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Tyrosine phosphorylation is a novel pathway for regulation of chloride secretion in shark rectal gland
1Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Abstract:
We used the specific tyrosine kinase inhibitor genistein to define the involvement of tyrosine phosphorylation in the regulation of chloride transport in the rectal gland of the dogfish shark, a model for chloride secretion via a cystic fibrosis transmembrane conductance regulator (CFTR)-like channel. In the perfused gland, genistein (100 microM) promptly increased chloride secretion from basal values of 159 +/- 36 to 966 +/- 49 mueq.h-1.g-1 (P < 0.0001). Bumentanide fully reversed genistein-induced secretion. In primary culture monolayers of rectal gland tubular cells, genistein, but not the inactive 7-glucoside form, genistin, increased short-circuit current in a dose-dependent manner, from basal values of 2.7 +/- 4.3 to 104 +/- 10 microA/cm2 (P < 0.0001). Apically applied genistein induced significantly greater chloride secretion than basolateral addition. Genistein did not increase the adenosine 3',5'-cyclic monophosphate (cAMP) content of either perfused glands or cultured monolayers. Using an anti-phosphotyrosine antibody, we observed phosphorylation of multiple proteins. Four peptides, with molecular masses of 250, 210, 55, and 53 kDa, responded to genistein treatment with a decrease in tyrosine phosphorylation. These data demonstrate the following: 1) genistein induces bumetanide-sensitive chloride secretion in both perfused rectal glands and cultured tubular cells; 2) these effects are not accompanied by an elevation of tissue cAMP, indicating that genistein-induced secretion is not mediated by the cAMP-protein kinase A pathway; and 3) genistein-sensitive peptides are present in the rectal gland cell and are candidates for involvement in the regulation of chloride secretion.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Genistein, a tyrosine kinase inhibitor, stimulates chloride secretion in shark rectal glands, independent of cAMP signaling. This suggests tyrosine phosphorylation plays a key role in regulating chloride transport via CFTR-like channels.
Area of Science:
- Physiology
- Molecular Biology
- Cell Biology
Background:
- The dogfish shark rectal gland serves as a model for studying chloride secretion.
- Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-like channels are involved in this process.
- The role of tyrosine phosphorylation in regulating chloride transport is not fully understood.
Purpose of the Study:
- To investigate the involvement of tyrosine phosphorylation in regulating chloride transport in the dogfish shark rectal gland.
- To determine if genistein, a tyrosine kinase inhibitor, affects chloride secretion.
- To explore the signaling pathways involved in genistein-induced chloride secretion.
Main Methods:
- Utilized perfused rectal glands and primary culture monolayers of rectal gland tubular cells.
- Administered genistein and bumetanide to assess chloride secretion.
- Measured short-circuit current and tissue cyclic adenosine monophosphate (cAMP) levels.
- Employed anti-phosphotyrosine antibodies to detect protein phosphorylation.
Main Results:
- Genistein significantly increased chloride secretion in both perfused glands and cell monolayers.
- Genistein-induced secretion was sensitive to bumetanide, indicating involvement of Na-K-2Cl cotransporters.
- Secretion was not associated with increased cAMP levels, suggesting a cAMP-independent pathway.
- Genistein treatment led to decreased tyrosine phosphorylation of specific proteins (250, 210, 55, and 53 kDa).
Conclusions:
- Genistein effectively induces bumetanide-sensitive chloride secretion in the shark rectal gland.
- The mechanism of genistein-induced secretion is independent of the cAMP-protein kinase A pathway.
- Specific tyrosine phosphorylated proteins in the rectal gland are likely involved in regulating chloride transport.