Hypotonic shock mediation by p38 MAPK, JNK, PKC, FAK, OSR1 and SPAK in osmosensing chloride secreting cells of killifish opercular epithelium
1 Department of Biology, St Francis Xavier University, PO Box 5000
Antigonish, Nova Scotia, Canada B2G 2W5
2 Department of Biochemistry, August Krogh Institute, University of
Copenhagen, 13 Universitetsparken, Copenhagen DK-2100, Denmark
* Author for correspondence (e-mail: bmarshal{at}stfx.ca)
Accepted 11 January 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Fundulus heteroclitus, protein kinase, protein phosphatase, SB203580, regulatory volume decrease, okadaic acid, chelerythrine, NKCC1, Na+, K+, 2Cl- cotransport, gill epithelium
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hypotonic shock is physiologically relevant because small estuarine fish
such as the common killifish (mummichog), Fundulus heteroclitus,
forage in shallow water following advancing tides and are exposed regularly to
freshwater (FW) microenvironments. Typically the salinity in these shallows is
low, 3% seawater (1.0 g l-1) or lower. Because the animals
return to high salinity between tides, only temporary `coping' mechanisms are
required, not permanent acclimation to FW
(Marshall, 2003
). In the first
few hours, blood osmolality and ion content are reduced, producing a
hyposmotic cue to ion transporting cells
(Marshall et al., 2000
).
Hypotonic shock in vitro rapidly inhibits the ion secretion by
mitochondria-rich chloride secreting cells of the gill and opercular
epithelium (Zadunaisky et al.,
1998
; Marshall et al.,
2000
), an effect mimicked by the protein tyrosine kinase inhibitor
genistein (Marshall et al.,
2000
). Surprisingly, the hypotonic inhibition was also exacerbated
by the phosphatase inhibitor calyculin A
(Zadunaisky et al., 1998
).
Killifish opercular epithelium and related teleost membranes are model
systems containing mitochondria-rich cells used to study the regulation of
salt transport. Reduction in ion transport after transfer to freshwater
includes an inhibition of active Cl- secretion and passive
diffusive ion loss in a three-stage process spanning approx. 30 min. There is
a combination of sympathetic neural reflex mediated by
2-adrenoceptors operating via intracellular
inositol trisphosphate through intracellular Ca2+ (Marshall et al.,
1993
,
1998
), a rapid cellular
hypotonic shock response (Marshall et al.,
2000
) and finally a covering over of ion secreting cells by
pavement cells (Daborn et al.,
2001
). These three steps effectively minimize salt loss in dilute
media. The upregulation of salt secretion on return to full strength seawater
(SW) may be via hormones (arginine vasotocin and urotensin I) and a
neurotransmitter (vasoactive intestinal polypeptide) in combination with
hypertonic shock (Hoffmann et al.,
2002
). Because in nature the rapid inhibition of Cl-
secretion is short lived (a few hours), voluntary (involving shoaling
behaviour) and mediated by autonomic reflex and neurohormones in combination
with direct effects of blood tonicity on ion transporting cells, the concept
of a nonstressful salinity change is put forward.
We hypothesized that the basolateral
Na+,K+,2Cl- cotransporter, NKCC1 isoform,
which is the mechanism for Cl- entry across the basolateral
membrane by secondary active transport, was the focal point of the regulation.
Cl- secretion by the isolated opercular membrane of killifish is
sensitive to serosal bumetanide, piretanide and furosemide (EC50 of
40, 52 and 295 µmol l-1, respectively) and insensitive to DIDS
and thiazide type drugs, indicating an operational NKCC-type cotransporter on
the basolateral membrane (Eriksson et al.,
1985; Eriksson and Wistrand,
1986
). Also Cl- secretion is rapidly blocked by
K+-free (Marshall and Bryson,
1998
) and Na+-free salines
(Marshall, 1981
), consistent
with NKCC mediation of Cl- transport. Finally, NKCC1 expression (by
qRT-PCR) and protein abundance (by western analysis) are increased
sequentially after transfer of killifish from brackish water to seawater but
not to freshwater (Scott et al.,
2004
), supporting NKCC1 as part of NaCl secretion in seawater.
NKCC is well known to be regulated by phosphorylation (reviewed by
Flatman, 2002) and is
activated by cell shrinkage in many cell types
(Hoffmann and Dunham, 1995
).
In the inhibition of NaCl secretion by hypotonic shock, it is unlikely that
transmural salt transport inhibition would act by blockade of solute exit at
the apical membrane, although the CFTR homologue is an anion channel that is
phosphorylated by protein kinase A
(Marshall et al., 1995
;
Singer et al., 1998
), because
this would exacerbate the cell swelling (whereas inhibition of NKCC would
actually aid in regulatory volume decrease). NKCC1 is found to be inactivated
after cell swelling in various cells, such as Ehrlich ascites tumour cells
(Krarup et al., 1998
), thus
hypotonicity would dephosphorylate NKCC and decrease NaCl entry, to reduce
cell swelling and inhibit transepithelial Cl- secretion. Presumably
hypotonic shock also initiates a regulatory volume decrease involving
activation of K+ and Cl- exit via independent
channels, as in Erhlich ascites tumour cells
(Hoffmann, 2000
) or KCl
cotransport, as in erythrocytes (Ellory et
al., 1998
). Conversely, hypertonic shock would shrink cells,
phosphorylate NKCC, evoke regulatory volume increase and increase
transepithelial Cl- secretion. The mechanisms for NKCC
phosphorylation/dephosphorylation are unknown, although several kinases have
been found to be involved in various cell types (see
Hoffmann and Dunham, 1995
). In
the killifish operculum myosin light chain kinase (MLCK) and protein kinase C
(PKC) were shown to be involved in the activation of NKCC1
(Hoffmann et al., 2002
) but
the PKC isoform involved was not determined.
Prostaglandins are known also to be involved in cellular osmotic responses.
In the vertebrate cell line Ehrlich ascites tumour cells, it has been
demonstrated that cytoplasmic phospholipase A2 (cPLA2)
is activated by hypotonic shock, resulting in increase of arachidonic acid
release and increased eicosanoid synthesis
(Hoffmann, 2000). The
eicosanoid prostaglandin E2 (PGE2) is known to rapidly
inhibit Cl- secretion in the opercular membrane of Fundulus
heteroclitus (Eriksson et al.,
1985
; Van Praag et al.,
1987
; Evans et al., 2003) and the tissue metabolizes several
different eicosanoids, including prostaglandins, leukotrienes and
hydroxyeicosatetraenoic acids (Van Praag
et al., 1987
). While thromboxanes and carbaprostacyclin (a PGI2
analogue) do not affect ion secretion rate, there appear to be both inhibitory
and stimulatory PGE2 receptors in the killifish opercular epithelium
(Evans et al., 2004
).
Cycloxygenase is immunolocalized not to the epithelial cells, but to the
vascular space in the gill filaments
(Evans, 2002
), a localization
more in tune with a vasoactive response, as has been suggested by Sundin and
Nilsson (2002
). The
prostaglandin effect may be downstream of endothelin and nitric oxide agonists
affecting cycloxygenase-2 (Evans et al., 2003). Therefore the opercular
epithelium was examined to see if hypotonic shock was affected by
prostaglandins.
