Protein tyrosine kinase involvement in the regulation of cardiac chloride channels.
Date
1996
Authors
Shuba, Lesya.
Journal Title
Journal ISSN
Volume Title
Publisher
Dalhousie University
Abstract
Description
Protein tyrosine kinase (PTK) inhibitor genistein (GST), inactive analogue daidzein, and phosphotyrosine phosphatase (PTPase) inhibitor Na$\sb3\rm VO\sb4(VO\sb4$) were used to modify tyrosine phosphorylation in guinea pig ventricular myocytes configured for recording of whole-cell Cl$\sp-$ current ($I\sb{\rm Cl}$).
Inactive daidzein failed to activate significant $I\sb{\rm Cl}$, and GST-activated $I\sb{\rm Cl}$ was rapidly and reversibly inhibited by 0.1-1 mM VO$\sb4$.
$I\sb{\rm Cl}$ activated by GST had biophysical and pharmacological properties similar to those of protein kinase A (PKA)-regulated, cystic fibrosis transmembrane conductance regulator (CFTR) $I\sb{\rm Cl}$ activated by forskolin (FSK). Both FSK-activated $I\sb{\rm Cl}$ and GST-activated $I\sb{\rm Cl}$ were absent in CFTR-lacking rat ventricular myocytes.
The concentration of GST required for half-maximal activation ($EC\sb{50}$) of Cl$\sp-$ conductance ($g\sb{\rm Cl}$) by GST was $\sim$93 $\mu$M, and $g\sb{\rm Cl}$ activated by 500 $\mu$M GST (9.4 $\pm$ 1.9 nS) was as large as that (8.3 $\pm$ 1 nS) activated by maximally-effective 5 $\mu$M FSK.
Submaximally-effective concentrations of GST and FSK activated $I\sb{\rm Cl}$ in a synergistic manner. GST (50 $\mu$M) incremented $I\sb{\rm Cl}$ pre-activated by 5 $\mu$M FSK or 1 mM cAMP dialysate, but not $I\sb{\rm Cl}$ pre-activated by FSK in myocytes treated with serine/threonin phosphatase (PSPase) inhibitor okadaic acid (OA). However, GST action was enhanced in non-FSK-treated OA-dialysed myocytes.
These findings, and others from experiments in which PKA activity prior to GST application was either stimulated or depressed, indicate that the activation of $I\sb{\rm Cl}$ by GST is not mediated via stimulation of the PKA pathway, or via inhibition of PSPase. The GST-induced reduction in tyrosine phosphorylation appears to promote CFTR channel opening by enhancing the phosphorylation of channel PKA-sites, and/or by relieving strong tonic inhibition of channel opening caused by tyrosine phosphorylation on a channel or extra-channel moiety.
Thesis (Ph.D.)--Dalhousie University (Canada), 1996.
Inactive daidzein failed to activate significant $I\sb{\rm Cl}$, and GST-activated $I\sb{\rm Cl}$ was rapidly and reversibly inhibited by 0.1-1 mM VO$\sb4$.
$I\sb{\rm Cl}$ activated by GST had biophysical and pharmacological properties similar to those of protein kinase A (PKA)-regulated, cystic fibrosis transmembrane conductance regulator (CFTR) $I\sb{\rm Cl}$ activated by forskolin (FSK). Both FSK-activated $I\sb{\rm Cl}$ and GST-activated $I\sb{\rm Cl}$ were absent in CFTR-lacking rat ventricular myocytes.
The concentration of GST required for half-maximal activation ($EC\sb{50}$) of Cl$\sp-$ conductance ($g\sb{\rm Cl}$) by GST was $\sim$93 $\mu$M, and $g\sb{\rm Cl}$ activated by 500 $\mu$M GST (9.4 $\pm$ 1.9 nS) was as large as that (8.3 $\pm$ 1 nS) activated by maximally-effective 5 $\mu$M FSK.
Submaximally-effective concentrations of GST and FSK activated $I\sb{\rm Cl}$ in a synergistic manner. GST (50 $\mu$M) incremented $I\sb{\rm Cl}$ pre-activated by 5 $\mu$M FSK or 1 mM cAMP dialysate, but not $I\sb{\rm Cl}$ pre-activated by FSK in myocytes treated with serine/threonin phosphatase (PSPase) inhibitor okadaic acid (OA). However, GST action was enhanced in non-FSK-treated OA-dialysed myocytes.
These findings, and others from experiments in which PKA activity prior to GST application was either stimulated or depressed, indicate that the activation of $I\sb{\rm Cl}$ by GST is not mediated via stimulation of the PKA pathway, or via inhibition of PSPase. The GST-induced reduction in tyrosine phosphorylation appears to promote CFTR channel opening by enhancing the phosphorylation of channel PKA-sites, and/or by relieving strong tonic inhibition of channel opening caused by tyrosine phosphorylation on a channel or extra-channel moiety.
Thesis (Ph.D.)--Dalhousie University (Canada), 1996.
Keywords
Biology, Animal Physiology.