Tyrosine phosphorylation is a novel pathway for regulation of chloride secretion in shark rectal gland.
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)
D000818:Animals; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology; D008297:Male; D010455:Peptides / Q000378:metabolism; D010477:Perfusion; D010766:Phosphorylation / Q000187:drug effects; D012491:Salt Gland / Q000378:metabolism*; D012754:Sharks / Q000378:metabolism*; D014443:Tyrosine / Q000037:antagonists & inhibitors / Q000378:metabolism*
D002712:Chlorides; D007529:Isoflavones; D010455:Peptides; D014443:Tyrosine; D019833:Genistein; D000242:Cyclic AMP