J. exp. Biol. 105, 83-94 (1983) 8 3Wanted in Great Britain © The Company of Biologists Limited 1983

PARDAXIN PRODUCES SODIUM INFLUX IN THETELEOST GILL-LIKE OPERCULAR EPITHELIA

BY NAFTALI PRIMOR*

Department of Physiology and Biophysics, New York University MedicalCenter, 550 First Avenue, New York, New York 10016, U.SA. and

Osborn Laboratories of Marine Sciences, New York Aquarium, New YorkZoological Society, Brooklyn, New York 11224, U.SA.

(Received 16 August 1982— Accepted 31 January 1983)

SUMMARY

1. Transport by the gill-like opercular epithelium of the teleost, Fun-dulus heteroclitus, was affected by pardaxin, a protein that is toxic to fish,isolated from the Red Sea flatfish Pardachirus marmoratus.

2. Administration of pardaxin to the mucosal (seawater) side of theisolated short-circuited opercular epithelium, caused a transient stimulationof the active transport of ions (Isc), followed by an inhibition. The Iscstimulation was abolished by ouabain or/and in Na+-free Ringer but not inCl~-or HCO3~-Ringer. When applied to the serosal (blood) side, pardaxindid not affect the Isc.

3. Pardaxin produced a net transient Na+ current from the mucosal sideto the serosal side of 2-2^equivcm~2h~'. It is concluded that this Na+

influx caused the Isc stimulation. The influx is suggested to be the mechan-ism of pardaxin's toxicity in fish.

INTRODUCTION

The Red Sea flatfish Pardachirus marmoratus exudes a fluid from more than 200glands located along its dorsal and anal fins (Clark & Chao, 1973) which is toxic to fish(Clark & George, 1979). The toxic component is a protein named pardaxin (Primor,Parness & Zlotkin, 1978). It is constructed of a monomeric chain, with a helicalstructure and four disulphide bridges (Primor & Tu, 1980).

Pardaxin probably acts on the gills in both teleosts and elasmobranchs and itstoxicity is elevated in fish preadapted to a medium of high salinity. Furthermore, itstoxicity decreases significantly when injected into the fish (Primor, Sabnay, Lavie &Zlotkin, 1980a; Primor, Zadunaisky, Murdaugh & Forrest, 19806).

The mode of action of pardaxin is examined here by studying its effect upontransport by the opercular epithelium of the killifish Fundulus heteroclitus, whoseion-osmoregulatory properties closely resemble those of teleost gills (Degnan, Kar-naky & Zadunaisky, 1977).

•Present address: Osborn Laboratories of Marine Sciences, New York Aquarium, New York ZoologicalSociety, Brooklyn, New York 11224 U.S.A.

words: Pardaxin, fish toxin, ion-osmoregulation, skin epithelium.

84 N. PRIMOR

MATERIALS AND METHODS

Pardachirus marmoratus fish (Pisces; Soleidae) were collected from The Red Sea(Eilat, Israel). P. marmoratus secretion was extracted by the method described byClark & Chao, (1973). Pardaxin (PX), the ichthyotoxic component isolated from thesecretion was obtained according to Primor et al. (1978). The molecular weight wasestimated by SDS gel electrophoresis and found to be correspondent to Mr = 13 100(N. Primor, in preparation).

Killifish, Fundulus heteroclitus, of both genders were collected from the estuariesnear the Osborn Laboratories of Marine Sciences, New York Aquarium, ConeyIsland, Brooklyn, New York. They were adapted to artificial sea water (Utility Chem-ical Co., Paterson, New Jersey) as described by Karnaky, Kinter, Kinter & Stirling(1976). The experiments were performed at room temperature (22-24°C) for a year.

