Báo cáo khoa học: Action of palytoxin on apical H+/K+-ATPase in rat colon pdf

Action of palytoxin on apical H
+
/K
+
-ATPase in rat colon
Georgios Scheiner-Bobis
1
, Thomas Hu¨ bschle
2
and Martin Diener
2
1
Institute for Biochemistry and Endocrinology,
2
Institute for Veterinary Physiology, Justus-Liebig-University Giessen, Germany
Palytoxin stimulated a cation-dependent short-circuit cur-
rent (Isc) in rat distal and proximal colon in a concen-
tration-dependent fashion when applied to the mucosal
surface of the tissue. The distal colon exhibited a higher
sensitivity to the toxin. The palytoxin-induced Isc was
blocked by vanadate but was resistant to ouabain or
scilliroside, suggesting the conversion of a vanadate-sen-
sitive H
+
/K
+
-ATPase into an electrogenic cation trans-
porter. Cation substitution experiments with basolaterally
depolarized tissues suggested an apparent permeability of
the palytoxin-induced conductance of Na
+

>K
+
>Li
+
.
Immunohistochemical control experiments confirmed the
absence of the Na
+
/K
+
-ATPase in the apical membrane.
Consequently, the pore-forming action of palytoxin is not
restricted to Na
+
/K
+
-ATPase but is also observed with
the colonic H
+
/K
+
-ATPase.
Keywords: ATPase; colon; palytoxin; Isc; ion channel.
P
2C
-type ATPases are oligomeric enzymes consisting of a
and b subunits [1]. The sodium pump from the plasma
membranes of animal cells, a member of this group,
generates a sodium gradient by pumping three Na
+

ions
out of the cell and two K
+
ions into the cell for each ATP
hydrolyzed [2]. This sodium gradient is the driving force of
all secondarily active transporters and a presupposition for
neuronal conduction of signals.
The closest relatives of the sodium pump are the proton
pumps from gastric and colon epithelial cells [3]. Although
these pumps are not identical, they both catalyze an active
secretion of protons driven by ATP hydrolysis. Unlike the
sodium pump, however, both proton pumps are electro-
neutral: each transports one K
+
ion from the luminal side
into the cytosol for each H
+
secreted.
Several naturally occurring toxins have been identified as
specific inhibitors of the sodium pump. Among them, the
so-called cardioactive steroids or cardiac glycosides are not
only known for their ability to selectively target the sodium
pump but are widely used as effective medication for
patients with heart failure or heart insufficiency [4]. Paly-
toxin, a toxin isolated from corals of the family Palythoa
(e.g. Palythoa caribaeorum), is also a highly specific inhibitor
of the sodium pump [5,6]. This most potent toxin (for
rodents the LD
50
is 10–250 ng per kg of body weight) of

animal origin can also be found together with ciguatoxin,
maitotoxin, or gambierol in fishes that contribute to
ciguatera poisonings [7,8]. Palytoxin is a rather unique
and large molecule with the structural formula
C
129
H
223
N
3
O
54
. The molecule can be divided into three
subdomains, each connected by peptide bonds: a large
N-terminal polyhydroxy x-amino acid followed by a
dehydro-b-alanine residue and an aminopropanol group.
The number of free hydroxyl groups is 42 [9,10]. Unlike the
cardioactive steroids, however, which inhibit both ATP
hydrolysis and ion conduction, palytoxin acts by arresting
the ionophore of the pump into a permanently open state.
Thus, in this case, inhibition of ATP hydrolysis is no longer
associated with inhibition of ion conductivity.
Yeast cells, which are usually insensitive to palytoxin,
display a palytoxin-induced K
+
efflux when they hetero-
logously express a and b subunits of the mammalian sodium
pump [11,12]. This flux is sensitive to ouabain (g-strophan-
thine), the most well known inhibitor of the pump. Based on
these and other experiments showing the formation of

palytoxin-induced ion channels in membranes containing
in vitro-translated Na
+
/K
+
-ATPase [13], it is widely
accepted that palytoxin specifically targets the sodium
pump and inhibits its catalytic activity by converting the
ATPase into an ion channel.
Palytoxin action on other P
2C
-type ATPases has not yet
been demonstrated. Thus, in the current investigation, we
describe the action of palytoxin on the H
+
/K
+
-ATPase
from the rat colon and demonstrate that the interaction of
this toxin with the enzyme results in specific currents that
are similar to those observed from its action on sodium
pumps.
EXPERIMENTAL PROCEDURES
Solutions
The Ussing chamber experiments were carried out in a
bathing solution containing according to Parsons &
Paterson [14] (mmolÆL
)1
): NaCl, 107; KCl, 4.5; NaHCO
3

