MULTIPLE MITOGEN-ACTIVATED PROTEIN-KINASES ARE REGULATED BY HYPEROSMOLALITY IN MOUSE IMCD CELLS

Inner medullary collecting duct (IMCD) cells adapt to a hypertonic env ironment by synthesizing transporters that allow for accumulation of o rganic osmolytes. To examine for activation of additional mitogen-acti vated protein (MAP) kinases, extracts of IMCD-3 cells subjected to a h ypertonic medium (600 mosmol/kgH(2)O) for 15 min were fractionated by Mono Q fast-performance liquid chromatography and assayed with the epi dermal growth factor receptor [EGFR-(662-681)] peptide as substrate. T hree peaks of activity were identified. Western blotting revealed that these peaks coincided with Jun NH2-terminal kinase (JNK), extracellul ar signal-regulated protein kinases, ERK1 and ERK2, and p38 MAP kinase . To assess the functional significance of ERK2 activation in IMCD-3 c ells, the effect of PD-098059, an inhibitor of the upstream regulatory protein kinase MAP/ERK kinase (MEK) was assessed. PD-098059 inhibited ERK activation by hypertonicity. Yet, the stimulation of inositol upt ake, a marker of adaptation, after 16 h was unaltered. Direct measurem ents of JNK activity [phosphorylation of GST-cJun-(1-79)] revealed a m arked (20- to 40-fold) increase in activity as medium osmolality was i ncreased from 300 to 900 mosmol/kgH(2)O with either NaCl or mannitol. Urea induced a more modest increase in activity. The response is promp t and detected as early as 2 min after exposure, reaching a maximum ac tivation at 10-15 min. Downregulation of cellular protein kinase C (PK C) 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 b y hyperosmolality does not prevent osmoregulatory increase in inositol transport. This is not consistent with a role for ERKs in the respons e. The roles for JNK and p38 have not been ruled out, and these pathwa ys may represent the initiating event in the subsequent transcription of organic osmolyte transporter genes and adaptation to extracellular hypertonicity.