Tissue preparation

The opercular epithelium was prepared and mounted in a Lucite chamber aspreviously described (Degnan e* al. 1977; Degnan & Zadunaisky, 1980a). Briefly, theopercular epithelium tissue was placed between two disks of polymerized Sylgard(Dow Corning, Midland, Michigan) with centrally located circular apertures. Twoepithelia from the same fish were mounted in matching chambers within 10-20 minof removing the fish from the seawater tank.

Solutions

The Ringer contained (in mM): NaCl, 135-0; KC1, 2-5; MgCl2, 1-0; CaCl2, 1-5;NaHCO3, 16-0; glucose, 5-0 and was gassed with 9 5 % 0 2 / 5 % C 0 2 (pH7-15). Inthe ion substitution experiments, equivalent substitution was made of sodium bycholine, potassium by sodium, and chloride by methylsulphate (Sigma Chemical Co.,St. Louis, Missouri). In HCC*3~-free Ringer experiments the chambers were gassedwith air. The sea water used to determine pardaxin's toxicity toF. heteroclitus consis-ted of (in mM): NaCl, 480; KC1, 10; CaCl2, 20; MgCl2, 24; and MgSO+F 28. Thelethal dose (LD50) was calculated by the method of Reed & Muench (1938).

Electrical and unidirectional fluxes: measurements and calculations

The procedures for measuring the transepithelial potential difference (p.d.), apply-ing the short-circuit current (Isc), and calculating the transepithelial d.c. resistance(R) or conductance (Gt), have been described previously (Degnan et al. 1977).^Na*, 36C1~ and 3H-inulin were obtained from New England Nuclear, Boston,Massachusetts. Concentrated isotope stock solutions were prepared in Ringer and10-50 pb aliquots, containing 5/iCi of ^Na* or ^Cl" or 20/iCi of 3H-inulin, wereadded to one side in the matching chambers and allowed to equilibrate for 60 min.Inulin fluxes were measured with Ringer containing 1 mM-inulin (Fisher Scientific,Fair Lawn, N.J.). Samples from the 'cold' side were taken at 10 or 20min intervalsand the activity determined by liquid scintillation. The 'cold' side volume was keptconstant by replacing the sample removed for counting with an equal volume ofRinger, or Ringer containing drug.

Pardaxin effect 85The serosal to mucosal flux (efflux, J«n) was measured in one preparation while the

mucosal to serosal flux (influx, Jm.) was measured in the paired preparation. The netCl~ flux was calculated as the average efflux minus the average influx.

The Na+ fluxes across the short-circuited opercular epithelium have been shownto be passive and traversing only one rate-limiting barrier (Degnan & Zadunaisky,1980a). This type of behaviour is attributed to movement through the paracellularshunt pathway (Bruus, Kristensen & Larsen, 1976).

The Na+ permeability is assumed to be independent of the voltage across themembrane. The relationship between the Na+ unidirectional flux (JN») and the per-meability (PNt) and tissue conductance (Gt) was applied as previously described byDegnan & Zadunaisky (19806) and summarized in Table 3.

RESULTS

The effect ofpardaxin on the electrical properties of the short-circuited killifish oper-cular epithelium

The isolated opercular epithelia of seawater-adapted fish, when bathed on bothsides with Ringer, displayed a mean short-circuit current (Isc) of 146-1 /iAcm"2, a

150 -

100-

50-

PX(9xlO-JmM

M

0 5 10 15

Time (min)

Fig. 1. The effect of pardaxin (PX) administered to the mucosal side (M) and to the serosal side (S)on the electrical properties of the isolated short-circuited opercular epithelium from seawater-adaptedF. heteroclitus in Cl~ and Na+-rich Ringer. PX (9 X 10rnM) was given on the serosal side. After5 min it was administered on the mucosal side. Isoprenaline (5 X 10"4 inn) was added to the serosalside. ( ), Tracing of continuous short-circuit current (Isc); (O O), transepithelial poten-tial difference (p.d.); ( 9 • ) , calculated resistance (R). Note PX first stimulates and secon-darily inhibits the Isc.