,
25; Na
2
HPO
4
,1.8;NaH
2
PO
4
,0.2;CaCl
2
,1.25;MgSO
4
,1;
andglucose,12.Thesolutionwasgassedwithamixtureof
5% CO
2
and 95% O
2
; the pH was 7.4. For depolarization of
the basolateral membrane, a modified bathing solution was
used in which NaCl was replaced by 111.5 mmolÆL
)1
KCl.
In the LiCl bathing solution, NaCl was replaced equimo-
larly by LiCl.
Correspondence to G. Scheiner-Bobis, Institut fu
¨
r Biochemie und
Endokrinologie, Justus-Liebig-Universita

¨
t Gießen, Frankfurter Str.
100, D-35392 Gießen, Germany.
Fax: + 49 641 99 38189, Tel.: + 49 641 99 38180,
E-mail:
Abbreviations:NMDG,N-methyl-
D
-glucamine; Gt, tissue
conductance; Isc, short-circuit current.
(Received 11 March 2002, revised 14 May 2002,
accepted 18 June 2002)
Eur. J. Biochem. 269, 3905–3911 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03056.x
Tissue preparation
Wistar rats were used with a weight of 180–220 g. The
animals had free access to water and food until the day of
the experiment. Animals were stunned by a blow on the
head and killed by exsanguination (approved by Regi-
erungspra
¨
sidium Giessen, Giessen, Germany). The serosa
and muscularis propria were stripped away by hand to
obtain the mucosa-submucosa preparation of the distal part
of the colon descendens. Two distal and two proximal
segments of the colon of each rat were prepared.
Short-circuit current measurement
The tissue was mounted in a modified Ussing chamber,
bathed with a volume of 3.5 mL (see above) on each side of
the mucosa and short-circuited by a voltage clamp (Ing.
Buero Mussler, Aachen, Germany) with correction for
solution resistance as described previously [15]. The exposed

surface of the tissue was 1 cm
2
. Short-circuit current (Isc)
was continuously recorded and tissue conductance (Gt) was
measured every min. Isc is expressed as lAÆh
)1
Æcm
)2
, i.e. the
flux of a monovalent ion per time and area with
1 lEqÆh
)1
Æcm
)2
¼ 26.9 lAÆcm
)2
. Tissues were left for 1 h
to stabilize the Isc before the effect of drugs was studied. The
baseline electrical parameters were determined as the mean
obtained during 3 min just before administration of a drug.
Immunohistochemical detection of the Na
+
/K
+
-ATPase
in colonic epithelium
Wistar rats (n ¼ 2) were anesthetized with sodium pento-
barbital (60 mgÆkg
)1
body weight; Narcoren, Merial

GmbH, Hallbergmoos, Germany) and transcardially per-
fused with 4% paraformaldehyde in 100 mmolÆL
)1
phos-
phate buffer (pH 7.2). The distal colon was removed and
postfixed in the same fixative for 1 h at room temperature
and then the tissue was cryoprotected in 20% sucrose in
phosphate buffer overnight at 4 °C. Tissue was cut the
following day.
Coronal 10–12 lm colonic sections were cut on a cryostat
(model HM 500, Microm, Walldorf, Germany). To detect
Na
+
/K
+
-ATPase immunoreactivity, a commercial tyra-
mide amplification kit (NEL700, NEN Life Science Prod-
ucts GmbH, Cologne, Germany), based on the catalyzed
reporter deposition method, was used. Tyramide amplifica-
tion staining was performed according to the kit description
in a phosphate buffer system (pH 7.2). In detail, sections
were placed in 10% fetal bovine serum containing 0.3%
Triton X-100 for 1 h at room temperature. Incubation with
the primary anti-(Na
+
/K
+
-ATPase) Ig (MA3-929, mon-
oclonal, mouse, a
1

