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Tyrosine kinase regulates epithelial sodium transport in A6 cells
P S Matsumoto1, A Ohara, P Duchatelle
1Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322.
Abstract:
Insulin increases epithelial Na+ reabsorption, and many of its actions involve tyrosine kinase. We used tyrosine kinase inhibitors to examine the role of tyrosine kinase in the action of insulin. Pretreatment of Na+ transporting cells with tyrosine kinase inhibitors attenuates the subsequent action of insulin, suggesting that the action of insulin on epithelial Na+ transport involves tyrosine kinase activity. In addition to their effect on insulin-induced Na+ transport, the tyrosine kinase inhibitors also significantly reduce Na+ transport in Na(+)-transporting epithelial cells, suggesting that there is a significant tonic tyrosine kinase activity that modulates epithelial Na+ transport. Using patch-clamp methods, we found that one inhibitor, genistein, reduces the number of active Na+ channels in cell-attached patches without significantly affecting the open probability of any remaining channels. The effects of the tyrosine kinase inhibitors are not due to inhibition of protein kinase A (PKA), since H89, a PKA inhibitor, does not affect Na+ transport of control cells (as the tyrosine kinase inhibitors do), and the tyrosine kinase inhibitor, genistein or tyrphostin 23, does not alter the stimulation of ion transport by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, a membrane-permeable adenosine 3',5'-cyclic monophosphate analogue (as H89 does).
Insights
Insulin
Area of Science:
- Cellular Biology
- Molecular Physiology
- Biochemistry
Background:
- Insulin is known to enhance epithelial sodium (Na+) reabsorption.
- Many cellular processes regulated by insulin involve tyrosine kinase signaling pathways.
Purpose of the Study:
- To investigate the role of tyrosine kinase activity in mediating insulin's effects on epithelial Na+ transport.
- To determine if tyrosine kinase activity is essential for insulin-stimulated Na+ reabsorption.
Main Methods:
- Utilized tyrosine kinase inhibitors (genistein, tyrphostin 23) to block tyrosine kinase activity in Na+-transporting epithelial cells.
- Employed patch-clamp electrophysiology to analyze Na+ channel activity.
- Investigated the involvement of protein kinase A (PKA) by using the PKA inhibitor H89 and a cAMP analogue.
Main Results:
- Tyrosine kinase inhibitors attenuated insulin's effect on Na+ reabsorption, indicating tyrosine kinase involvement.
- These inhibitors also reduced basal Na+ transport, suggesting tonic tyrosine kinase activity.
- Genistein decreased the number of active Na+ channels without altering their open probability.
- Results demonstrated that tyrosine kinase inhibitors do not affect PKA-mediated ion transport.
Conclusions:
- Insulin-stimulated epithelial Na+ reabsorption critically depends on tyrosine kinase activity.
- A tonic level of tyrosine kinase activity continuously modulates epithelial Na+ transport.
- The mechanism involves regulating the number of active Na+ channels, not their gating properties.