00

Tab

le 1

. The

eff

ect o

fpar

daxi

n on

the

sho

rt-c

ircu

it (

he),

tr

anse

pith

elia

l pot

enti

al d

iffe

renc

e (p

.d.)

and

res

ista

nce

(R)

acro

ss th

eis

olat

ed o

perc

ular

epi

thel

ia fr

om t

he s

eaw

ater

-ada

pted

tel

eost

, Fun

dulu

s he

tero

clit

us

Tim

e (m

in)

20%

cha

nge

40%

cha

nge

60%

cha

nge

80%

cha

nge

Con

trol

Isc

p.d.

1461

±18

-6

17-5

±5

-6

1330

+ 2

01

161

±7-

6

122-

5 ±

17-6

15

-2 ±

6-5

101-

6 +

21-4

13

-6±6

-8

R

119-

7

1210

124-

1

133-

8

3 X

10"

3 mM

(N=

10)

Isc

p.d.

112-

1 ±

20

0 12

1 ±

3-6

-23-

2 -3

0-8

110-

6± 1

8-4

14

-9±

51

-16-

8 -7

-4

98-1

±18

-9

13-7

±4-

0-1

9-9

-9-8

99-6

±20

-5

101

±3-

9-1

-9

-25-

7

The

Isc

is g

iven

in u

nits

of

juA

cm"2, t

he p

.d. i

n m

V a

nd th

e re

sist

ance

in Q

cm2.

mea

n ±

S.E.

M. a

nd th

e nu

mbe

r of

pai

red

expe

rim

ents

are

giv

en in

par

enth

esis

.

R

107-

9-9

-8

134-

7-J-

11-3

139-

6+

12-4

101-

4-2

4-2

Isc

22-6

±

9--8

4-5

Pard

axin

6 X

10"

3 mM

(N=

Z0)

p.d.

6 4-

9 ±

1-9

-72

0

18-2

± 7-

8 4-

2± 1

-7-8

6-3

-73-

9

16-5

+ 1

1-8

6-5

14-6

+

9-8

5-6

5 3-

5 ±

10

-76-

9

7 2-

9 ±

0-6

-78-

6

Pard

axin

was

add

ed t

o th

e m

ucos

al

R

216-

8+

81

1

230-

7+9

0-6

212-

1+7

0-9

198-

6+4

8-4

Isc

22-3

+ 4

-1-8

4-7

17-6

±5-9

-86-

7

11-5

+ 3

-1-9

0-6

9-6

+ 3

-2-9

0-5

(sea

wat

er)

side

onl

y. r

9x

10"3m

M(A

T =

10)

p.d.

40

+ 11

-77-

13-

6 ±

0-9

-77-

1

3-1

±0

-7-7

9-6

2-2

±0

-5-8

3-8

R

179-

3+4

9-7

204-

5+6

9-0

269-

5+

1171

229-

1+7

1-2

The

data

are

exp

ress

ed a

s

i—i

JL *\RIMC

Pardaxin effect 87an transepithelial potential difference (p.d.)of 17-5 mV (serosal 9ide positive), and

a mean transepithelial d.c. resistance of 119-7Qcm2 (Table 1).Pardaxin (PX) at 3 X 10"3mM (40/igml"1) 6 X 10 ~3 HIM (80/igmr1) and 9 x

10~3mM (120/igml"1) administered into the mucosal (seawater) side of the opercularepithelium elicited a dual effect on the Isc. At first, the Isc was transiently stimulated,and then, within 20min inhibited (Fig. 1). At 3 X 10~3 mM the Isc recovered within80min, but at higher concentrations the Isc was irreversibly inhibited (Table 1).Following PX action, the epithelium was capable of responding to isoprenaline sti-mulation (5 X 10~4 mM, on the serosal side). This finding suggests that PX does notdestroy the epithelium's response to /S-adrenergic activation of Cl~ secretion (Degnan& Zadunaisky, 1979; Mendelson, Cherksey & Degnan, 1981).