Because p38 MAPK and JNK have been implicated in volume responses of human
leukemia cells (Pandey et al.,
1999), Ehrlich ascites tumour cells
(Pedersen et al., 2002
) and
rat hepatocytes (vom Dahl et al.,
2001
), we examined blockers of p38 MAPK and sought to identify
this kinase using antibodies directed to human phosphorylated p38 MAPK and
phosphorylated JNK. In Fundulus heteroclitus gill cells from animals
exposed to osmotic shock, Kültz and Avila
(2001
) observed changes in
activity and abundance of stress-associated protein kinase 1 (SAPK1, also
known as Jun N-terminal kinase, JNK) and SAPK2 (p38 MAPK). In addition, a
recently identified kinase that co-immunoprecipitates with NKCC1, a stress
associated, Ste20/sps1-related proline-alanine-rich protein kinase SPAK (also
known as PASK; Piechotta et al.,
2002
) is a prime candidate for involvement in NKCC1 regulation. We
investigated expression of SPAK using an antibody directed against human SPAK.
This heterologous approach is viable in this case, because the degree
of sequence similarity at the amino acid level between teleost (from genome
libraries for Fugu, Takifugu rubripes and zebrafish, Danio
rerio) and human protein kinases is greater than 90%, yet subclasses of
protein kinases from one species have very low similarity, only 40-50%. As a
result, we expected that the human directed antibodies should still detect the
correct target enzymes in teleosts. Protein tyrosine kinase inhibition mimics
the hypotonic response (Marshall et al.,
2000
) and it is well known that tyrosine kinases are involved in
the RVD response in other cell types, e.g. Ehrlich ascites tumour cells
(Hoffmann, 2000
), thus we
examined further the role of this enzyme in volume responses and extended this
to include focal adhesion kinase (FAK), a ubiquitous scaffolding protein with
a tyrosine phosphorylation site (Pandey et
al., 1999
). Hence the goal of the study was to begin uncovering
the mechanisms of transport regulation, particularly of NKCC1, in chloride
cells in response to osmotic stress using a combination of pharmacology,
immunoblot and immunocytochemistry.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bathing solutions
Both opercular epithelia were dissected for electrophysiology of ion
transport (below) and placed in a modified Cortland's saline (composition in
mmol l-1: NaCl 160, KCl 2.55, CaCl2 1.56,
MgSO4 0.93, NaHCO3 17.85, NaH2PO4
2.97 and glucose 5.55, pH 7.8, when equilibrated with a 99% O2/1%
CO2 gas mixture. Hypotonic shock was applied by flushing of the
hemi-chambers with diluted Cortland saline at 244 mOsm kg-1, a
reduction of 60 mOsm kg-1 from the normal 304 mOsm kg-1.
Hypertonic shock, used in some cases, was applied by flow through of regular
Cortland saline with 60 mmol l-1 mannitol added to increase
osmolality to 364 mOsm kg-1. The solution changes were performed
symmetrically to avoid possible effects of asymmetrical solutions.
Electrophysiology
The opercular epithelium was removed and mounted in a modified Ussing
chamber as described previously (Marshall
et al., 1998) except that the epithelium was dissected without the
nerve supply. The epithelium was supported by a nylon mesh and pinned out over
the circular aperture (0.125 cm2) with the rim area lightly greased
and bevelled to minimize edge damage. In the membrane chambers, the following
epithelial electrophysiological variables were monitored: transepithelial
potential Vt (mV), transepithelial resistance
Rt (
cm2) and short-circuit current
Isc µAcm-2). Isc is
expressed as positive for secretion of anions and is equivalent to the net
secretion of Cl- (Degnan et al.,
1977
). Epithelia were left at open circuit and were clamped to 0
mV for short periods to allow recording of Isc. A
current-voltage clamp (D. Lee Co., Sunnyvale, CA. USA or WP Instruments
Sarasota, FL, USA; DVC 1000) was used to measure the epithelial variables. A
control period of 1 h established the resting Isc, after
which drug additions and hypotonic shock tests were performed.
Pharmaceuticals
The protein kinase A inhibitor
N-2-P-bromocinnamyl-aminoethyl-5-isoquinolinesulfoniamide (H-89), the protein
kinase C inhibitor that is selective for subtypes and ß
(Way et al., 2000
) Gö6976
[12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole]
and the protein kinase C inhibitor chelerythrine chloride were obtained from
LC Laboratories, Woburn MA, USA and were dissolved in water (H-89 only) or a
minimal volume of dimethylsulfoxide (DMSO; solvent for Gö6976 and
chelerythrine). The ß-adrenergic agonist isoproterenol (10 µmol
l-1 in saline, serosal side) was used to test the efficacy of the
PKA inhibitor H-89. H-89 (1.0 µmol l-1) was added to the basal
side, incubated for 30 min, then 10 µmol l-1 isoproterenol was
added; parallel control membranes received no H-89. The p38 MAPK inhibitor
SB203580, the protein phosphatase inhibitor okadaic acid and a second batch of
Gö6976 were obtained from Calbiochem (San Diego, USA) and were all
dissolved in a minimal volume of DMSO. Maximum amount of DMSO added with drug
was 0.75% of chamber volume, a level without noticeable effect on the control
membranes. Each drug was added to the serosal side of the opercular membrane,
while the drug vehicle was added to a parallel running paired control membrane
from the same animal.
Preparation of extracts
Three pairs of opercular- and gill epithelia, from three different animals,
were used in preparation of each sample. Dissected opercular and gill
epithelia were transferred to boiling sodium dodecyl sulphate (SDS) lysis
buffer (10 mmol l-1 Tris-HCl pH 7.4, 1% SDS) and minced, using
scissors. The minced tissue was homogenized using a homogenizer, then boiled,
frozen at -80°C, thawed and sonicated. The extracts were centrifuged for 5
min at 12,000 g and 5°C. Protein concentration was
determined spectrophotometrically using the detergent-compatible Bio-Rad DC
Protein Assay Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA).
Proteins were isolated from the crude extract by trichloroacetic acid (TCA) precipitation. TCA were purchased from Sigma-Aldrich Inc. (St Louis, MO, USA). To 1.0 ml samples containing equal amounts of protein were added 200 µl of ice cold 100% TCA solution and the samples incubated on ice for 30 min. Precipitated protein was collected by centrifugation for 10 min at 12 000 g. The supernatant was discarded and the pellet washed three times in 500 µl ice-cold acetone, then air dried. The dry pellet was resuspended in NuPAGE 4 x lauryl dodecyl sulphate (LDS) sample buffer (Invitrogen, Carlsbad, CA, USA) supplemented with dithiothreitol (DTT) to 0.05 M final concentration.
SDS-PAGE and western blotting
Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE; precast NuPAGE 10% Bis-Tris gels and all NuPAGE
products were purchased from Invitrogen) and electro-transferred to
nitrocellulose membranes (Invitrogen), using NuPAGE transfer buffer. After
transfer, the nitrocellulose membranes were incubated in blocking buffer (pH
7.5 Tris-buffered saline plus 0.1% Tween 20 (TBS-T), supplemented with 5%
non-fat dry milk) for 1 h at room temperature or overnight at 4°C. The
primary, polyclonal antibodies against p38 MAPK, phospho-p38 MAPK and
phospho-JNK were purchased from Cell Signalling Technology Inc. (Beverly, MA,
USA). The SPAK and OSR1 antibodies were a kind gift from Dr E. Delpire
(Piechotta et al., 2002). All
antibodies were diluted 1:100 and applied overnight at 4°C. The membrane
was then washed four times in TBS-T for 5-15 min each. The goat anti-rabbit,
alkaline phosphatase-conjugated secondary antibody (Jackson Immunoresearch
Laboratories Inc., West Grove, PA, USA) was diluted 1:500 in blocking buffer
and applied for 1 h at room temperature. The membranes were then washed as
described above. Immunoreactive bands were detected using
5-bromo,4-chloro,3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT) membrane
phosphatase substrate (KPL, Gaithersburg, MD, USA). A HP Scanjet 4600 (Hewlett
Packard, Palo Alto, CA, USA) and the UN-SCAN-IT gel version 5.1 for Windows
(Silk Scientific Corp., Salt Lake City, Utah, USA) software were used for
quantification of immunoreactive bands and estimation of molecular masses.