-subunit, Affinity BioReagents, Golden,
CO, USA) was performed for 24–36 h at 4 °C at a dilution
of 1 : 150 to 1 : 5000). The primary antibody was then
detected with a secondary biotinylated anti-(mouse IgG) Ig
(1 : 200, Vector BA-2001, Linaris Biologische Produkte,
Wertheim-Bettingen, Germany) for 1 h at room tempera-
ture. After amplification, the immunohistochemical
processing was finished with 1 : 200 fluorescein (FITC)-
conjugated avidin D (Vector, Linaris Biologische Produkte,
Wertheim-Bettingen, Germany). In order to demonstrate
the overall morphology of the colonic epithelium, parallel
series of sections adjacent to cryosections of the immuno-
fluorescent-stained series were cut for light-microscopic
analysis and consequently counterstained using cresylviolet.
Finally, these sections were cover slipped with Entellan
(Merck, Darmstadt, Germany) while immunofluorescent
sections were cover slipped with crystal/mount (Biomedia,
FosterCity, USA).
Microscopic analysis
Sections were analyzed using a an Olympus BX50 light/
fluorescent microscope (Olympus Optical Co., Hamburg,
Germany). For light microscopy, digital images were
taken with an Olympus Camedia 3030 camera using the
Olympus
CAMEDIA MASTER
software package (Olympus
Optical Co., Hamburg, Germany). For fluorescent
microscopy, digital images were taken with a Visicam
(PCO Computer Optics, Kehlheim, Germany) using the
METAMORPH/METAFLUOR

software package (Visitron
Systems, Puchheim, Germany). Image editing software
(
ADOBE PHOTOSHOP
) was used to adjust brightness and
contrast and to combine the individual images into the
greyscale mode figure plate.
Drugs
Palytoxin (purchased from L. Be
´
ress, Institute for Toxicol-
ogy, University of Kiel, Germany) was dissolved in
10 mmolÆL
)1
Hepes, 0.5 mmolÆL
)1
Tris, 1 mmolÆL
)1
CaCl
2
and 1 gÆL
)1
BSA. Sodium orthovanadate (Calbiochem, Bad
Soden, Germany) was dissolved in an aqueous stock
solution. Ouabain was dissolved in dimethylsulfoxide (final
concentration 2.5 lLÆmL
)1
), scilliroside (Sandoz, Basel,
Switzerland) was dissolved in methanol (final concentration
2.5 lLÆmL

)1
). If not indicated differently, drugs were from
Sigma, Deisenhofen, Germany.
Statistics
Results are given as means ± SEM. When the means of
several groups were compared, an analysis of variances was
first performed. If the analysis of variances indicated
significant differences between the groups investigated,
further comparison was carried out by a Student’s t-test
(paired or unpaired as appropriate) or by the Mann–
Whitney U-test. An F-test was applied to decide which test
method was to be used.
RESULTS
Basal effects of palytoxin
Palytoxin (10
)8
molÆL
)1
on the mucosal side) induced an
increase in short-circuit current (Isc) in rat distal and
proximal colon (Fig. 1A). The response started immediately
after administration of the toxin and was stable at least for
30 min The effect was concentration-dependent (Fig. 1B).
A first, significant increase in Isc occurred at a concentration
of 10
)10
molÆL
)1
. Similar effects were observed in the distal
and proximal colon, although the potency of palytoxin

appeared to be higher in the distal than in the proximal
colon. The increase in Isc was concomitant with a rise in
tissue conductance (Gt). At the highest concentration of
palytoxin tested (5 · 10
)8
molÆL
)1
), Gt increased by
3906 G. Scheiner-Bobis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
7.1 ± 1.7 msÆcm
)2
in the distal and by 8.9 ± 2.7 msÆcm
)2
in the proximal colon (n ¼ 6–8, p < 0.05 for both colonic
segments).
The effect of palytoxin was enhanced in the presence of
mucosal borate (0.5 mmolÆL
)1
), especially in the proximal
colon (Table 1). Similar observations were made in the past
concerning palytoxin effects on erythrocytes, neurosyna-
ptosomes or yeast cells that express mammalian sodium
pumps [6,11]. Although no real evidence exists about the
role of borate, borate alone does not induce any cation
fluxes from erythrocytes [6] or from yeast expressing the
mammalian sodium pump [11] or from the colon tissues
investigated here. It is possible that borate interacts with
some of the 42 free hydroxyl groups of palytoxin, similarly
to the way it interacts with carbohydrates. It also might be
that it interacts with the carbohydrates of the strongly