To identify the ion(s) involved in the elevation of the Isc, various ions were sub-stituted by others. In Cl~-free Ringer, PX (9 X 10~3 HIM) administered to the mucosalside elicited a large transient Isc stimulation. This effect was not observed when PXwas added to the serosal side (Fig. 2). In the opercular epithelium, the rate of Cl~secretion has been shown to be dependent upon the HCCh" concentration (Degnanetal. 1977). In HCCV-free Ringer, PX (9 X 10~3mM) on the mucosal side producedIsc stimulation similar to that observed in Cl~-free Ringer. PX on the serosal side hadno effect. In addition, the presence of 10~3M-acetazolamide (carbonic anhydraseinhibitor) did not depress the PX-induced Isc stimulation in HCC>3~-free Ringer(data not shown). In Na+-free Ringer, PX (9 X 10~3

ITIM) on the mucosal side did notstimulate the Isc (data not shown). Removal of K+ resulted in a decrease of Isc from180/iAcm~2 to 30^Acm~2; and addition of PX (9 X 10~3mM) on the mucosal sidethen caused an elevation of Isc (Fig. 3).

J80

40

20

120

-80

-40

aa:

0 10 20

Time (min)

30 40

Fig. 2. The effect of pardaxin (PX) administered to the mucosal side (M) and to the serosal side (S)on the electrical properties of the isolated short-circuited opercular epithelium from seawater-adaptedF. heteroclitus in Cl~-free Ringer. ( ), Tracing of continuous short-circuit current (Isc);(O O), transepithelial potential difference (p.d.); ( 9 • ) , calculated resistance (R). NotePX (9 X 10~3 mM) stimulates the Isc only when given on the mucosal side.

N. PRIMOR

200-

100-

100% N2

\

\PX \(9xlO"3m»iKto mucosal N.

K+-Free ^

Ouabain (10~2

to serosal

\

mM)

PX(9Xl0"3mM)to mucosal

- T AIV40 80

Time (min)

120

Fig. 3. The effects of ouabain, anoxia and removal of K+ on pardaxin (PX)-induced ahort-circuitcurrent (Isc) stimulation in the opercular epithelium from seawater-adapted F. heteroclitus in Cl" andNa+-rich Ringer. Ouabain (10 mM) was added to the aerosal side. PX (9 X 10~3 OIM) waa given onthe mucosal side. Anoxia waa introduced by gassing with 100 % Nj . Note that ouabain prevented thePX-induced Isc stimulation.

Anoxia has been shown to inhibit, by 83 %, the Isc in the operculum (Degnan etal. 1977), indicating that the active transport of ions is dependent on metabolicenergy. The dual effect of PX (Isc stimulation followed by inhibition) waa againobserved when the Ringer was gassed with 100 % N2 instead of 95 % O2/S % CO 2 •

Pretreatment on the serosal side with ouabain (10~2mM) the classical inhibitor ofNa+, K+-ATPase, prevented PX (9 X 10~3 mM) on the mucosal side from stimulatingthe Isc in both the CF-Ringer (Fig. 3) and the Cl~-free Ringer (Fig. 4). The mostsimple interpretation of this experiment is that ouabain abolishes the ion gradientbetween the cells and the bathing medium and prevents PX from inducing a netsodium influx across the epithelium.

The above results indicate that Na+ is directly involved in PX-induced activetransport stimulation but that Cl", HCCb" and K+ are not.

The effect of PX on Cl ~, Na + and inulin fluxes across the short-circuited killifishopercular epithelium

In the short-circuited epithelia of seawater-adapted fish, bathed on both sides withRinger, the mean Cl~ serosal side to mucosal side flux (efflux) was 130-8 fiA cm"2 andthe mean Cl~ mucosal side to serosal side flux (influx) was 28-5 juAcm"2. This resul-ted in a net Cl~ serosal to mucosal flux of 104-9 fiA cm"2. On the other hand, the meanNa+ efflux was 169-6 ̂ A cm"2 and the mean Na+ influx was 192-9/iAcm~2(Table2).Despite the relatively large Na+ fluxes there is no significant net flux (Degnan &Zadunaisky, 1980a).

