Abstract
Bradykinin (BK) has been implicated in the regulation of renal function. Activation of extracellular signal-regulated protein kinase (ERK1/2) has been demonstrated in several models of toxic or proliferative renal injury. We studied activation of ERK1/2 by BK in a cell model of the most distal part of the nephron, inner medullary collecting duct (mIMCD-3) cells. Exposure of mIMCD-3 cells to BK (10−10–10−5 M) resulted in a concentration-dependent increase in tyrosine phosphorylation of ERK1/2, with maximal effect at 10−8 M BK. ERK1/2 activation by BK was observed as early as 1 min, peaked at 5 min, and was sustained at least for 1 h. The effect of BK was mediated by the B2receptor and was pertussis toxin-independent. Inhibition of phospholipase C, protein kinase C, or phosphatidylinositol 3-kinase did not alter ERK1/2 activation by BK. BK-induced ERK1/2 activation was Ca2+-calmodulin-independent but was sensitive to genistein, an inhibitor of tyrosine kinase(s). AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) kinase, completely blocked the effect of BK, suggesting an essential role of EGFR in ERK1/2 activation by BK. Immunoprecipitation/Western blot studies revealed that BK stimulated tyrosine phosphorylation of EGFR, its association with an adapter molecule Grb2, and complex formation between Grb2 and the adapter protein Shc. Activation studies of monomeric G protein Ras showed that BK-induced stimulation of Ras was dependent on EGFR tyrosine kinase activity. These studies demonstrate that BK stimulates Ras-dependent activation of ERK1/2 in mIMCD-3 cells via transactivation of EGFR through a novel mechanism.
Footnotes
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This work was supported by grants from the Department of Veterans Affairs (Merit Award to M.N.G. and an REAP Award to Y.V.M. and M.N.G.), and by Grant K01-DK02694 from the National Institutes of Health (to Y.V.M.).
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DOI: 10.1124/jpet.102.043943
- Abbreviations:
- BK
- bradykinin
- IMCD
- inner medullary collecting duct
- MAPK
- mitogen-activated protein kinase
- ERK
- extracellular signal-regulated protein kinase
- RTK
- receptor tyrosine kinase
- GPCR
- G protein-coupled receptor
- MEK
- mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
- PKC
- protein kinase C
- CaM
- calmodulin
- DMEM
- Dulbecco's modified Eagle's medium
- EGF
- epidermal growth factor
- EGFR
- epidermal growth factor receptor
- F-12
- Ham's F-12 nutrient mixture
- Grb2
- growth factor receptor binding protein 2
- Jak2
- Janus kinase 2
- LPA
- lysophosphatidic acid
- PI3K
- phosphatidylinositol 3-kinase
- PLC
- phospholipase C
- PMA
- phorbol 12-myristate 13-acetate
- PTX
- pertussis toxin
- RIPA
- radioimmune precipitation buffer
- Shc
- Src homology and collagen protein
- Sos
- son of sevenless (Ras exchange factor)
- VSMC
- vascular smooth muscle cells
- PAGE
- polacrylamide gel electrophoresis
- BAPTA
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
- TxA2
- thromboxane A2
- Received September 4, 2002.
- Accepted October 31, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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