glycosylated b subunits of the P
2C
-type ATPases. These
possible complexes might induce a particular conformation
of the palytoxin molecule or of the enzyme that favors
mutual interaction between the two reactants. Therefore, all
subsequent experiments were carried out with borate in the
mucosal solution using a palytoxin concentration of
10
)8
molÆL
)1
.
For theoretical reasons it is not possible that admin-
istration of palytoxin to the basolateral side of an
epithelium can induce an Isc. If the toxin converts the
Na
+
/K
+
-pump into a cation channel, the cytosolic Na
+
concentration will increase and finally reach the extracel-
lular concentration, whereas the cytosolic K
+
concentra-
tion will fall to the level at the extracellular side. Thus
there is no more driving force for any active ion
movement, i.e. there will be no short-circuit current
response. Therefore, as expected, when applied at the

serosal side, in six independent experiments the toxin had
no effect on Isc (data not shown).
Sensitivity against inhibitors of ATPases
The effect of palytoxin (10
)8
molÆL
)1
at the mucosal side)
was resistant to mucosal ouabain (10
)3
molÆL
)1
) or scill-
iroside (10
)4
molÆL
)1
at the mucosal side) (Table 1), a
potent blocker of the Na
+
/K
+
-pumpinrattissue[16].All
pump inhibitors were administered 1 h prior to palytoxin;
for effects of the blockers on baseline Isc, see Table 2. In
contrast, pretreatment with sodium orthovanadate
(10
)4
molÆL
)1

at the mucosal side) nearly suppressed the
action of palytoxin (Table 1).
Table 1. Effect of palytoxin on Isc under different conditions. The increase in Isc evoked by palytoxin (10
)8
molÆL
)1
at the mucosal side) was
measured in the absence of any drugs or in the presence of Tris borate (10
)4
molÆL
)1
at the mucosal side; pretreated for 15 min), ouabain
(10
)3
molÆL
)1
at the mucosal side; pretreated for 1 h), scilliroside (10
)4
molÆL
)1
at the mucosal side; pretreated for 1 h), or vanadate (10
)4
molÆL
)1
at the mucosal side; pretreated for 1 h), or after replacement of NaCl by NMDG chloride (107 mmolÆL
)1
NMDG chloride buffer at the mucosal
side). *p < 0.05 vs. baseline, p < 0.05 vs. response to palytoxin in the absence of any drugs.
Distal colon
D Isc (lEqÆh

)1
Æcm
)2
)
Proximal colon
D Isc (lEqÆh
)1
Æcm
)2
) n
Palytoxin 2.7 ± 0.6* 0.3 ± 0.2 5–8
Palytoxin + borate 4.2 ± 0.7* 1.9 ± 0.4*  5–9
Palytoxin 2.8 ± 0.8* 2.0 ± 0.9 5–9
Palytoxin + ouabain 2.3 ± 0.7* 2.2 ± 0.6* 6–9
Palytoxin + vanadate 0.4 ± 0.1* 0.3 ± 0.1 6
Palytoxin + scilliroside 2.1 ± 0.6* 1.1 ± 0.5 6–8
Palytoxin, Na ± free 0.1 ± 0.1 0.1 ± 0.1 6
Basolateral depolarization 0.5 ± 0.1* 0.1 ± 0.1 5–6
(apical Na
+
)
Basolateral depolarization )1.7 ± 0.5* )0.9 ± 0.2* 6–8
(apical Li
+
)
Fig. 1. Induction of a short-circuit current in rat distal and proximal
colon by palytoxin. (A) Typical Isc response evoked by palytoxin
(10
)8
molÆL