Immunocytochemistry
The primary antibody for detection of
Na+,K+,2Cl- cotransporter (NKCC) was T4
(Lytle et al., 1992;
Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA),
an antibody to the carboxyl region of NKCC that has been shown to bind to
several isoforms of NKCC across several species
(Haas and Forbush, 1998
;
Wilson et al., 2000
) including
killifish opercular membrane (Marshall et
al., 2002
). The secondary antibodies were goat polyclonal
anti-mouse IgG conjugated to an Oregon Green 488 fluorophore and the same
antibody conjugated to one of Alexa Fluor 546 or Alexa Fluor 594 (Molecular
Probes, Eugene, OR, USA). Primary antibody against SPAK was a rabbit
polyclonal anti-mouse SPAK (residues 424-556; a gift of E. Delpire)
(Piechotta et al., 2002
).
Primary antibody against phosphorylated FAK was rabbit polyclonal
anti-hFAK[pY407] (Biosource Int. Camarillo CA, USA) where the
highly conserved epitope region is known to be 100% similar among human,
mouse, rat, chicken and Takifugu. Secondary antibodies were goat
anti-rabbit polyclonal conjugated with Oregon Green 488 and the same antibody
conjugated with either Alexa Fluor 546 or 594 (Molecular probes, Eugene OR,
USA).
Opercular epithelia were dissected without the dermal chromatophore layer and pinned to modeller's wax. For genistein pretreatment before pFAK antibody detection, paired membranes were incubated in aerated Cortland's saline for 2 h with 0.14% v/v DMSO vehicle or 100 mmol l-1 genistein in an equivalent volume of DMSO. Preparations were rinsed three times in rinsing buffer comprising 0.1% bovine serum albumin (BSA) and 0.05% Tween 20 in PBS (TPBS), where PBS is phosphate-buffered saline, composition in mmol l-1: NaCl 137, KCl 2.7, Na2HPO4 4.3, KH2PO4 1.4, pH 7.4. The membranes were fixed for 3 h at -20°C in a formaldehyde-free 80% methanol/20% dimethyl sulfoxide (DMSO) fixative. The methanol was used as a dehydrating agent and the DMSO as a cryoprotective agent. The membranes were rinsed three times, then immersed in a blocking solution with 5% normal goat serum (NGS)/0.1% BSA/0.05% TPBS, pH 7.4 for 30 min at room temperature in the dark and incubated in the primary antibody (8 µg ml-1 in 0.5% BSA in PBS) overnight at 4°C; parallel control tissues received no primary antibody. Control and test membranes were then rinsed three times and exposed to the secondary antibody (1:50 in 0.5% BSA in PBS), singly and in combination for 5 h at 4°C. After three final rinses the membranes were mounted in mounting medium (Geltol, Immunon Thermo Shandon, Pittsburgh, PA, USA). Mitochondria-rich cells were identified, using bright-field DIC microscopy, as large spheroidal cells with finely granular cytoplasm and centrally placed nucleus, confirmed by mitochondrial dye (Mitotracker, Molecular Probes, Eugene OR, USA). Slides were viewed in single blind fashion and images collected with a laser confocal microscope (Olympus, Markham, ON, Canada; model FV300). In each opercular membrane, randomly selected Z-stack series were collected at 40x, zoom of 3.0 and with optical sections of 1.0±0.05 µm.
Statistical analyses
Data are expressed as the mean ± 1
S.E.M. Statistical significance was
determined by paired t-tests between test and control membranes with
significance ascribed if P<0.05, in a two-tailed test. For the
immunoblot results, a one sample t-test, two-tailed, was applied
comparing fold-activation relative to a reference and normalized for
nonspecific kinase activation using controls incubated for the same time
interval.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Prostaglandin and PKA
We tested whether the cycloxygenase inhibitor indomethacin could prevent
the initial current decrease in Isc and the secondary
increase in resistance after hypotonic stress. However, indomethacin had no
detectable impact on the initial decrease in Isc or
resistance increase (Fig. 2),
thus involvement of prostaglandins in the volume response is unlikely.
|
The PKA inhibitor (H89, 1.0 µmol l-1) significantly reduced
the augmentation of chloride secretion produced by 10.0 µmol l-1
isoproterenol in opercular membranes from 130±22 µA cm-2
to 21±3.3 µA cm-2 (P<0.001, N=5),
thus confirming the efficacy of this kinase inhibitor in teleost systems.
However, previous cAMP assays (Marshall et
al., 2000) showed no change in cAMP levels with hypotonic shock,
thus PKA appears not to be involved in the volume response.
Protein kinase C
The general PKC inhibitor chelerythrine significantly inhibited the resting
membrane current but had little effect on the hypotonic shock response
(Fig. 3). Chelerythrine greatly
reduced the recovery of the membrane current when isotonic solutions were
restored (Fig. 3). There was no
significant change in transepithelial resistance with chelerythrine (data not
shown). To help determine the isoform of PKC that might be involved, we used a
more specific PKC inhibitor, Gö6976, which selectively inhibits the
calcium-dependent conventional PKC (cPKC) isoforms ( and ß1)
(Way et al., 2000
). However,
Gö6976 at 1.0 and 10 µmol l-1 did not reduce the resting
Isc and it did not block the hypotonic response (current
reduction in vehicle was -81.6±19.6 versus -100.3±30.7
µA cm-2, N=3 animals on three experiment days) and it
did not block the recovery afterwards when isotonic solutions were introduced.
There was no significant change in transepithelial resistance with Gö6976
(data not shown).
|
p38 mitogen activated protein kinase
The p38 MAPK inhibitor SB 203580 is considered to be highly specific
(Davies et al., 2000;
Bain et al., 2003
) and we have
used this inhibitor to investigate whether this MAPK is involved in hypotonic
inhibition and hypertonic activation of the Cl- current. Whereas
SB203580 had no effect on resting current
(Fig. 4), the decrease in
current was significantly larger in the SB203580-treated membranes
(P<0.05). There was a decrease in the current overshoot when
isotonic solutions were replaced (P<0.05) and a trend toward a
smaller increase in current when hypertonic conditions were invoked.
