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
*
Author for correspondence (e-mail:
bmarshal{at}stfx.ca)
Accepted 11 January 2005
Hypotonic shock rapidly inhibits Cl- secretion by chloride
cells, an effect that is osmotic and not produced by NaCl-depleted isosmotic
solutions, yet the mechanism for the inhibition and its recovery are not
known. We exposed isolated opercular epithelia, mounted in Ussing chambers, to
hypotonic shock in the presence of a variety of chemicals: a general protein
kinase C (PKC) inhibitor chelerythrine, Gö6976 that selectively blocks
PKC
and ß subtypes, H-89 that blocks PKA, SB203580 that blocks p38
mitogen-activated protein kinase (MAPK), as well as serine/threonine protein
phosphatase (PP1 and 2A) inhibitor okadaic acid, and finally tamoxifen, a
blocker of volume-activated anion channels (VSOAC). Chelerythrine has no
effect on hypotonic inhibition but blocked the recovery, indicating PKC
involvement in stimulation. Gö6976 had little effect, suggesting that
PKC and PKCß subtypes are not involved. H-89 did not block
hypotonic inhibition but decreased the recovery, indicating PKA may be
involved in the recovery and overshoot (after restoration of isotonic
conditions). SB203580 significantly enhanced the decrease in current by
hypotonic shock, suggesting an inhibitory role of p38 MAPK in the hypotonic
inhibition. Okadaic acid increased the steady state current, slowed the
hypotonic inhibition but made the decrease in current larger; also the
recovery and overshoot were completely blocked. Hypotonic stress rapidly and
transiently increased phosphorylated p38 MAPK (pp38) MAPK (measured by western
analysis) by eightfold at 5 min, then more slowly again to sevenfold at 60
min. Hypertonic shock slowly increased p38 by sevenfold at 60 min.
Phosphorylated JNK kinase was increased by 40-50% by both hypotonic and
hypertonic shock and was still elevated at 30 min in hypertonic medium. By
immunoblot analysis it was found that the stress protein kinase (SPAK) and
oxidation stress response kinase 1 (OSR1) were present in salt and freshwater
acclimated fish with higher expression in freshwater. By immunocytochemistry,
SPAK, OSR1 and phosphorylated focal adhesion kinase (pFAK) were colocalized
with NKCC at the basolateral membrane. The protein tyrosine kinase inhibitor
genistein (100 µmol l-1) inhibited Cl- secretion that
was high, increased Cl- secretion that was low and reduced
immunocytochemical staining for phosphorylated FAK. We present a model for
rapid control of CFTR and NKCC in chloride cells that includes: (1) activation
of NKCC and CFTR via cAMP/PKA, (2) activation of NKCC by PKC, myosin
light chain kinase (MLCK), p38, OSR1 and SPAK, (3) deactivation of NKCC by
hypotonic cell swelling, Ca2+ and an as yet unidentified protein
phosphatase and (4) involvement of protein tyrosine kinase (PTK) acting on FAK
to set levels of NKCC activity.
Key words: Fundulus heteroclitus, protein kinase, protein phosphatase, SB203580, regulatory volume decrease, okadaic acid, chelerythrine, NKCC1, Na+, K+, 2Cl- cotransport, gill epithelium
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