First published online March 14, 2005
Journal of Experimental Biology 208, 1063-1077 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01491
Hypotonic shock mediation by p38 MAPK, JNK, PKC, FAK, OSR1 and SPAK in osmosensing chloride secreting cells of killifish opercular epithelium
W. S. Marshall1,*,
C. G. Ossum2 and
E. K. Hoffmann2
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
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Fig. 1. Effect of hypotonic shock in isolated opercular epithelia mounted in
symmetrical saline. (A) Example trace of time course of hypotonic shock on
Vt, a 1 h duration period in hypotonic conditions and
overshoot after restoration of isotonic conditions. (B) The size of the
overshoot is a function of the period of hypotonic exposure; short periods
yield no overshoot, longer periods yield larger overshoot in current,
indicative of regulatory volume decrease during long hypotonic exposure.
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Fig. 2. The cycloxygenase inhibitor indomethacin (0.1 mmol l-1) had no
detectable effect on the hypotonic response (HYPO) and no effect on the
current rebound when isotonic conditions were restored (ISO).
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Fig. 3. The protein kinase C inhibitor chelerythrine (Chel) significantly inhibited
membrane current compared to vehicle (Veh) treatment
(**P<0.01, paired t-test) and significantly
reduced the recovery of current after isotonic (ISO) solutions were restored
(*P<0.05, paired t-test).
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Fig. 4. The p38 MAPK inhibitor SB203580 had no effect on current (A) compared to
the vehicle-treated membranes (Drug/Veh), but SB203580 significantly enhanced
the inhibition of current by hypotonic shock (HYPO) and significantly reduced
the recovery of current after restoration of isotonic conditions (ISO;
*P<0.05, paired t-test). The transepithelial
resistance (B) change mirrors the current changes, with SB203580 increasing
the resistance rise compared to vehicle (*P<0.05,
paired t-test) seen with hypotonic shock.
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Fig. 5. Immunoblot of protein form opercular epithelia treated for different times
with isotonic solution (control), hypotonic saline (Hypo) and hypertonic
saline (Hyper). (A) Upper panel: p38 MAPK (38-40 kDa) was detected by antibody
to any form of the protein. Lower panel: p38 MAPK detected by the antibody
specific to the phosphorylated form of the protein. (B) Bar charts of
quantitative scans averaged over three immunoblots where p38 MAPK
phosphorylation was enhanced significantly (*P<0.05,
paired t-test compared to isotonic control level at the respective
time, N=3) by hypotonic shock at 5 min, by hypertonic shock at 30 min
and by both hypertonic and hypotonic shock in a second phase response at 60
min.
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Fig. 6. The p38 MAPK is present in opercular epithelium and gill epithelial tissue
in fully acclimated freshwater and seawater animals. Scans averaged over three
immunoblots (top) indicate a lower level of expression of p38 MAPK (bottom) in
seawater relative to freshwater levels in both gill and opercular epithelium
(*P<0.05, t-test, N=3).
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Fig. 7. The protein phosphatase inhibitor okadaic acid significantly increased
membrane current initially (*P<0.05, paired
t-test), had a marginal potentiating effect on the magnitude of the
hypotonic inhibition of current (*P<0.05, paired
t-test) and entirely blocked the recovery of current
(**P<0.02, paired t-test) after restoration of
isosmotic solutions.
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Fig. 9. (A) Immunoblots of phosphorylated JNK (phospho-JNK) were similar to those
for p38 MAPK (Fig. 5) but were
normalized to the control values to remove a tendency for pJNK to increase
with time. (B) There was a significant rise in pJNK in both hyper- (bottom)
and hypotonic (top) shock at 5 min (*P<0.05,
t-test, N=3) and with hypertonic shock at 30 min
(*P<0.05), compared to the isotonic controls at the
same time.
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Fig. 10. Immunoblot of the stress-associated proteins OSR1 (A) and SPAK (B),
comparing expression in freshwater (FW)- and seawater (SW)-acclimated animals.
Quantitative western analysis revealed significantly higher level of OSR1 and
SPAK expression in FW- as compared to SW-acclimated animals for the gill and
opercular epithelium (oe; N=3, *P<0.05) with
the difference in OSR1 expression being more marked than that for SPAK.
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Fig. 11. Immunocytochemistry for NKCC (T4 mouse anti-hNKCC antibody with goat
anti-mouse Oregon Green 488) and for SPAK (rabbit anti-SPAK polyclonal with
goat anti-rabbit Alexa Fluor 594) visualized by confocal microscopy. (A)
Anti-NKCC alone (green) (B) Anti-SPAK alone (red) (C) Same field as A and B
but with both channels activated (red/green); exact colocalization is yellow.
NKCC and SPAK appear in the same mitochondria-rich cells, not in the apical
membrane but lower in the cell (here at the plane of the nuclei) and, in most
areas are exactly colocalized (yellow in C). (D) A line scan of fluorescence
intensity (arbitrary units) versus distance in µm across a cell
(indicated by the arrow in C) shows good correspondence in the colocalization
of SPAK (red line) and NKCC (green line) in the cytosol (peaks) and not in the
nucleus (central region). (E-H) As A-D except that the primary antibody for
the kinase was rabbit anti-OSR1 polyclonal, not SPAK. Bars, 20 µm.
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Fig. 12. Immunocytochemistry for phosphorylated focal adhesion kinase (pFAK)
(primary: rabbit anti-phosphorylated human FAK; secondary: goat anti-rabbit
Oregon Green 488) and for NKCC (primary: T4 mouse anti-hNKCC; secondary: goat
anti-mouse Alexa Fluor 594). (A) pFAK immunofluorescence was present in all
mitochondria-rich cells (green). (B) NKCC immunofluorescence (red) had a
similar distribution to pFAK. (C) There was a high degree of colocalization of
NKCC and phosphorylated pFAK (yellow). (D) Genistein pretreatment (100 µmol
l-1, for 1 h) to inhibit tyrosine kinase before addition of the
primary anti-pFAK antibody eliminated pFAK immunofluorescence. Bar, 20
µm.
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© The Company of Biologists Ltd 2005
2 adrenergic agonist,
1.0 µmol l-1), genistein instead increased the current to an
intermediate level (*P<0.05). The inactive analogue
daidzein (0.1 mmol l-1) has no inhibitory effect
(Marshall et al., 2000