Urea signaling to ERK phosphorylation in renal medullary cells requires extracellular calcium but not calcium entry

The renal cell line mIMCD3 exhibits markedly upregulated phosphorylation of the extracellular signal-regulated kinase (ERK) 1 and 2 in response to ure a treatment (200 mM for 5 min). Previous data have suggested the involvemen t of a classical protein kinase C (cPKC)-dependent pathway in downstream ev ents related to urea signaling. We now show that urea-inducible ERK activat ion requires extracellular calcium; unexpectedly, it occurs independently o f activation of cPKC isoforms. Pharmacological inhibitors of known intracel lular calcium release pathways and extracellular calcium entry pathways fai l to inhibit ERK activation by urea. Fura 2 ratiometry was used to assess t he effect of urea treatment on intracellular calcium mobilization. In singl e-cell analyses using subconfluent monolayers and in population-wide analys es using both confluent monolayers and cells in suspension, urea failed to increase intracellular calcium concentration. Taken together, these data in dicate that urea-inducible ERK activation requires calcium action but not c alcium entry. Although direct evidence is lacking, one possible explanation could include involvement of a calcium-dependent extracellular moiety of a cell surface-associated protein.