)1
at the mucosal side in the presence of 0.5 mmolÆL
)1
Na
borate at the mucosal side). (B) Concentration-dependent increase in
Iscabovebaseline(D Isc) evoked by palytoxin in the distal (closed
circles) and proximal (open rectangles) rat colon. Palytoxin was
administered cumulatively at the mucosal side in the presence of
0.5 mmolÆL
)1
Na borate. Values are means ± SEM, n ¼ 6–8.
Ó FEBS 2002 Palytoxin action on colonic H
+
/K
+
-ATPase (Eur. J. Biochem. 269) 3907
Ionic selectivity of the palytoxin-induced pore
Assuming that palytoxin might induce cation-permeable
pores in the apical membrane, the Isc evoked by the toxin
should consist of an influx of Na
+
, the prevalent cation in
the mucosal solution, into the cell with the consequence of
the stimulation of a pump current generated by the
basolateral Na
+
/K
+
-ATPase [17]. In order to test this
hypothesis, NaCl was replaced by NMDG chloride in the

buffer solution. Under these conditions, palytoxin
(10
)8
molÆL
)1
at the mucosal side) no longer had any
significant effect on Isc (Table 1).
In order to elucidate the cationic selectivity of the
palytoxin-induced pore, a protocol was used in which the
basolateral membrane was electrically eliminated by a
basolateral depolarization. The basolateral membrane was
depolarized by a high K
+
solution (111.5 mmolÆL
)1
KCl at
the serosal side). Due to the high basolateral K
+
perme-
ability, the electrical properties of the tissue, which are
normally characterized by two batteries in series, are then
expected to be dominated by the apical membrane [18].
Consequently, the current evoked by palytoxin in the
presence of different monovalent cations should not be
affected by the ionic selectivity of the basolateral Na
+
/K
+
-
ATPase.

Depolarization of the basolateral membrane induces a
negative current across the tissue (Fig. 2A) due to
diffusion of K
+
across the apical membrane into the
mucosal compartment, which is driven by the applied K
+
gradient as reported previously [17]. When palytoxin
(10
)8
molÆL
)1
at the mucosal side) was administered in the
presence of mucosal Na
+
(107 mmolÆL
)1
), the toxin
induced a prompt increase in Isc, especially in the distal
colon (Fig. 2A, Table 1). This suggests that the pore
induced by palytoxin has a permeability for Na
+
that is
higher compared with that for K
+
, leading to a flux of
Na
+
from the mucosal to the serosal compartment and
an increase in Isc.

An opposite effect was observed when LiCl was
present in the mucosal solution. Under these conditions,
palytoxin (10
)8
molÆL
)1
at the mucosal side) stimulated a
negative current (Fig. 2B), suggesting that the pores
formed by palytoxin in the apical membrane have a
permeability for K
+
that is higher than that for Li
+
,
thereby mediating a flux of K
+
from the serosal into the
mucosal compartment driven by the chemical gradient.
Similar experiments were performed with CsCl in the
mucosal compartment; however, this solution proved to
be toxic for the tissue as indicated by a massive increase
in Gt. Nevertheless, this series of experiments suggests a
permeability sequence of the palytoxin-formed pore of
Na
+
>K
+
>Li
+
.

Morphological control experiments
ANa
+
/K
+
-stimulated ATPase activity has been found in
vesicles isolated from the apical membrane of rat distal
colon [19]. Therefore, using an immunohistochemical tech-
nique we investigated whether we could find evidence for a
Na
+
/K
+
-ATPase in the apical membrane using a mono-
clonal antibody against the murine a
1
-subunit of this pump.
The overall morphology of rat distal colonic crypts was
demonstrated in cresylviolet counterstained sections
(Fig. 3A). In adjacent immunhistochemically processed
sections immunoreactivity of the a
1
-subunit of the
Table 2. Effect of inhibitors/activators on baseline Isc. Concentration of drugs were: Tris borate (10
)4
molÆL
)1
at the mucosal side), ouabain
(10
)3

molÆL
)1
at the mucosal side), vanadate (10
)4
molÆL
)1
at the mucosal side). *p < 0.05 vs. baseline.
Distal colon
D Isc (lEqÆh
)1
Æcm
)2
)
Proximal colon
D Isc (lEqÆh
)1
Æcm
)2
) n
Borate 0.4 ± 0.2 0.7 ± 0.7 5–9
Ouabain )0.2 ± 0.3 )0.6 ± 0.4 6–9
Vanadate )0.6 ± 0.4 )1.0 ± 0.2* 6
Fig. 2. Action of palytoxin (10
)8
molÆL
)1
at the mucosal side) under
conditions in which the basolateral membrane was depolarized by a high
concentration of K
+