Transepithelial resistance increases with hypotonic shock and, with SB203580,
the increase in resistance is larger (P<0.05), reflecting the
larger decrease in current. This general pattern holds, so resistance results
are not shown for the other treatments.
|
Activation of p38 MAPK was measured by western blot analysis in triplicate in tissues that were dissected and placed in hypotonic or isotonic bathing media for different times and scanned with reference to tissues incubated in isotonic conditions for the same time (Fig. 5). The antibody to p38 MAPK and that to the phosphorylated form of p38 MAPK detected a single band in the expected molecular mass range, 38-40 kDa. The p38 MAPK phosphorylation was significantly higher than controls at 5 min incubation in hypotonic media (eightfold, P<0.05, paired t-test compared to isotonic control level at each time), threefold at 30 min incubation in hypertonic media and eightfold by both hypertonic and hypotonic shock in a second phase response at 1 h (P<0.05) (Fig. 5A,B). Equal amounts of p38 MAPK were expressed in each sample of opercular epithelium, as measured with antibody to unphosphorylated p38 MAPK (Fig. 5A).
|
Gill and opercular epithelium tissue from long-term acclimated freshwater and seawater killifish yielded detectable expression of p38 MAPK western immunoblots for both epithelia and in both salinities (Fig. 6). There was significantly lower expression in the gill and opercular membranes from seawater animals relative to tissue from freshwater acclimated fish.
|
Okadaic acid and protein phosphatase
Okadaic acid is an inhibitor of serine/threonine protein phosphatases (PP)
that binds to the active site (Huang et
al., 1997). It has highest affinity for PP2A and to a lesser
extent for PP1 (Takai et al., 1993) and in intact cell systems also can act on
PP4 and PP5 (Millward et al.,
1999
). It was previously shown in opercular epithelium that the
PP1 and PP2A inhibitor calyculin A increased the steady state current
significantly (Hoffmann et al.,
2002
) and potentiated the decrease in current after hypotonic
shock (Zadunaiski et al., 1997). Application of okadaic acid significantly
increased membrane current, compared to vehicle controls
(Fig. 7). In addition, we found
that okadaic acid had a marginal potentiating effect on the decrease in
current after hypotonic shock. The current in control and test membranes
remained inhibited for the hour. However, restoration of isotonic conditions
did not restore the membrane current in the okadaic acid-treated membranes,
but controls had a normal overshoot in current
(Fig. 7).
|
Protein tyrosine kinase
The protein tyrosine kinase (PTK) inhibitor genistein, but not the inactive
analogue daidzein (Marshall et al.,
2000) inhibit chloride secretion in seawater killifish opercular
epithelia (P<0.01, Fig.
8; Marshall et al.,
2000
). However, the effect is level dependent. If the membranes
are first inhibited with the
-adrenergic agonist clonidine (1.0 µmol
l-1), genistein instead increases chloride secretion
(P<0.05), as Isc
(Fig. 8). Thus the effect of
genistein is to release the membrane from steady-state stimulated or inhibited
states, allowing the current to move to an intermediate level.
|
Jun N-terminal kinase
Antibody to the phosphorylated form of JNK (pJNK) was used in western
analysis to determine if JNK was activated by hyper and hypotonic shock
(Fig. 9). With time, there
appeared to be an increase in pJNK in the isotonic controls, probably an
effect of repeated disturbance of the fish before tissue collection.
Therefore, the time controls in isotonic media were ascribed a normalized 100%
and the treated tissues (from animals sampled at the same time) were compared
to this control. There was a modest activation of pJNK by hyper- and hypotonic
shock at 5 min (P<0.05) and again at 30 minincubation in
hypertonic media, compared to parallel running control tissues in isotonic
conditions. The 63 kDa isoform detected here is the same protein described as
SAPK1 by Kültz and Avila
(2001).
|
The stress associated protein kinases SPAK and OSR1
Western analysis of OSR1 and SPAK in gill and opercular membrane epithelial
cells demonstrated that OSR1, the 66 kDa isoform and a truncated form, were
present in freshwater and seawater gill and opercular epithelium
(Fig. 10A), and that
expression in freshwater-acclimated animals was significantly greater than in
seawater-acclimated animals, by two- to fivefold for gill and opercular
membrane, respectively. There were positive indications of SPAK expression in
seawater and freshwater opercular epithelium and gill tissue
(Fig. 10B) with modestly
higher expression of SPAK in freshwater-acclimated animals.
Immunocytochemistry demonstrated that NKCC is present in the mid to lower
levels of mitochondria-rich cells in the opercular membrane, at the level of
the nucleus and to the basal side of the cells
(Fig. 11A). This distribution
is similar to the distribution of SPAK immunofluorescence
(Fig. 11B) and in most cases
there is exact colocalization of NKCC and SPAK (yellow colour in
Fig. 11C; line scan in
Fig. 11D), consistent with
juxtaposition of the two proteins (N=8 animals on 6 experiment days).
In seawater opercular epithelia, there was also colocalization of NKCC and
OSR1 (Fig. 11E-H) with all
mitochondria-rich cells showing colocalized immunofluorescence of the two
proteins (N=3 animals on 3 experiment days).
|
|
Focal adhesion kinase
Immunocytochemistry using antibody for the phosphorylated form of the
scaffolding protein focal adhesion kinase (pFAK) revealed in 11 experiments on
11 animals, positive staining for pFAK (green fluorescence in
Fig. 12A) with the
cotransporter NKCC (red fluorescence in B; four experiments on four animals)
in mitochondria-rich cells of seawater killifish opercular membranes.
Furthermore, FAK appeared in all mitochondria-rich cells and was highly
colocalized with NKCC (four experiments on four animals) in all
mitochondria-rich cells in the membrane (yellow fluorescence in
Fig. 12C). Genistein
pretreatment (100 µmol l-1) of opercular epithelia before
addition of the anti-pFAK antibody completely eliminated pFAK fluorescence
(Fig. 12D) while the genistein
control tissue that received 0.14% DMSO vehicle had positive pFAK fluorescence
(not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
p38 MAPK and JNK
Multiple protein kinases are activated following hypertonic stress in
mammalian cells including the MAPK subfamilies Erk1, Erk2, p38 MAPK and JNK
(Pandey et al., 1999;
Roger et al., 1999
;
Duzgun et al., 2000
;
Pedersen et al., 2002
). The
p38 MAPK has been shown to be induced by hypertonic shock in corneal
epithelial cells (Bilden et al., 2003). As well, p38 MAPK is involved in the
regulatory volume increase process (Roger
et al., 1999
; Sheikh-Hamad et
al., 1998
).
In killifish, gill SAPK2 (=p38 MAPK) is enhanced by salinity transfer with
a fivefold increase in the expression of SAPK2 1 h after transfer from
freshwater to seawater (Kültz and
Avila, 2001). Our data confirm the activation of SAPK2/p38 MAPK in
teleost gill at 1 h and extend previous results by revealing an early, 5 min
eightfold activation by hypotonic shock only. The differential activation of
p38 MAPK with hypotonic shock at 5 min implicates this kinase in the
swelling-induced inhibition of chloride secretion that is happening
simultaneously. But the p38 kinase inhibitor SB203580 exacerbates the
hypotonic response in the opercular membranes, suggesting that p38 kinase is
still involved with activation of the Cl- secretion. Consistent
with this, the p38 inhibitor also blocks the overshoot after an hour when
isotonic solutions are restored and p38 activation is again elevated.
Protein kinase C and myosin light chain kinase
Our results reported here showing inhibition of the hypertonic response by
chelerythrine confirm our previous findings
(Hoffmann et al., 2002) and
support the idea of PKC involvement in volume responses. In eel intestine,
chelerythrine and calyculin A were potent inhibitors of the activation of
NKCC-mediated ion uptake in an RVI response to hypertonic shock
(Lionetto et al., 2002
). In
human cervical cancer cells, hypotonic shock evokes activation of
extracellular signal activated kinase (ERK1 and ERK2) and p38 MAPK through a
PKC-dependent pathway that also involves intracellular calcium
(Shen et al., 2001
). In rabbit
proximal tubule cells, PKC is translocated to the plasma membrane following
hypotonic shock (Liu et al.,
2003
). Meanwhile, hyperosmolality activates some PKC isoforms in
NIH/3T3 cells (Zhuang et al.,
2000
).
