heatlsy (1) has

shown that the guanidine moiety of amilorids is essential far

the inhibitory effect of amiloride on sodium transport in the toad bladder but that guanidine itself ie 1,000 times lees active than emiloride . The

rsLtionahipa between the structural compoaenta of amiloride and their

effects on transepithslial sodium tranepoz~t were studied on the iaolatad

akin

of Rang eeculenta and thin paper is a preliminary exposition of our results. Methods Ths ventral

abdominal akin of

double-pithed iroga was rsaoved and ®ousted

between two lucits chambers, the ezpoesd akin eurfaoe being 7.5

cm.

The

caer

position of the Ringer need was as follows (in mM) :RC1, 2.5 ; NaCl, 83 ; NaHC03, 24 ;Na~04, 4.0; KH2P04, 1.3 ; Mg$04, bubbled

with

4;

CaC12, 4; glucose, 11 .This solution wan

a mixture of C02 (9%) and 02

directional sodium fluzsa were

(997é)

and its pH was 7.4 .The uni -

measured in open circuit conditions using

(0 .4m0i/1) .Sodium net fluxes were measured by short-circuit current (SCC) (2) . Reeulta and Diacusaion The

addition

of guanidine

to 1 x 10~M to

the external

chamber had

no

measurable effect on the SCC whereas Hsntley(1) found a ~ inhibition in toad bladder

preparations . It aseabd possible that this difference in reapona! re

Elects hietological differences of the two tissues, the stratum aorneum of the 539

540

Stimulation of Na Transport by 2-Imidazolines

akin acting

sa a

Vol . 15, No . 3

barrier to the penetration of guaaidias .For this reason two

guanidine compounds more lipophylic than guanidine were chosen for study . The

first compound

tented wan

2-guanidinbenzimidazole (GBI), which, when

added to the external chamber immediately stimulated the SCC .Table I summarizes the results. Evsa at 10~ there was noes effect and at 103M the 3CC wan more than twice that of the control . TABLE I Effects

of Düfersnt Doaee of GBI oa the Short-Circuit Current of the

In Vitro

Rang esoulenta

Skin in Ringer Solution . Ratios are that

of

GBI-Treated Period to Control Period + Oms Standard Error. DOSE

n

RATIO

M

5

1.05 + 0 .02

lô

M

6

1.36 + 0.08

low*

M

11

1.84 + 0 .06

lÔ3 M

4

2.41 + 0 .14

low 5

Table II recorda the stimulating effect of GBI on the sodium influxes measured with ~Na in open circuit. TABLE II Effect of Different Doses of GBI Solution in the In Vitro asq x h1 x

cm2

lô

lo~

Rang ssculenta

Skin . Fluxsa

Expireseed in

± One Standard Error. n

CONTROL

GBI

DIFF . §

M

8

779 _+ 158

1004 _+ 219

225 _+ 96§§

M

5

810 + 229

1319 ~ 259

509 + 93§§§

DOSE 5

on Sodium Influxes from a Ringer~e

§ : Mean of difference of paired data ± oas Standard Error . §§ : P< 0.05

ldhsa GBI was added

to the internal

chamber (doses of 10~M to 10~M)

the

3CC remained unaffected . The second guanidinic compound studied

was

N,N-diphenylguamidine. Although

more lipophylic than GBI its effects on the SCC were negligible . It would therefore

that the

guanidine

group is

not responsible

for the

seem

activating

541

Stimulation of Na Transport by 2-Imidâzolinea

Vol . 15, No . 3

action of GBLTo test whether the imidazols group is the activating substance, the following

oospounds, all containing an imidazole group, were tented on the

SCCs Phentolamiaa (Regions), beazimidazols and 2-(Hydroxybenzyl)-bsnzisidazole (HBBI) .

c>

2~3uanidinbenzimidazole

~N ~

~N~OH

Bsnzimidazole Phentolamins

OH

Phentolemins

2-(Hydroxybenzil)-bsnzimidazols

was found to have

as even greater stimulating effect on the

9CC than GBI,the SCC ratio between experimental sad control periods, at a dose of 2.6 z lÔ

5

H, being 1.48 ± 0.04 (a = 10) for GBI and 1 .89 + 0.08 (n a 5 .)for

phentolamine . At the aa~e dose benzisidazole

1.08 ± 0.01

HHBI

gave a ratio

of 1.10 + 0 .01 (n = 7)

and

(n = 5) . Ths stimulation caused by these substances

was, as far GBI, very rapid and sustained . Of the substances tested the moat active were phentolamins and GBI,ths common feature of

which is an imidazole

that the stimulating

group substituted is position 2, suggesting

effect observed ie dun to the imidazole group . If at the

experiments the trivalent nitrogen bound to the benzene groups of

pH of these

the phentolamine molecule is in part protonated the molecule would carry a posinus oharge same distance froo the imidazole group. This wou]dfurniah a semblance

to

the

GHI

re-

molecule with its positive charge due to the guanidine

moiety .Thia positive region of the molecule may augment the etisulating action of the imidazole port site .

group by forming an attaçhemsnt on ar near the sodium

trans-

Ths fact that both GBI and phentolaeine show effects only when ad-

ded

to

the external medium suggests that they act on the external barrier of

the

sodiur transport campartesat . Experiments are being ends to determine the

Stimulation of Na Transport by 2-imidazolines

542

relationships

between

Vol. 15, No . 3

the sites of action of GBI and phentolamine on the one

hand and the amiloride oa the other. I wish to thank the Ciba Laboratories for providing phentolamine (Regitiae Ciba) . 1 . P.J .BENThEY, J .Physio1 .1~9~ 317-330 (1968) . 2. H.A .IISSIIQG and K .ZFRAHN, Acta Phyaiol.8cand .~ 110-127 (1951) .