Evidence That Tyrphostins AG10 and AG18 Are Mitochondrial Uncouplers That Alter Phosphorylation-dependent Cell Signaling

Abstract

Receptor agonists that initiate fluid secretion in salivary gland epithelial cells also increase protein phosphorylation. To assess contributions of tyrosine phosphorylation to secretion, changes in muscarinic receptor-initiated secretion (estimated from sodium pump-dependent increases in oxygen consumption) were measured in parotid acinar cells exposed to tyrosine kinase inhibitors. However, like the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, tyrphostins AG10 and AG18 increased the rate of oxygen consumption and reduced cellular ATP by ϳ90% in the absence of the muscarinic agonist carbachol, indicating that these tyrphostins uncouple mitochondria. Exposure of isolated mitochondria to five structurally related tyrphostins demonstrated that their relative potencies as uncouplers differed from their in vitro kinase-inhibitory potencies due to different molecular requirements for the two effects. AG10 and AG18 blocked parotid phosphorylation events only at concentrations that reduced ATP content. The tyrosine kinase inhibitor genistein reduced ATP content by 15-20% and weakly uncoupled isolated mitochondria, but its inhibition of carbachol-mediated protein kinase C␦ tyrosine phosphorylation and ERK1/2 activation appeared attributable to blocking tyrosine kinases directly. Carbachol itself rapidly reduced ATP content by 15-20%. Carbachol, 3-O-(4-benzoyl)benzoyl adenosine 5-triphosphate (P2X 7 receptor agonist), AG10, AG18, and carbonyl cyanide p-trifluoromethoxyphenyl hydrazone rapidly activated the fuel sensor AMP-activated protein kinase (AMPK); however, only AMPK activation by carbachol and BzATP was due to sodium pump stimulation. AG10 and AG18 also activated AMPK and/or uncoupled mitochondria in PC12, HeLa, and HEK293 cells. These studies demonstrate that some tyrosine kinase inhibitors produce cellular effects that are mechanistically different from their primary in vitro characterizations and, as do salivary secretory stimuli, promote rapid metabolic alterations that initiate secondary signaling events.Signaling by G protein-coupled receptors engages a variety of downstream effectors and signaling cascades that rely on alterations in the phosphorylation of proteins on tyrosine and other residues. In salivary gland epithelial cells, secretagogues (i.e. receptor ligands that promote secretion), including the muscarinic receptor ligand carbachol, increase the tyrosine phosphorylation of various proteins (1-4). Therefore, a likely hypothesis is that secretagogue-dependent changes in tyrosine phosphorylation contribute to fluid secretion in the parotid gland, one of the three main salivary glands. To examine this possibility, experiments were designed to block tyrosine phosphorylation using chemical inhibitors of tyrosine kinases, including AG18, a member of the tyrphostin (tyrosine phosphorylation inhibitor) family, and genistein. To evaluate the action of kinase inhibition on fluid secretion, the effects of these inhibitors were measured on carbachol-stimulated increases in the ...

AMP-activated protein kinase (AMPK) activating agents cause dephosphorylation of Akt and glycogen synthase kinase-3

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“…Stimulation of muscarinic receptors with the cholinergic agonist carbachol previously was reported to activate AMPK in rat parotid acinar cells [40]. Since SH-SY5Y cells endogenously express muscarinic receptors, predominantly of the M3 subtype coupled to the phosphoinositide signal transduction system [41], we tested if AMPK was activated by muscarinic receptor stimulation in SH-SY5Y cells and if it caused dephosphorylation of Akt and GSK3.…”

β-Adrenoceptors, but not α-adrenoceptors, stimulate AMP-activated protein kinase in brown adipocytes independently of uncoupling protein-1

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Regulation of ERK1/2 by ouabain and Na-K-ATPase-dependent energy utilization and AMPK activation in parotid acinar cells

“…ATP was measured in parotid acinar cells as described previously (32). In general, cells suspended in solution A were equilibrated for ϳ10 min at 37°C before treatment with various agents or vehicles (water or 0.1-0.2% DMSO), as indicated.…”

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