There has been some discussion about the specificity and efficacy of
chelerythrine, that it is effective in vivo against cPKC,ß,
but less so against nPKC isoforms (Keenan
et al., 1997
) and that it is ineffective in some in vitro systems
(Lee et al., 1998
). We
therefore also tested a more selective PKC inhibitor, Gö6976, which
specifically inhibits the calcium-dependent conventional PKC (cPKC) isoforms
(
and ß1) (Way et al.,
2000
). Because Gö6976 inhibited neither the hypotonic
response nor its recovery, it appears that the cPKC isoforms (
and
ß1) are not involved, rather that other isoforms of cPKC or members of
the novel PKC (nPKC) family are involved. It has been shown in NIH/3T3 cells
that hyperosmolarity activates both cPKC and nPKC families of kinases
(Zhuang et al., 2000
).
Hypotonic stress in rabbit proximal tubule cells specifically stimulates
translocation of certain isoforms of PKC to the plasma membrane, notably
PKC
, PKC
and PKC
(Liu
et al., 2003
). NKCC is activated by PKC in rabbit tracheal
epithelium cells (Liedtke and Thomas,
1996
) and the isoform is the PKC
(Liedtke et al., 2003
). While
it is clear that PKC is involved in chloride cell volume responses, it is
possible that PKC
, PKC
and/or PKC
are the forms involved
with chloride cells, but this awaits positive identification of the PKC
isoform(s). Of particular interest is PKC
, which colocalizes with actin
and NKCC1 in the periphery of airway epithelial cells. This complex could be
represent the osmosensing mechanism
(Liedtke et al., 2003
).
Myosin light chain kinase (MLCK) inhibitors block the augmentation of
Cl- secretion by the opercular epithelium
(Hoffmann et al., 2002)
Stress-associated protein kinases, SPAK and OSR1
The stress-associated Ste20/SPS1-related proline-alanine-rich kinase (SPAK)
and proline-alanine-rich Ste20-related kinase (PASK) are homologous
serine-threonine kinases cloned from human brain
(Johnston et al., 2000) and
rat brain (Ushiro et al.,
1998
), respectively. SPAK and PASK interact, co-immunoprecipitate
and specifically activate p38 MAPK
(Johnston et al., 2000
;
Piechotta et al., 2002
) but
apparently not JNK or ERK (Johnston et
al., 2000
). The kinases SPAK/PASK and OSR1 also
co-immunoprecipitate with NKCC1 (Dowd and
Forbush, 2002
; Johnston et
al., 2000
; Piechotta et al.,
2002
), as well as other cation chloride cotransporters, such as
KCC3 (Piechotta et al., 2002
).
These cellular stress proteins, together with the demonstration of SPAK
interactions with p38 MAPK and NKCC1, point to SPAK and OSR1 functioning as
stress responsive scaffolding proteins. In support of this notion, Piechotta
et al. (2003
) demonstrated
that binding of SPAK to NKCC1 and p38 MAPK decreases during cellular stress.
The amount of p38 MAPK coimmunoprecipitated with SPAK and the cotransporter
significantly decreases upon cellular stress, while the binding of SPAK to
NKCC1 remains unchanged.
In teleost fish there are similar genes. There are homologs for hOSR1 in zebrafish (Danio rerio) and puffer fish (Takifugu rubripes) genomes that are 81% and 77% identical at the amino acid level, respectively, and for hSPAK that are 74% and 73% identical, respectively. However, the nearest match to hSPAK in zebrafish is in fact the OSR1 homologue, so the zebrafish (freshwater stenohaline) may lack a SPAK homologue. It is also of interest that the marine puffer fish OSR1 homologue has a seven amino acid deletion in the centre of the catalytic region (VLMLTLQ at position 235-241), while the SPAK homologue is exactly homologous in the same region and there is a truncated (366 aa) SPAK homologue with 85% identity to the human SPAK that includes the seven amino acid sequence. This analysis suggests that SPAK may be more important in seawater osmoregulation.
In the mitochondria-rich chloride cell, SPAK and NKCC1 colocalize by
immunocytochemistry and SPAK expression is detectable by immunoblotting in
gill and opercular epithelial cells of seawater and freshwater acclimated
animals, suggesting a role of the protein in NKCC1 regulation in salinity
acclimation. There is significantly more SPAK expression in freshwater animals
compared to seawater fish, consistent with the general trend for higher
expression of kinases in freshwater
(Kültz and Avila, 2001).
Importantly, co-expression of wild-type SPAK/PASK with either human or shark
NKCC1 increased cotransport activity in HEK cells, affecting phosphorylation
of the regulatory residues T184/T189 in shark NKCC1
(Darman and Forbush, 2002
;
Dowd and Forbush, 2002
),
indicating a relationship between SPAK and NKCC1 activation in fish. Our
results confirm this relationship and extend it to teleost chloride cells in
both freshwater and seawater.
OSR1 is expressed in seawater opercular epithelium and gill, but at much
lower levels than in freshwater-acclimated animals. Our OSR1 results are
consistent with previous findings, where transfer of killifish to freshwater
appears to upregulate expression of all MAP kinases, while transfer to
hyperosmotic conditions does the reverse
(Kültz and Avila, 2001).
There was a clear colocalization of OSR1 with NKCC by immunocytochemistry,
suggesting a strong role for OSR1 in seawater osmoregulation and its higher
expression suggests also a role in freshwater osmoregulation. The lack of a
SPAK homologue in zebrafish (above) also implicates OSR1 as being more
important in stenohaline freshwater teleosts.
Protein phosphatase
While it is clear there is serine/threonine protein phosphatase (PP)
involvement in the recovery from hypotonic shock, the subtype could be PP2a,
PP1 or possibly PP4 and PP5, because the concentration of Okadaic acid used
here on whole tissue was high enough to potentially affect all these
phosphatase subtypes (Millward et al.,
1999).
The cotransporter in shark rectal gland epithelium, a salt secreting
epithelium that shares many functional aspects with teleostean salt-secreting
cells of the gill and opercular membrane, has a protein phosphatase 1 (PP1)
binding site in the amino terminus at residues 107-112, and PP1
co-immunoprecipitates with NKCC1 (Darman et
al., 2001). Thus it is probable that PP1 may also be involved in
dephosphorylation of NKCC1 in teleosts. In support of this notion, okadaic
acid that blocks PP1 and related protein phosphatases, causes an increase in
membrane current in the opercular epithelium. The effect of okadaic acid in
blocking recovery from hypotonic shock requires an alternative explanation.
The lack of recovery after blockade of protein phosphatases implies that there
is a protein phosphatase whose activity is needed to unlock or release the
regulatory complex from steady state inhibition.
Protein tyrosine kinase
Tyrosine kinases are activated during RVD in several different cell systems
and a PTK inhibitor genistein inhibits RVD (for a review, see
Hoffmann, 2000) and tyrosine
kinases appears to play a role in the volume sensing mechanism. Genistein
alters chloride secretion by opercular membranes in a level-dependent fashion,
increasing chloride secretion of inhibited tissues (inhibited by
adrenergic agonist) and decreasing secretion in salt water stimulated tissues.
Genistein appears to unlock the transport from steady state `on' or `off'
conditions, yielding a moderate or intermediate level. Daidzein, the analogue
of genistein that is not active on kinases, is without effect on the opercular
membrane (Marshall et al., 2001). In a related system, the shark rectal gland
(that normally secretes Cl- at a low rate), genistein has been
shown to augment chloride secretion without activating cAMP
(Lehrich and Forrest, 1995
).
Genistein instead evokes trafficking of cystic fibrosis transmembrane
conductance regulator (CFTR) into the apical membrane
(Lehrich et al., 1998
), but
the mechanism is not clear. CFTR has numerous Ser/Thr phosphorylation sites as
well as a tyrosine site that might be involved
(Dahan et al., 2001
). NKCC
isoforms have two threonine residues that are phosphorylation sites and
phosphorylation of these sites, detected by antibody directed against the
phosphorylated form, occurs when NKCC is activated by cAMP via PKA
(Flemmer et al., 2002
).
