Abstract
Genistein, a protein tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) in transfected NIH-3T3 fibroblasts that express the CFTR (3T3-CFTR). CFTR activity was assayed by 125I efflux and by patch clamping in the cell-attached mode. Both forskolin and genistein stimulated 125I efflux and activated a 9-10 pS anion channel in 3T3-CFTR cells but failed to activate 125I efflux in mock-transfected NIH-3T3 cells. Genistein, unlike forskolin and 3-isobutyl- 1-methylxanthine, did not increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) above control levels. This demonstrates that genistein- dependent activation does not involve inhibition of phosphodiesterase activity and suggests that stimulation does not involve a direct activation of protein kinase A. Genistein stimulated 125I efflux to ~50% of the maximal rate with forskolin. Genistein did not increase 125I efflux at saturating forskolin but decreased the concentration of forskolin required for half-maximal stimulation. Orthovanadate (VO4), a phosphotyrosine phosphatase inhibitor, inhibited genistein-induced channel activation with an inhibition constant of approximately 20 μM. These effects suggest that, in addition to activation by protein kinase A, the CFTR is regulated by a tyrosine kinase-dependent pathway.
| Original language | English (US) |
|---|---|
| Journal | American Journal of Physiology - Cell Physiology |
| Volume | 268 |
| Issue number | 4 37-4 |
| State | Published - 1995 |
| Externally published | Yes |
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Keywords
- 3-isobutyl-1- methylxanthine
- forskolin
- orthovanadate
- phosphodiesterase
- protein kinase A
ASJC Scopus subject areas
- Clinical Biochemistry
- Cell Biology
- Physiology
- Agricultural and Biological Sciences(all)
Cite this
cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein. / Illek, B.; Fischer, H.; Santos, G. F.; Widdicombe, Jonathan; Machen, T. E.; Reenstra, W. W.
In: American Journal of Physiology - Cell Physiology, Vol. 268, No. 4 37-4, 1995.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein
AU - Illek, B.
AU - Fischer, H.
AU - Santos, G. F.
AU - Widdicombe, Jonathan
AU - Machen, T. E.
AU - Reenstra, W. W.
PY - 1995
Y1 - 1995
N2 - Genistein, a protein tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) in transfected NIH-3T3 fibroblasts that express the CFTR (3T3-CFTR). CFTR activity was assayed by 125I efflux and by patch clamping in the cell-attached mode. Both forskolin and genistein stimulated 125I efflux and activated a 9-10 pS anion channel in 3T3-CFTR cells but failed to activate 125I efflux in mock-transfected NIH-3T3 cells. Genistein, unlike forskolin and 3-isobutyl- 1-methylxanthine, did not increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) above control levels. This demonstrates that genistein- dependent activation does not involve inhibition of phosphodiesterase activity and suggests that stimulation does not involve a direct activation of protein kinase A. Genistein stimulated 125I efflux to ~50% of the maximal rate with forskolin. Genistein did not increase 125I efflux at saturating forskolin but decreased the concentration of forskolin required for half-maximal stimulation. Orthovanadate (VO4), a phosphotyrosine phosphatase inhibitor, inhibited genistein-induced channel activation with an inhibition constant of approximately 20 μM. These effects suggest that, in addition to activation by protein kinase A, the CFTR is regulated by a tyrosine kinase-dependent pathway.
AB - Genistein, a protein tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) in transfected NIH-3T3 fibroblasts that express the CFTR (3T3-CFTR). CFTR activity was assayed by 125I efflux and by patch clamping in the cell-attached mode. Both forskolin and genistein stimulated 125I efflux and activated a 9-10 pS anion channel in 3T3-CFTR cells but failed to activate 125I efflux in mock-transfected NIH-3T3 cells. Genistein, unlike forskolin and 3-isobutyl- 1-methylxanthine, did not increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) above control levels. This demonstrates that genistein- dependent activation does not involve inhibition of phosphodiesterase activity and suggests that stimulation does not involve a direct activation of protein kinase A. Genistein stimulated 125I efflux to ~50% of the maximal rate with forskolin. Genistein did not increase 125I efflux at saturating forskolin but decreased the concentration of forskolin required for half-maximal stimulation. Orthovanadate (VO4), a phosphotyrosine phosphatase inhibitor, inhibited genistein-induced channel activation with an inhibition constant of approximately 20 μM. These effects suggest that, in addition to activation by protein kinase A, the CFTR is regulated by a tyrosine kinase-dependent pathway.
KW - 3-isobutyl-1- methylxanthine
KW - forskolin
KW - orthovanadate
KW - phosphodiesterase
KW - protein kinase A
UR - http://www.scopus.com/inward/record.url?scp=0028928015&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028928015&partnerID=8YFLogxK
M3 - Article
C2 - 7537452
AN - SCOPUS:0028928015
VL - 268
JO - American Journal of Physiology - Renal Fluid and Electrolyte Physiology
JF - American Journal of Physiology - Renal Fluid and Electrolyte Physiology
SN - 1931-857X
IS - 4 37-4
ER -