Abstract
The biochemical cascade linking activation of phospholipase C-coupled thyrotropin-releasing hormone (TRH) receptors to rat ERG (r-ERG) channel modulation was studied in situ using perforated-patch clamped adenohypophysial GH3 cells and pharmacological inhibitors. To check the recent suggestion that Rho kinase is involved in the TRH-induced r-ERG current suppression, the hormonal effects were studied in cells pretreated with the Rho kinase inhibitors Y-27632 and HA-1077. The TRH-induced r-ERG inhibition was not significantly modified in the presence of the inhibitors. Surprisingly, the hormonal effects became irreversible in the presence of HA-1077 but not in the presence of the more potent Rho kinase inhibitor Y-27632. Further experiments indicated that the effect of HA-1077 correlated with its ability to inhibit protein kinase C (PKC). The hormonal effects also became irreversible in cells in which PKC activity was selectively impaired with GF109203X, Gö6976 or long-term incubation with phorbol esters. Furthermore, the reversal of the effects of TRH, but not its ability to suppress r-ERG currents, was blocked if diacylglycerol generation was prevented by blocking phospholipase C activity with U-73122. Our results suggest that a pathway involving an as yet unidentified protein kinase is the main cause of r-ERG inhibition in perforated-patch clamped GH3 cells. Furthermore, they demonstrate that although not necessary to trigger the ERG current reductions induced by TRH, an intracellular signal cascade involving phosphatidylinositol-4,5-bisphosphate hydrolysis by phospholipase C, activation of an alpha/betaII conventional PKC and one or more dephosphorylation steps catalysed by protein phosphatase 2A, mediates recovery of ERG currents following TRH withdrawal.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / analogs & derivatives*
-
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / pharmacology
-
Amides / pharmacology
-
Animals
-
Cells, Cultured
-
ERG1 Potassium Channel
-
Enzyme Inhibitors / pharmacology
-
Estrenes / pharmacology
-
Ether-A-Go-Go Potassium Channels
-
Hydrolysis
-
Indoles / pharmacology
-
Intracellular Signaling Peptides and Proteins
-
Isoenzymes / antagonists & inhibitors
-
Isoenzymes / metabolism*
-
Maleimides / pharmacology
-
Membrane Potentials / drug effects
-
Membrane Potentials / physiology
-
Patch-Clamp Techniques
-
Phosphatidylinositol 4,5-Diphosphate / metabolism
-
Phosphoprotein Phosphatases / metabolism
-
Phosphorylation
-
Pituitary Gland, Anterior / cytology
-
Pituitary Gland, Anterior / enzymology*
-
Potassium Channels / metabolism*
-
Potassium Channels, Voltage-Gated*
-
Protein Kinase C / antagonists & inhibitors
-
Protein Kinase C / metabolism*
-
Protein Kinase C beta
-
Protein Kinase C-alpha
-
Protein Phosphatase 2
-
Protein Serine-Threonine Kinases / antagonists & inhibitors
-
Protein Serine-Threonine Kinases / metabolism
-
Pyridines / pharmacology
-
Pyrrolidinones / pharmacology
-
Rats
-
Signal Transduction / drug effects
-
Signal Transduction / physiology*
-
Thyrotropin-Releasing Hormone / pharmacology*
-
rho-Associated Kinases
Substances
-
Amides
-
ERG1 Potassium Channel
-
Enzyme Inhibitors
-
Estrenes
-
Ether-A-Go-Go Potassium Channels
-
Indoles
-
Intracellular Signaling Peptides and Proteins
-
Isoenzymes
-
Maleimides
-
Phosphatidylinositol 4,5-Diphosphate
-
Potassium Channels
-
Potassium Channels, Voltage-Gated
-
Pyridines
-
Pyrrolidinones
-
1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione
-
Y 27632
-
Thyrotropin-Releasing Hormone
-
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
-
Protein Serine-Threonine Kinases
-
rho-Associated Kinases
-
Protein Kinase C
-
Protein Kinase C beta
-
Protein Kinase C-alpha
-
Phosphoprotein Phosphatases
-
Protein Phosphatase 2
-
bisindolylmaleimide I
-
fasudil