However, because there is no tyrosine phosphorylation site on NKCC, any action
of tyrosine kinase must be indirect.
Focal adhesion kinase
The focal adhesion kinases are involved in membrane protein regulation and
especially in cellular motility. FAK signalling is connected to its ability to
become phosphorylated in response to integrin-mediated adhesion to Tyr-397.
Integrin-FAK interaction permits further interactions with a number of
different signalling effectors containing Src homology 2 (SH2) domains
(Hanks et al., 2003;
Gelman, 2003
). FAK Tyr-397 is
an autophosphorylation site that, when phosphorylated, defines an interaction
surface for SH2 domains. Proteins found to interact with Tyr-397 include Src,
Grb7, Shc, PLC
and the p85 subunit of phosphoinositide 3-kinase. When
Src is recruited to this site, additional tyrosine residues are
phosphorylated. FAK associates with integrins via its N-terminal FERM
domain and to talin and paxillin via the C-terminal focal adhesion
targeting (FAT) domain (Gelman,
2003
; Hauck et al.,
2002
). In hepatocytes, hypotonic shock activates a number of
kinases including p38 MAPK, as in the opercular membrane, but does not affect
phosphorylation of FAK (vom Dahl et al.,
2003
). Conversely, in fibroblasts, stretch-induced phosphorylation
of FAK at Tyr-397 is critical to activation of p38 MAPK in response to cyclic
stretch (Wang et al., 2001
).
Our results indicate FAK and NKCC colocalization is very close and
correspondent in every mitochondria-rich cell in the tissue. Furthermore,
genistein, which decreases Cl- secretion in membranes with high
current and increases Isc in previously inhibited
membranes, results in FAK dephosphorylation
(Fig. 12). Our results suggest
dephosphorylation of FAK on hypotonic shock connected to the rapid
dephosphorylation and deactivation of NKCC. We speculate that FAK
phosphorylation, presumably at Tyr-397, locks NKCC in steady state high or low
activity levels and that FAK dephosphorylation is permissive to rapid changes
in NKCC phosphorylation and activity level.
We observed rapid activation through phosphorylation of JNK in response to
hypotonic shock and this observation is consistent with previous results of
interactions between FAK and JNK. The FAK7/p130Cas complex can
mediate anchorage-dependent activation of JNK
(Almeida et al., 2000;
Oktay et al., 1999
). Also, FAK
and JNK co-localization was observed
(Almeida et al., 2000
). Igishi
et al. (1999
) suggest that
activation of JNK by FAK is independent of the kinase activity of FAK. Rather,
FAK-mediated recruitment of paxillin to the plasma membrane is sufficient for
JNK activation.
A tentative model
Whereas there is no tyrosine phosphorylation site on NKCC1, FAK has a
tyrosine phosphorylation site and is known to operate as a scaffolding
protein, associated with membrane proteins and the cytoskeleton. The almost
exact colocalization of NKCC with phosphorylated FAK demonstrated here
supports the notion of FAK being a scaffolding protein in NKCC regulation in
teleost mitochondria-rich cells. For instance colocalization of FAK with actin
has implicated FAK in normal spreading of cultured intestinal epithelial cells
(Ray et al., 2001). Thus FAK
could be part of the regulatory complex with NKCC1, OSR1 and SPAK.
Dephosphorylation of a tyrosine apparently `unlocks' the complex, allowing
NKCC1 to be phosphorylated and turned on (consistent with hypertonic shock and
RVI) or, if the transport is already activated, allowing NKCC1 to be
dephosphorylated and turned off (consistent with hypotonic shock and RVD). We
hypothesize then, that phosphorylated FAK (phosphorylated on Tyr-397) somehow
occludes phosphorylation sites on NKCC and locks the cotransporter in its
present phosphorylation state. When FAK is dephosphorylated, the complex
enters a dynamic state where phosphorylation or dephosphorylation is allowed.
The activation of NKCC1 (that contributes to RVI) probably involves p38 MAPK,
MLCK, PKC, OSR1 and SPAK terminating with phosphorylation of the two Ser/Thr
sites on NKCC1. Conversely, the cascade that turns off NKCC1 (and contributes
to RVD) probably involves JNK because it is activated by hypotonicity and a
terminal protein phosphatase, possibly PP2A, as a means to dephosphorylate and
deactivate NKCC1.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Almeida, E. A., Ilic, D., Han, Q., Hauck, C. R., Jin, F.,
Kawakatsu, H., Schlaepfer, D. D. and Damsky, C. H. (2000).
Matrix survival signaling: from fibronectin via focal adhesion kinase
to c-Jun NH(2)-terminal kinase. J. Cell Biol.
149,741
-754.
Bain, J., McLauchlan, H., Elliott, M. and Cohen, P. (2003). The specificities of protein kinase inhibitors: an update. Biochem. J. 371,199 -204.[CrossRef][Medline]
Bildin, V. N., Wang, Z., Iserovich, P. and Reinach, P. S. (2003). Hypertonicity-induced p38 MAPK activation elicits recovery of corneal epithelial cell volume and layer integrity. J. Membr. Biol. 193,1 -13.[CrossRef][Medline]
Daborn, K., Cozzi, R. F. F. and Marshall, W. S.
(2001). Dynamics of pavement cell-chloride cell interactions
during abrupt salinity change in Fundulus heteroclitus J.
Exp. Biol. 204,1889
-1899.
Dahan, D., Evagelidis, A., Hanrahan, J. W., Hinkson, D. A., Jia, Y., Luo, J. and Zhu, T. (2001). Regulation of the CFTR channel by phosphorylation. Pflügers Arch. 443, Suppl. 1,S92 -S96.[CrossRef][Medline]
Darman, R. B., Flemmer, A. and Forbush, B.
(2001). Modulation of ion transport by direct targeting of
protein phosphatase type 1 to the Na-K-Cl cotransporter. J. Biol.
Chem. 276,34359
-34362.
Darman, R. B. and Forbush, B. (2002) A
regulatory locus of phosphorylation in the N terminus of the Na-K-Cl
cotransporter, NKCC1. J. Biol. Chem.
277,37542
-37550.
Davies, S. P., Reddy, H., Caivano, M. and Cohen, P. (2000). Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351,95 -105.[CrossRef][Medline]
Degnan, K. J., Karnaky, K. J., Jr and Zadunaisky, J. A. (1977). Active chloride transport in the in vitro opercular skin of a teleost (Fundulus heteroclitus), a gill-like epithelium rich in chloride cells. J. Physiol. Lond. 271,155 -191.[Medline]
Dowd, B. F. X. and Forbush, B. (2002). PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na-K-Cl cotransporter (NKCC1). J. Biol. Chem. 278,27347 -27353.[CrossRef]
Duzgun, S. A., Rasque, H., Kito, H., Azuma, N., Li, W., Basson, M. D., Gahtan, V., Dudrick, S. J. and Sumpio, B. E. (2000). Mitogen-activated protein phosphorylation in endothelial cells exposed to hyperosmolar conditions. J. Cell Biochem. 76,567 -571.[CrossRef][Medline]
Ellory, J. C., Gibson, J. S. and Stewart, G. W. (1998). Pathophysiology of abnormal cell volume in human red cells. Contrib. Nephrol. 123,220 -239.[Medline]
Eriksson, Ö. and Wistrand, P. J. (1986). Chloride transport inhibition by various types of `loop' diuretics in fish opercular epithelium. Acta Physiol. Scand. 126,93 -101.[Medline]
Eriksson, Ö., Mayer-Gostan, N. and Wistrand, P. J. (1985). The use of isolated fish opercular epithelium as a model tissue for studying intrinsic activities of loop diuretics. Acta Physiol. Scand. 125,55 -66.[Medline]
Evans, D. H. (2002). Cell signalling and ion transport across the fish gill epithelium. J. Exp. Zool. 293,336 -347.[CrossRef][Medline]
Evans, D. H., Rose, R. E., Roeser, J. M. and Stidham, J. D. (2004). NaCl transport across the opercular epithelium of Fundulus heteroclitus is inhibited by an endothelin to NO, superoxide, and prostanoid signaling axis. Am. J. Physiol. 286,R560 -R568.