After administration of PX (9 X 10~3 min) to the mucosal side, the net flux of Cl"was found to be inhibited by 488 % (Table 2). Sodium efflux was inhibited, resulting

Pardaxin effect 89

30-

2 0 - 2

10 1

PX(9xl0-3m«to mucosal

10 5030Time (mm)

Fig. 4. The effect of ouabain on pardaxin (PX)-induced short-circuit current (Isc) stimulation inCl~-free Ringer of the isolated opercular epithelium from seawater-adapted F. heteroclitus. Followingthe addition of ouabain (10"2 HIM) to the serosal side, PX (9 X 10"3 m ) was given to the mucosal aide.Note that ouabain prevented the PX-induced Isc stimulation. ( ), Tracing of continuous Isc;(O O), transepithelial potential difference (p.d.).

in a significant (P = 0-005-0-01) net sodium influx of 58-9/iAcm"2 (Table 2) whichis equal to 2-2^equivcm~2h~1 (Table 3).

Sodium permeability (PN») and conductance (GN») were calculated from JN« valuesas obtained for sodium unidirectional fluxes. The tissue conductance (Gt) was mostlydue to the contribution of the sodium conductance (Table 3). Therefore the effect ofPX on the tissue conductance reflected mostly a change in GN» • The JN« , PNE andGN» for 20 min and 40 min were considerably larger from the mucosal to the serosalside than the opposite direction (Table 3).

Gill permeability was assessed with inulin as has been used for teleosts (Kirschner,1980a; Isaia, 1982). The unidirectional inulin fluxes were found to be 4—5 orders ofmagnitude lower than the sodium fluxes. Within 20 min after PX application, theinulin fluxes reached a maximum of 2-2 X 10~3/iequivcm~2h~1 which is 10—13 timeshigher than the control (0-16X 10~3/iequivcm~2h~1) and then decayed. They werestill 3 3 times higher than the control after 50 min (Table 4).

Toxicity ofPX to the teleost F. heteroclitus adapted to sea water and Ringer medium

For a period of 3 weeks, F. heteroclitus fish were adapted to artificial sea water andto the fish Ringer (Materials and Methods). The lethal dose (LDso) of PX using 40F. heteroclitus (weighing 0-2—0-3 g) adapted to sea water was found to be 30-0 figml"1 g body weight"1, as determined after 20 min. The LD50 in fish adapted to thefish Ringer was much higher at SOO/igml^g"1. Thus the PX toxicity in F.heteroclitus is dependent upon the medium concentration as previously noted forAphanius dispar (Primore/ al. 1980a) and the effects elicited by PX in the operculum

in Ringer solution were produced by a sub-lethal dose.

Tab

le 2

. The

eff

ect o

fpar

daxi

n (P

X)

on u

nidi

rect

iona

l36C

l an

d22N

a+ f

luxe

s an

d th

e sh

ort-

circ

uit c

urre

nt (

Isc)

acr

oss

the

isol

ated

oper

cula

r ep

ithe

lia

from

the

sea

wat

er-a

dapt

ed t

eleo

st, F

undu

lus

hete

rocl

itus

Eff

lux

36

C1-

(N=

10

)In

flux

Net

flu

x/

= 2

0)Is

cE

fflu

xIn

flux

fiA

cm

Mea

n co

ntro

l20

min

aft

er P

X%

cha

nge

(com

pare

d to

con

trol

)P %

cha

nge,

inf

lux

to e

fflu

xP T

he f

luxe

s ar

e gi

ven

in u

nits

of

curr

ent

(Ia,

IN.)

for

com

pari

son

wit

h th

e Is

c. P

X w

as a

dded

to

the

muc

osal

sid

e at

a c

once

ntra

tion

of

9-1

X 1

0~3m

M.