(111.5 mmolÆL
)1
KCl solution at the serosal side;
black bar) either in the presence of mucosal Na
+
(107 mmolÆL
)1
;A)or
mucosal Li
+
(107 mmolÆL
)1
;B).The two schematic drawings sum-
marize the experimental conditions. The line tracings are typical for
5–8 experiments with similar results; for statistical evaluation, see
Table 1.
3908 G. Scheiner-Bobis et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Na
+
/K
+
-ATPase was restricted to the basolateral mem-
brane of rat colonic epithelial cells, as shown for the sagital
(Fig. 3C) and coronal plane (Fig. 3D). The specificity of this
immunohistochemical signal was further analyzed in con-
trol experiments. Omission of the primary antibody led to a
dramatically reduced immunoreactivity in particular at the
basolateral membrane of the colonic epithelial cells (com-
pare Fig. 3B with Fig. 3C).
DISCUSSION

Palytoxin is produced from corals of the genus Palythoa.It
is the strongest toxin produced by animals, with an LD
50
for
rodents of 10–250 ngÆkg
)1
body weight. The toxin is
without any effect on bacterial or native yeast cells. In
erythrocytes and other animal cells, however, it induces an
efflux of K
+
ions from the cytosol and a series of secondary
effects that are most likely associated with the cell depolar-
ization induced by the K
+
loss.
Using yeast as a heterologous expression system, the
sodium pump was shown to be the target of palytoxin
[11,12], which, as verified in experiments involving in vitro
translation and integration of the expressed proteins in
membranes [13], is converted by the toxin into an ion
channel [20]. This channel, very much like natural ion
channels, allows ions to flow through it following their
electrochemical gradients. Apparently, nature has devel-
oped here a highly effective toxic principle; the conversion of
a pump into a channel, most likely by arresting the natural
ionophore of the pump into a permanently open state.
Palytoxin acts by binding to extracellular sites [6,21] of
the sodium pump. Considering the high degree of homology
between the various P

2C
-type ATPases, which approaches
65% [3], we were interested in investigating the interactions
of the toxin with the H
+
/K
+
-ATPase from rat colon. The
experiments were carried out in an Ussing chamber, which,
compared to the single-cell paradigm, has the advantage of
allowing one to asses at any time of the measurement either
the apical or the basolateral surfaces of the epithelial cell
membrane.
Administration of palytoxin on the apical membrane
results in the generation of a current (Fig. 1A), which is
dependent on the presence of Na
+
ions (Table 1). The most
plausible explanation for this observation is that palytoxin
induces the formation of cation channels in the apical
membrane mediating the influx of Na
+
,which,when
extruded by the electrogenic basolateral Na
+
/K
+
-ATPase,
leads to the generation of a transepithelial current. The
Fig. 3. Immunohistochemical detection of the a

1
-subunit of the Na
+
/K
+
-ATPase in rat colonic crypts. Photomicrographs of rat colonic crypts are
shown in the sagital (A–C) and coronal (D) plane. The morphology of sagital colonic crypts can be detected from a cresylviolet counterstained
cryosection (10–12 lm) as shown in A. The immunofluorescent detection of the primary antibody MA3-929 (Affinity BioReagents, dilution
1 : 500) is shown in (C) and (D). Note the intense immunoreactivity at the basolateral membrane of the proliferating cryptic cells [see white
arrowheads in (C) and inset in (B,D)] as compared to the basal immunohistochemical signal detected in an adjacent control cryosection (B) in which
the primary antibody was omitted [see white arrowheads in (B)]. The dotted frame area in (D) shows a coronal view of a single colonic crypt, which
is also shown at higher magnification in the inset (B,D). Scale bar (A–D), 50 lm. Scale bar inset (B,D), 20 lm.
Ó FEBS 2002 Palytoxin action on colonic H
+
/K
+
-ATPase (Eur. J. Biochem. 269) 3909
pores induced by palytoxin have an apparent permeability
of Na
+
>K
+
>Li
+
as suggested by cation experiments in
which the basolateral membrane was by-passed by a
basolateral depolarization (Fig. 2).
The inhibition of palytoxin-induced currents by vanadate,
a known blocker of apical H
+