Flatman, P. W. (2002). Regulation of Na-K-2Cl cotransport by phosphorylation and protein-protein interactions. Biochim. Biophys. Acta 1566,140 -151.[Medline]
Flemmer, A. W., Gimenez, I., Dowd, B. F., Darman, R. B. and
Forbush, B. (2002). Activation of the Na-K-Cl cotransporter
NKCC1 detected with a phospho-specific antibody. J. Biol.
Chem. 277,37551
-37558.
Gelman, I. H. (2003). Pyk 2 FAKs, any two FAKs. Cell Biol. Int. 27,507 -510.[CrossRef][Medline]
Haas, M. and Forbush, B., 3rd (1998). The Na-K-Cl cotransporters. J. Bioenerget. Biomemb. 30,161 -172.[CrossRef][Medline]
Hanks, S. K., Ryzhova, L., Shin, N. Y. and Brabek, J. (2003) Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility. Front. Biosci. 8,d982 -d996.[Medline]
Hauck, C. R., Hsia, D. A. and Schlaepfer, D. D. (2002). The focal adhesion kinase - a regulator of cell migration and invasion. IUBMB Life 53,115 -119.[Medline]
Hoffmann, E. K. (2000). Intracellular signaling involved in volume regulatory decrease. Cell Physiol. Biochem. 10,273 -288.[CrossRef][Medline]
Hoffmann, E. K. and Dunham, P. B. (1995). Membrane mechanisms and intracellular signaling in cell volume regulation. Int. Rev. Cytol. 162,173 -262.
Hoffmann, E. K. and Simonsen, L. O. (1989)
Membrane mechanisms in volume and pH regulaton in vertebrate cells.
Physiol. Rev. 69,315
-382.
Hoffmann, E. K., Hoffmann, E., Lang, F. and Zadunaisky, J. A. (2002). Control of Cl- transport in the opercular epithelium of Fundulus heteroclitus: long- and short-term salinity adaptation. Biochim. Biophys. Acta 1566,129 -139.[Medline]
Huang, H. B., Horiuchi, A., Goldberg, J., Greengard, P. and
Nairn, A. C. (1997). Site-directed mutagenesis of amino acid
residues of protein phosphatase 1 involved in catalysis and inhibitor binding.
Proc. Natl. Acad. Sci. USA
94,3530
-3535.
Igishi, T., Fukuhara, S., Patel, V., Katz, B. Z., Yamada, K. M.
and Gutkind, J. S. (1999). Divergent signaling pathways link
focal adhesion kinase to mitogen-activated protein kinase cascades. Evidence
for a role of paxillin in c-Jun NH(2)-terminal kinase activation.
J. Biol. Chem. 274,30738
-30746.
Johnston, A. M., Naselli, G., Gonez, L. J., Martin, R. M., Harrison, L. C. and DiAizpurua, H. J. (2000). SPAK, a STE20/SPS1-related kinase that activates the p38 pathway. Oncogene 19,4290 -4297.[CrossRef][Medline]
Keenan, C., Goode, N. and Pears, C. (1997). Isoform specificity of activators and inhibitors of protein kinase C gamma and delta. FEBS Lett. 415,101 -108.[CrossRef][Medline]
Krarup, T., Jakobsen, L. D., Jensen, B. S. and Hoffmann, E. K. (1998). Na-K-2Cl cotransport in Ehrlich cells: Regulation by protein phosphatases and kinases. Am. J. Physiol. 275,C239 -C250.[Medline]
Kültz, D. and Avila, K. (2001). Mitogen activated protein kinases are in vivo transducers of osmosensory signals in fish gill cells. Comp. Biochem. Physiol. B 129,821 -829.[CrossRef][Medline]
Lee, S. K., Qing, W. G., Mar, W., Luyengi, L., Mehta, R. G.,
Kawanishi, K., Fong, H. H., Beecher, C. W., Kinghorn, A. D. and Pezzuto, J.
M. (1998). Angoline and chelerythrine, benzophenanthridine
alkaloids that do not inhibit protein kinase C. J. Biol.
Chem. 273,19829
-19833.
Lehrich, R. W. and Forrest, J. N., Jr (1995). Tyrosine phosphorylation is a novel pathway for regulation of chloride secretion in shark rectal gland. Am. J. Physiol. 269,F594 -F600.[Medline]
Lehrich, R. W., Aller, S. G., Webster, P., Marino, C. R. and
Forrest, J. N., Jr (1998). Vasoactive intestinal peptide,
forskolin, and genistein increase apical CFTR trafficking in the rectal gland
of the spiny dogfish, Squalus acanthias. Acute regulation of CFTR
trafficking in an intact epithelium. J. Clin. Invest.
101,737
-745.
Liedtke, C. M. and Thomas, L. (1996). Phorbol ester and okadaic acid regulation of Na-2Cl-K cotransport in rabbit tracheal epithelial cells. Am. J. Physiol. 271,C338 -C346.[Medline]
Liedtke, C. M., Hubbard, M. and Wang, X. (2003). Stability of actin cytoskeleton and PKC-delta binding to actin regulate NKCC1 function in airway epithelial cells. Am. J. Physiol. 284,C487 -C496.
Lionetto, M. G., Pedersen, S. F., Hoffmann, E. K., Giordano, M. E. and Schettino, T. (2002). Roles of the cytoskeleton and of protein phosphorylation events in the osmotic stress response in eel intestinal epithelium. Cell Physiol. Biochem. 12,163 -178.[CrossRef][Medline]
Liu, X., Zhang, M. I., Peterson, L. B. and O'Neil, R. G. (2003). Osmomechanical stress selectively regulates translocation of protein kinase C isoforms. FEBS Lett. 538,101 -106.[CrossRef][Medline]
Lytle, C., Xu, J. C., Biemsderfer, D., Haas, M. and Forbush, B.,
3rd (1992). The Na-K-Cl cotransport protein of shark rectal
gland. I. Development of monoclonal antibodies, immunoaffinity purification
and partial biochemical characterization. J. Biol.
Chem. 267,25428
-25437.
Marshall, W. S. (1981). Sodium dependency of active chloride transport across isolated fish skin (Gillichthys mirabilis). J. Physiol. Lond. 319,165 -178.[Abstract]
Marshall, W. S. (2003). Rapid regulation of NaCl secretion by estuarine teleost fish: Coping strategies for short duration fresh water exposures. Biochim. Biophys. Acta 1618,95 -105.[Medline]
Marshall, W. S. and Bryson, S. E. (1998). Transport mechanisms of seawater chloride cells: An inclusive model of a multifunctional cell. Comp. Biochem. Physiol. 119A,97 -106.
Marshall, W. S., Bryson, S. E. and Garg, D.
(1993). 2-adrenergic inhibition of chloride
transport by opercular epithelium is mediated by intracellular
Ca2+. Proc. Natl. Acad. Sci. USA
90,5504
-5508.