Th

e da

ta a

reex

pres

sed

as m

ean

± S

.E.M

. an

d th

e nu

mbe

r of

pai

red

expe

rim

ents

are

giv

en i

n pa

rent

hesi

s.

130-

8115

-680

-1±

20-

5-3

8-8

00

1-0

02

28-5

± 1

26-4

± 1

-7-3

0-4

0-0

•0 •7 •50

104-

9 ±

16

053

-7 ±

21

-7-4

8-8

0-2

5-0

-05

126-

5 ±

16

-822

-6 ±

9-

6-8

4-5

00

01

169-

6 ±

12

-714

7-1

± 1

1-4

-13

-20

05

-01

0

192-

9 ±

12-

82

06

0 ±

24-

2+

6-8

0-4

0-0

-50

40

00

-00

5-0

-01

o

Pardaxin effect 91

Table 3. The effect of pardaxin (PX) on Na + fluxes (JNa), permeabilities (PNa) andtissue conductances (GJ across short-circuited opercular epithelia from the seawater-

adapted teleost, Fundulus heteroclitus

Control20 min after PX40 min after PX60 min after PX80 min after PX

JN.Efflux(/ttquivcm

6-325-483-573142-44

Influx-2h"')

7-197-68»5-383-602-99

Efflux(io-

15-3513-308-717-66604

PN.Influx

-"cms"1)

17-4518-6813-078-777-29

The results are means calculated from unidirectional a Na + fluxes.•The difference between JN. influx and JN. efflux is a net flux of 2-

Gn.Efflux Influx

(mmho cm"2)

6-645-683-753-302-53

2/iequivcm

7-568075-653-78314

-2h"' (P

G,

7-225023031-831-46

GN./G,

0-911171-231-801-73

= 0-005-0-01).

Table 4. The effect of pardaxin on 3H-inulin fluxes across short-circuited isolatedopercular epithelia from the seawater-adapted teleost, Fundulus heteroclitus

Mean control10 min% ChangeP20 min% ChangeP30 min* ChangeP50 min% ChangeP60 min% ChangeP

The data are expressed as /icquiv of inulin and are mean 1 S.E.M. of five paired experiments. Pardaxin wasadded to the mucosal side only at a concentration of 9 X 10~3 nw.

DISCUSSION

Pardaxin applied to the mucosal side of the opercular epithelium caused a transientstimulation of active ion transport and then an inhibition, as indicated by the Isc (Fig.1). The mechanism of the stimulation was found to be sodium-dependent. Examina-tion of the ^Na* fluxes (Table 2) shows that PX elicited a significant (/) = 0-01-0-005) net Na+ influx of about 60 /iA cm"2. A similar increase in current was producedby pardaxin in the absence of chloride (Fig. 2). Examination of Na+ (Table 3), inulin(Table 4), and Isc stimulation (Fig. 2) reveals a transient action in spite of thecontinuous presence of PX. The finding that ouabain (10~2mM added to the serosal

abolished the PX-induced Isc stimulation (Figs 3, 4) supports the view that the

Efflux

016510-031-36810-53

741-3005-010

2-36 ±0-861315-9

0-05-0-101-64 10-52

895-20-025-0-05

0-55 ±0-26234-5

0-10-0-200-60 10-23

265-8005-0-10

InulinInflux

uivcm~2h-') X 10~3

016410-040-86110-42

425-10-01-0-02

1-90 10-691061-2

0-05-0-101-55 10-41

1001-40-05-0-10

0-54 10-15230-5

0-025-0-050-52 10-17

218-20-05-0-10

92 N. PRIMOR

net movement of Na+ across the epithelium increased the Isc. Degnan & Zadunais^P(1980a) have shown that in opercular epithelium Na+ movements are passive (dif-fusional) and paracellular while PX stimulation is active and ouabain-sensitive.Further, the stimulation of Isc strongly suggests that the induced Na+ influx iscellular rather than paracellular. Therefore, this result may indicate that PX acts onor produces an active cellular Na+ pathway.