/K
+
-ATPase [22], suggests
that this enzyme is involved inthe formation of the palytoxin-
induced channels. The enzymatic activity [19], the amount of
mRNA, and the protein expression of the H
+
/K
+
-ATPase
are higher in the distal compared to the proximal colon [23].
Therefore, it seems reasonable to assume that this segmental
heterogeneity might be responsible for the higher sensitivity
of the distal colon to palytoxin when compared with the
proximal part of this organ (Fig. 1B).
In the experiments described here, ouabain did not have
any effect on the palytoxin-induced current across the apical
membrane of colon epithelial cells. Ouabain inhibition of
the colonic H
+
/K
+
-ATPase has been a matter of contro-
versy in many investigations. Early experiments involving
ATPase measurements on apical membrane preparations
appeared to indicate the presence of at least two types of
H
+
/K
+

-ATPase, denoted ouabain-sensitive and ouabain-
resistant. These results were supported by expression
cloning experiments showing a ouabain-sensitive form when
the a subunit of colonic H
+
/K
+
-ATPase was expressed
together in Xenopus oocytes with the bsubunit of the H
+
/
K
+
-ATPase from toad bladder or with either the b subunit
of gastric H
+
/K
+
-ATPase or the b subunit of Na
+
/K
+
-
ATPase in HEK293 cells [24,25]. In Sf9 cells, however,
expression of H
+
/K
+
-ATPase a subunit without a corre-
sponding b subunit produces a ouabain-insensitive H

+
/K
+
-
ATPase [26]. Finally, coexpression of cDNAs encoding the
H
+
/K
+
-ATPase a and b subunits also results in ouabain-
resistant enzymes [22]. These, however, remain highly
sensitive to orthovanadate [22]. Our measurements indi-
rectly support these latter findings, as the palytoxin-induced
conductance was insensitive to 1 m
M
ouabain while being
inhibited at low concentrations of orthovanadate.
Similar findings showing a palytoxin-induced K
+
efflux
that is resistant to ouabain have also been demonstrated
using rat erythrocytes, which contain Na
+
/K
+
-ATPase but
not H
+
/K
+

-ATPase [21]. Thus, in order to exclude a
possible involvement of the sodium pump in the currents
observed here, the localization of the sodium pump a
subunit was investigated by an immunohistochemical
method applied to sagital and coronal segments of the rat
distal colonic crypts. As shown in Fig. 3, the presence of
Na
+
/K
+
-ATPase a
1
subunit in the apical membranes can
be excluded, allowing us to conclude that the previously
measured Na
+
-andK
+
-stimulated ATPase activity found
in the apical membranes of the colon is not due to Na
+
/
K
+
-ATPase, but could be associated with some form(s) of
the colonic H
+
/K
+
-ATPase, as suggested previously

[27,28].
The overall conclusion of the investigation presented here
is that palytoxin targets not only the sodium pump but also
the colonic H
+
/K
+
-ATPase. Apparently, as with the Na
+
/
K
+
-ATPase, the toxin converts the H
+
/K
+
-ATPase into
an ion channel that allows cations to pass down their
electrochemical gradients. This ion channel may be the
ionophore of the pump, which under physiological condi-
tions is only accessible from one side of the plasma
membrane and becomes arrested in a permanently open
state upon interaction with the toxin. This conclusion is
indirectly supported by the fact that monovalent cations can
penetrate the channel, whereas large or divalent cations fail
to be conducted.
Taking into consideration the relatively high homology of
the Na
+
/K

+
-ATPase and colonic H
+
/K
+
-ATPase with
the gastric H
+
/K
+
-ATPase, one might expect to obtain
similar palytoxin effects with the latter enzyme. Although
this has yet to be investigated, the fact that other P
2
-type
cation pumps such as the yeast Na
+
-ATPase or Ca
2+
-
ATPase are not sensitive to palytoxin suggests that P
2C
-type
ATPases are exclusive targets of the toxin. Furthermore,
because the b subunit has been shown to influence the
enzyme kinetic properties of these cation pumps, this
subunit might be required for and closely associated with
the formation of the palytoxin-induced cation channel.
ACKNOWLEDGEMENTS
We wish to acknowledge the diligent care of B. Bru

¨
ck, A. Metternich,
B. Schmidt and E. Haas. G. S. B. was supported through the Deutsche
Forschungsgemeinschaft grant Sche 307/5-1 and M. D. through the
Deutsche Forschungsgemeinschaft grant Di 388/3-4.
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Ó FEBS 2002 Palytoxin action on colonic H
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-ATPase (Eur. J. Biochem. 269) 3911