Marshall, W. S., Bryson, S. E., Midelfart, A. and Hamilton, W. F. (1995). Low conductance anion channel activated by cAMP in teleost Cl- secreting cells. Am. J. Physiol. 268,R963 -R969.[Medline]
Marshall, W. S., Duquesnay, R. M., Gillis, J. M., Bryson, S. E.
and Liedtke, C. M. (1998). Neural modulation of salt
secretion in teleost opercular epithelium by 2-adrenergic
receptors and inositol 1,4,5-trisphosphate. J. Exp.
Biol. 201,1959
-1965.
Marshall, W. S., Bryson, S. E. and Luby, T.
(2000). Control of epithelial Cl- secretion by
basolateral osmolality in euryhaline teleost Fundulus heteroclitus.
J. Exp. Biol. 203,1897
-1905.
Marshall, W. S., Lynch, E. M. and Cozzi, R. R. F.
(2002). Redistribution of immunofluorescence of CFTR anion
channel and NKCC cotransporter in chloride cells during adaptation of the
killifish Fundulus heteroclitus to sea water. J. Exp.
Biol. 205,1265
-1273.
Millward, T. A., Zolnierowicz, S. and Hemmings, B. A. (1999). Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem. Sci. 24,186 -191.[CrossRef][Medline]
Oktay, M., Wary, K. K., Dans, M., Birge, R. B. and Giancotti, F.
G. (1999). Integrin-mediated activation of focal adhesion
kinase is required for signaling to Jun NH2-terminal kinase and progression
through the G1 phase of the cell cycle. J. Cell Biol.
145,1461
-1469.
Pandey, P., Avraham, S., Kumar, S., Nakazawa, A., Place, A.,
Ghanem, L., Rana, A., Kumar, V., Majumder, P. K., Avraham, H., Davis, R. J.
and Kharbanda, S. (1999). Activation of p38 mitogen-activated
protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent
mechanism. J. Biol. Chem.
274,10140
-10144.
Pedersen, S. F., Varming, G., Christensen, S. T. and Hoffmann, E. K. (2002). Mechanisms of activation of NHE by cell shrinkage and by Calyculin A in Ehrlich ascites tumour cells. J. Memb. Biol. 189,67 -81.[CrossRef][Medline]
Piechotta, K., Lu, J. and Delpire, E. (2002).
Cation chloride cotransporters interact with the stress-related kinase
Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response
1 (OSR1). J. Biol. Chem.
277,50812
-50819.
Piechotta, K., Garbarini, N., England, R. and Delpire, E.
(2003). Characterization of the interaction of the stress kinase
SPAK with the Na+-K+-2Cl- cotransporter in
the nervous system: evidence for a scaffolding role of the kinase.
J. Biol. Chem. 278,52848
-52856.
Ray, R. M., Viar, M. J., McCormack, S. A. and Johnson, L. R. (2001). Focal adhesion kinase signaling is decreased in polyamine-depleted IEC-6 cells. Am. J. Physiol. 281,C475 -C485.
Roger, F., Martin, P. Y., Rousselot, M., Favre, H. and Feraille,
E. (1999). Cell shrinkage triggers the activation of
mitogen-activated protein kinases by hypertonicity in the rat kidney medullary
thick ascending limb of the Henle's loop. Requirement of p38 kinase for the
regulatory volume increase response. J. Biol. Chem.
274,34103
-34110.
Scott, G. R., Richards, J. G., Forbush, B., Isenring, P. and Schulte, P. M. (2004). Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer. Am. J. Physiol. 287,C300 -C309.[CrossRef]
Sheikh-Hamad, D., di Mari, J., Suki, W. N., Safirstein, R.,
Watts, B. A. 3rd and Rouse, D. (1998). p38 kinase activity is
essential for osmotic induction of mRNAs for HSP70 and transporter for organic
solute betaine in Madin-Darby canine kidney cells. J. Biol.
Chem. 273,1832
-1837.
Shen, M. R., Chou, C. Y., Browning, J. A., Wilkins, R. J. and
Ellory, J. C. (2001). Human cervical cancer cells use
Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in
regulatory volume decrease. J. Physiol.
537,347
-362.
Singer, T. D., Tucker, S. J., Marshall, W. S. and Higgins, C. F. (1998). A divergent CFTR homologue: Highly regulated salt transport in the euryhaline teleost Fundulus heteroclitus. Am. J. Physiol. 274,C715 -C723[Medline]
Sundin, L. and Nilsson, S. (2002). Branchial innervation J. Exp. Zool. 293,232 -248.[CrossRef][Medline]
Takei, M., Mitsui, H. and Endo, K. (1993). Effect of okadaic acid on histamine release from rat peritoneal mast cells activated by anti-IgE. J. Pharm. Pharmacol. 45,750 -752.[Medline]
Ushiro, H., Tsutsumi, T., Suzuki, K., Kayahara, T. and Nakano, K. (1998). Molecular cloning and characterization of a novel Ste20-related protein kinase in neurons and transporting epithelia. Arch. Biochem. Biophys. 355,233 -240.[CrossRef][Medline]
Van Praag, D., Farber, S. J., Minkin, E. and Primor, N. (1987). Production of eicosanoids by the killifish gills and opercular epithelia and their effect on active transport of ions. Gen. Comp. Endocrinol. 67, 50-57.[Medline]
vom Dahl, S., Schliess, F., Graf, D. and Haussinger, D. (2001). Role of p38(MAPK) in cell volume regulation of perfused rat liver. Cell Physiol. Biochem. 11,285 -294.[CrossRef][Medline]
vom Dahl, S., Schliess, F., Reissmann, R., Gorg, B.,
Weiergraber, O., Kocalkova, M., Dombrowski, F. and Haussinger, D.
(2003). Involvement of integrins in osmosensing and signaling
toward autophagic proteolysis in rat liver. J. Biol.
Chem. 278,27088
-27095.
Wang, J. G., Miyazu, M., Matsushita, E., Sokabe, M. and Naruse, K. (2001). Uniaxial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation. Biochem. Biophys. Res. Commun. 288,356 -361.[CrossRef][Medline]
Way, K. L., Chou, E. and King, G. L. (2000). Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol. Sci. 21,181 -187.[CrossRef][Medline]
Wilson, J. M., Randall, D. J., Donowitz, M., Vogl, A. W. and Ip,
A. K. Y. (2000). Immunolocalization of ion transport proteins
to branchial epithelium mitochondria-rich cells in the mudskipper
(Periophthalmodon schlosseri). J. Exp. Biol.
203,2297
-2310.
Zadunaisky, J. A., Cardona, S., Au, L., Roberts, D. M., Fisher, B. Lowenstein, B., Cragoe, E. J., Jr and Spring, K. R. (1995). Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus. J. Memb. Biol. 143,207 -217.[Medline]
Zadunaisky, J. A., Balla, M. and Colon, D. E. (1997). A reduction in chloride secretion by lowered osmolality in chloride cells of Fundulus heteroclitus. Bull. Mt. Desert Isl. Biol. Lab. 24,52 (abstract).
Zadunaisky, J. A., Hoffmann, E. K., Colon, E., Hoffmann, E. and Einhorn, J. (1998). Volume activated chloride secretion in operculum epithelium of Fundulus heteroclitus. Bull. Mt. Desert Isl. Biol. Lab. 37,74 -75.
Zhuang, S., Hirai, S. I. and Ohno, S. (2000). Hyperosmolality induces activation of cPKC and nPKC, a requirement for ERK1/2 activation in NIH/3T3 cells. Am. J. Physiol. 278,C102 -C109.