Multiple mitogen-activated protein kinases are regulated by
hyperosmolality in mouse imcd cells.
Berl, Tomas, Gamini Siriwardana, Lili Ao, Laura M. Butterfield, and
Lynn E. Heasley.
Department of Medicine, University of Colorado School of Medicine,
Denver, Colorado 80262
APStracts 3:0203F, 1996.
Inner medullary collecting duct (IMCD) cells adapt to a hypertonic
environment by synthesizing transporters that allow for accumulation
of organic osmolytes. To examine for activation of additional MAP
kinases, extracts of IMCD-3 cells subjected to a hypertonic medium
(600 mOsm) for 15 min were fractionated by Mono Q FPLC and assayed
with the EGFR662-681 peptide as substrate. Three peaks of activity
were identified. Western blotting revealed that these peaks coincided
with Jun N-terminal kinase (JNK), ERK1 and ERK2 and p38 MAP kinase.
To assess the functional significance of ERK2 activation in IMCD-3
cells, the effect of PD098059, an inhibitor of the upstream
regulatory protein kinase MAP/ERK kinase (MEK), was assessed.
PD098059 inhibited ERK activation by hypertonicity. Yet, the
stimulation of inositol uptake, a marker of adaptation, after 16 hr
was unaltered. Direct measurements of JNK activity (phosphorylation
of GST-cJun 1-79) revealed a marked (20 to 40-fold) increase in
activity as medium osmolality was increased from 300 to 900 mOsm with
either NaCl or mannitol. Urea induced a more modest increase in
activity. The response is prompt and detected as early as 2 min after
exposure, reaching a maximum activation at 10-15 min. Downregulation
of cellular PKC by chronic exposure to phorbol esters only minimally
attenuated the JNK response to hyperosmolality, indicating a lack of
involvement of PKC. We conclude that in IMCD-3 cells, inhibition of
ERK activation by hyperosmolality does not prevent osmoregulatory
increase in inositol transport. This is not consistent with a role
for ERKs in the response. The roles for JNK and p38 have not been
ruled out and these pathways may represent the initiating event in
the subsequent transcription of organic osmolyte transporter genes
and adaptation to extracellular hypertonicity.
Received 26 June 1996; accepted in final form 28 October 1996.
APS Manuscript Number F181-6.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 13 November 1996