Action of palytoxin on apical H+/K+-ATPase in rat colonGeorgios Scheiner-Bobis1, Thomas Hu¨bschle2and Martin Diener2 1 Institute for Biochemistry and Endocrinology, 2 Institute for Veter
Trang 1Action of palytoxin on apical H+/K+-ATPase in rat colon
Georgios Scheiner-Bobis1, Thomas Hu¨bschle2and Martin Diener2
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
P2C-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 LD50 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
C129H223N3O54 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 P2C-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
E X P E R I M E N T A L P R O C E D U R E S 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; NaHCO3, 25; Na2HPO4, 1.8; NaH2PO4, 0.2; CaCl2, 1.25; MgSO4, 1; and glucose, 12 The solution was gassed with a mixture of 5% CO2and 95% O2; 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)1KCl
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: Georgios.Scheiner-Bobis@vetmed.uni-giessen.de
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)
Trang 2Tissue 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 cm2 Short-circuit current (Isc)
was continuously recorded and tissue conductance (Gt) was
measured every min Isc is expressed as lAÆh)1Æcm)2, i.e the
flux of a monovalent ion per time and area with
1 lEqÆh)1Æcm)2¼ 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 (n¼ 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 4C 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 descripamplifica-tion
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, a1-subunit, Affinity BioReagents, Golden,
CO, USA) was performed for 24–36 h at 4C 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)1Hepes, 0.5 mmolÆL)1Tris, 1 mmolÆL)1CaCl2
and 1 gÆL)1BSA 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
R E S U L T S Basal effects of palytoxin Palytoxin (10)8molÆL)1on 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)10molÆL)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)8molÆL)1), Gt increased by
Trang 37.1 ± 1.7 msÆcm)2in the distal and by 8.9 ± 2.7 msÆcm)2
in the proximal colon (n¼ 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 P2C-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)8molÆL)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)8molÆL)1at the mucosal side) was resistant to mucosal ouabain (10)3molÆL)1) or scill-iroside (10)4molÆL)1 at the mucosal side) (Table 1), a potent blocker of the Na+/K+
-pump in rat tissue [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)4molÆL)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)8molÆL)1at the mucosal side) was measured in the absence of any drugs or in the presence of Tris borate (10)4molÆL)1at the mucosal side; pretreated for 15 min), ouabain (10)3molÆL)1at the mucosal side; pretreated for 1 h), scilliroside (10)4molÆL)1at the mucosal side; pretreated for 1 h), or vanadate (10)4molÆL)1
at the mucosal side; pretreated for 1 h), or after replacement of NaCl by NMDG chloride (107 mmolÆL)1NMDG 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)1Æcm)2)
Proximal colon
D Isc (lEqÆh)1Æcm)2) n
(apical Na + )
(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)8molÆL)1at the mucosal side in the presence of 0.5 mmolÆL)1Na
borate at the mucosal side) (B) Concentration-dependent increase in
Isc above baseline (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)1Na borate Values are means ± SEM, n ¼ 6–8.
Trang 4Ionic 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)8molÆL)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)1KCl 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)8molÆL)1at 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)8molÆL)1at 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
A Na+/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 a1-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-subunit of the
Table 2 Effect of inhibitors/activators on baseline Isc Concentration of drugs were: Tris borate (10)4molÆL)1at the mucosal side), ouabain (10)3molÆL)1at the mucosal side), vanadate (10)4molÆL)1at the mucosal side) *p < 0.05 vs baseline.
Distal colon
D Isc (lEqÆh)1Æcm)2)
Proximal colon
D Isc (lEqÆh)1Æcm)2) n
Fig 2 Action of palytoxin (10)8molÆL)1at the mucosal side) under conditions in which the basolateral membrane was depolarized by a high concentration of K+(111.5 mmolÆL)1KCl 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.
Trang 5Na+/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)
D I S C U S S I O N
Palytoxin is produced from corals of the genus Palythoa It
is the strongest toxin produced by animals, with an LD50for
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 P2C-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.
Trang 6pores 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 in the 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 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 b subunit 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 mMouabain 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 a1subunit in the apical membranes can
be excluded, allowing us to conclude that the previously
measured Na+- and K+-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 P2-type cation pumps such as the yeast Na+
-ATPase or Ca2+ -ATPase are not sensitive to palytoxin suggests that P2C-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
A C K N O W L E D G E M E N T S
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.
R E F E R E N C E S
1 Axelsen, K.B & Palmgren, M.G (1998) Evolution of substrate specificities in the P-type ATPase superfamily J Mol Evol 46, 84–101.
2 Scheiner-Bobis, G (2002) The sodium pump: its molecular properties and mechanics of ion transport Eur J Biochem 269, 2424–2433.
3 Jaisser, F & Beggah, A.T (1999) The nongastric H+-K+ -ATPases: molecular and functional properties Am J Physiol.
276, F812–F824.
4 Hansen, O (1984) Interaction of cardiac glycosides with (Na+,K+)-activated ATPase Biochemical linkto digitalis-in-duced inotropy Pharmacol Rev 36, 143–163.
5 Ishida, Y., Tagaki, K., Takahashi, M., Satake, N & Shibata, S (1983) Palytoxin isolated from marine coelenterates: the inhibitory action on (Na,K)-ATPase J Biol Chem 258, 7900–7902.