In the opercular epithelium the Isc is chiefly produced by net Cl~ secretion (Degnanet al. 1977). PX inhibition of the Isc following its initial stimulation, may thus haveresulted from inhibition of Cl~ secretion as indicated by inhibition of net flux (Table 2).

Inulin is believed to cross the teleost gill by a paracellular route among the chloridecells (Kirschner, 1980a; Isaia, 1982). The increase in inulin permeability induced inthe killifish opercular epithelium by PX (Table 4) may thus indicate an increasedparacellular leak. However, PX might also damage the membranes of the epithelialcells, since it increases permeability of red cells (Primor & Lazarovici, 1981), the viralenvelope (Pal, Bernholz& Wagner, 1981a,6),andlipidbilayers(Moran,Primor&Zlot-kin, 1977; Korchak, 1979) and vesicles (Zlotkin & Bernholz, 1983). An analysis of^Na"1" and 36C1~ fluxes (Table 2) shows, however, that during the first 20 min there is noincrease in the unidirectional permeability. From 40 min to 80 min after adding PX, theNa+ conductance (as calculated from the unidirectional fluxes) dropped gradually(Table 3). This finding is correlated with an increase in transepithelial resistance (Fig.1). While PX administered to the serosal side did not elicit Isc stimulation, it did notinhibit Isc stimulation when PX was later added to the mucosal side (Figs 1,2).

The results indicate that PX did not cause a substantial leak to Na+ or to Cl~ buta transient Na+ influx (Table 3). The increase in inulin permeability (Table 4) isabout three orders of magnitude lower than the value of the net Na+ influx createdby PX (Table 4). Therefore an increase in paracellular or cellular leak throughnecrotic cells is only a small fraction of its activity. The most marked effect is theproduction of a net Na+ influx.

The mode of action of PX in molecular terms remains unknown. However, itshould be noted that it has a surfactant effect (Primor, Zadunaisky & Tu, 1983;Zlotkin & Bernholz, 1983).

The Isc was stimulated by PX only when administration was made on the mucosalside. Similarly, during PX toxicity tests in dogfish, an inhibition of the spiracular rateand an extensive struggle, as well as the production of a transient leak to urea andsodium, were elicited when PX was administered to the medium and were not obser-ved when it was injected into the dorsal artery (Primor et al. 1980a,6).

In teleosts the serosal side of the branchial epithelium is known to be directlyinvolved in active transport of ions (Pic, 1978; Kirschner, 19806; Karnaky et al.1976). However, ion and acid-base modulation are thought to be located on themucosal side of the branchial chloride cells (Girard & Payan, 1980; Perry, Haswell,Randall & Farrell, 1981). In addition, the apical membrane is involved in active Na+

secretion (Bradley, 1981). It is suggested that PX interrupts the process of Na+

extrusion causing its inhibition which finally leads to a net increase in Na+ influx.In a high salinity medium, the Na+ influx can be expected to be larger than in

Ringer solution. PX-enhanced toxicity in a high salinity medium further supports thenotion that a net Na+ influx is the mechanism of PX toxicity in fish.

Pardaxin effect 93This author is grateful to Dr Kevin Degnan for introducing me to the operculum

preparation techniques and valuable discussions. Appreciation is expressed to Dr JoseA. Zadunaisky for his help in performing part of this work in his laboratory. I wouldlike to thank Dr George D. Ruggieri for providing some of the fish for these studiesand to Esta Van Tuyl for excellent typing of the manuscript. Financial support from:The Department of Defense, Office of Naval Research, Research Grants No.N00014-81-K-0667 and N00014-82-C-0435 is acknowledged.

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