6 Habermann, E (1989) Palytoxin acts through Na + /K + -ATPase Toxicon 27, 1171–1187.
7 Kodama, A.M., Hokama, Y., Yasumoto, T., Fukui, M., Manea, S.J & Sutherland, N (1989) Clinical and laboratory findings implicating palytoxin as cause of ciguatera poisoning due to Decapterus macrosoma (mackerel) Toxicon 27, 1051–1053.
8 Wachi, K.M., Hokama, Y., Haga, L.S., Shiraki, A., Takenaka, W.E., Bignami, G.S & Levine, L (2000) Evidence for palytoxin as one of the sheep erythrocyte lytic in lytic factors in crude extracts
of ciguateric and non-ciguateric reef fish tissue J Nat Toxins 9, 139–146.
9 Moore, R.E & Bartolini, G.J (1981) Structure of palytoxin.
J Am Chem Soc 103, 2491–2494.
10 Cha, J.K., Christ, W.J., Finan, J.M., Fuijoka, M., Kishi, J., Klein, L.L., Ko, S.S., Leder, J., McWhorter, W.W., Pfaff, K.-P., Yonaga, M., Uemura, D & Hirata, Y (1982) Stereochemistry of palytoxin.
4 Complete structure J Am Chem Soc 104, 7369–7371.
11 Scheiner-Bobis, G., Meyer zu Heringdorf, D., Christ, M & Habermann, E (1994) Palytoxin induces K + efflux from yeast cells expressing the mammalian sodium pump Mol Pharmacol.
45, 1132–1136.
12 Redondo, J., Fiedler, B & Scheiner-Bobis, G (1996) Palytoxin-induced Na + influx into yeast cells expressing the mammalian sodium pump is due to the formation of a channel within the enzyme Mol Pharmacol 49, 49–57.
Trang 713 Hirsh, J.K & Wu, C.H (1997) Palytoxin-induced single-channel
currents from the sodium pump synthesized by in vitro expression.
Toxicon 35, 169–176.
14 Parsons, D.S & Paterson, C.R (1965) Fluid and solute transport
across rat colonic mucosa Quart J Exp Physiol 50, 220–231.
15 Reuss, L (2001) Ussing’s two-membrane hypothesis: the model
and half a century of progress J Membr Biol 184, 211–217.
16 Robinson, J.W (1970) The difference in sensitivity to cardiac
steroids of (Na++K+)-stimulated ATPase and amino acid
transport in the intestinal mucosa of the rat and other species.
J Physiol 206, 41–60.
17 Schultheiss, G & Diener, M (1997) Regulation of apical and
basolateral K + conductances in rat colon Br J Pharmacol 122,
87–94.
18 Fuchs, W., Larsen, E.H & Lindemann, B (1977) Current-voltage
curve of sodium channels and concentration-dependence of
sodium permeability in frog skin J Physiol 267, 137–166.
19 Del Castillo, J.R., Rajendran, V.M & Binder, H.J (1991)
Apical membrane localization of ouabain-sensitive K + -activated
ATPase activities in rat distal colon Am J Physiol 261, G1005–
G1011.
20 Scheiner-Bobis, G (1998) Ion-transporting ATPases as ion
channels Naunyn-Schmiedeberg’s Arch Pharmacol 357, 477–482.
21 Habermann, E., Hudel, M & Dauzenroth, M.-E (1989) Palytoxin
promotes potassium outflow from erythrocytes, Hela and bovine
adrenomedullary cells through its interaction with Na+/K+
-ATPase Toxicon 27, 419–430.
22 Sangan, P., Thevananther, S., Sangan, S., Rajendran, V.M & Binder, H.J (2000) Colonic H-K-ATPase alpha- and beta-sub-units express ouabain-insensitive H-K-ATPase Am J Physiol.
278, C182–C189.
23 Sangan, P., Rajendran, V.M., Mann, A.S., Kashgarian, M & Binder, H.J (1997) Regulation of colonic H-K-ATPase in large intestine and kidney by dietary Na depletion and dietary K depletion Am J Physiol 272, C685–C696.
24 Cougnon, M., Planelles, G., Crowson, M.S., Shull, G.E., Rossier, B.C & Jaisser, F (1996) The rat distal colon P-ATPase alpha subunit encodes a ouabain-sensitive H + , K + -ATPase J Biol Chem 271, 7277–7280.
25 Asano, S., Hoshina, S., Nakaie, Y., Watanabe, T., Sato, M., Suzuki, Y & Takeguchi, N (1998) Functional expression of putative H + -K + -ATPase from guinea pig distal colon Am.
J Physiol 275, C669–C674.
26 Lee, J., Rajendran, V.M., Mann, A.S., Kashgarian, M & Binder, H.J (1995) Functional expression and segmental localization of rat colonic K-adenosine triphosphatase J Clin Invest 96, 2002– 2008.
27 Cougnon, M., Bouyer, P., Planelles, G & Jaisser, F (1998) Does the colonic H,K-ATPase also act as an Na,K-ATPase? Proc Natl Acad Sci U.S.A 95, 6516–6520.
28 Rajendran, V.M., Sangan, P., Geibel, J & Binder, H.J (2000) Ouabain-sensitive H,K-ATPase functions as Na,K-ATPase in apical membranes of rat distal colon J Biol Chem 275, 13035– 13040.