Use of U-73122 as an Inhibitor of Phospholipase C-Dependent Processes

doi:10.1006/ncmn.1993.1046

Neuroprotocols

Volume 3, Issue 2, October 1993, Pages 125-133

https://doi.org/10.1006/ncmn.1993.1046 Get rights and content

Abstract

1-[6-[[17β-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]-hexyl]1H-pyrrole-2,5-dione (U-73122) is an aminosteroid that was identified initially as a potent inhibitor of platelet activation by receptor-specific agonists. U-73122 inhibits receptor-coupled generation of inositol 1,4,5-trisphosphate (but not cyclic AMP) and intracellular mobilization of Ca ²⁺ in a variety of cell types. U-73122 inhibits phosphoinositide-specific phospholipase C (PI-PLC) activity in cell-free systems, but exhibits little or no direct inhibition of phospholipases A₂ and D. Structure-activity analysis revealed that the maleimide group of U-73122 is essential, but not sufficient, for inhibitory activity. The succinimide analog of U-73122 (U-73343) has negligible inhibitory activity and is a useful control compound. On the basis of information derived from the use of U-73122 in a variety of cell types, procedures for storing, dissolving, and presenting U-73122 to cells are recommended. While knowledge of the mechanism of action of U-73122 would extend the utility of this compound, U-73122 has already been employed successfully to examine PI-PLC involvement in a variety of cellular processes. The application of U-73122 in an investigation of muscarinic receptor sequestration in SK-N-SH neuroblastoma cells is illustrated.

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In contrast, C4P did not affect the level of ROS in cells. ROS production was almost completely abrogated in cells treated with DSC4P-1, SA-3, or 8FC4P in the presence of each inhibitor of Rho proteins (rhosin), PLC (D609, U73122),53 PKC (rottlerin, UCN-01),54,55 and NOX (diphenyleneiodonium (DPI), apocynin),56,57 all of which are inhibitors with no effects on ROS generation, cytochrome c release from mitochondria into the cytosol, caspase activation, or cell viability (Figures 4C and S33). These findings are taken as a clear indication that ROS are generated upon treatment with the synthetic ion transporters of this study and that this occurs via the sequential activation of Rho proteins PLC, PKC, and NOX.
Recently, we showed that synthetic anion transporters DSC4P-1 and SA-3 had activity related to cancer cell death. They were found to increase intracellular chloride and sodium ion concentrations. They were also found to induce apoptosis (DSC4P-1) and both induce apoptosis and inhibit autophagy (SA-3). However, determinants underlying these phenomenological findings were not elucidated. The absence of mechanistic understanding has limited the development of yet-improved systems. Here, we show that three synthetic anion transporters, DSC4P-1, SA-3, and 8FC4P, induce osmotic stress in cells by increasing intracellular ion concentrations. This triggers the generation of reactive oxygen species via a sequential process and promotes caspase-dependent apoptosis. In addition, two of the transporters, SA-3 and 8FC4P, induce autophagy by increasing the cytosolic calcium ion concentration promoted by osmotic stress. However, they eventually inhibit the autophagy process as a result of their ability to disrupt lysosome function through a transporter-mediated decrease in a lysosomal chloride ion concentration and an increase in the lysosomal pH.
Histamine H<inf>3</inf> receptor activation stimulates calcium mobilization in a subpopulation of rat striatal neurons in primary culture, but not in synaptosomes
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These results indicate that in striatal nerve terminals H3R activation stimulated PLC activity. Of note, the H3R-mediated increase in IP3 formation was modestly (−30%), but significantly reduced by U-73343, reported to be an inactive analogue of U-73122 (Bleasdale and Fisher, 1993). However, a previous report (Thompson et al., 1991) indicates that in SK-N-SH cells both U-73122 and U-73343 (10 μM) inhibited inositol phosphate release induced by the stimulation of muscarinic receptors, although to a different extent (−86 ± 3% and −25 ± 8%, respectively).
The histamine H₃ receptor (H₃R) is abundantly expressed in the Central Nervous System where it regulates several functions pre and postsynaptically. H₃Rs couple to Gα_i/o proteins and trigger or modulate several intracellular signaling pathways, including the cAMP/PKA pathway and the opening of N- and P/Q-type voltage-gated Ca²⁺ channels. In transfected cells, activation of the human H₃R of 445 amino acids (hH₃R₄₄₅) results in phospholipase C (PLC) stimulation and release of Ca²⁺ from intracellular stores. In this work we have studied whether H₃R activation induces Ca²⁺ mobilization from intracellular stores in native systems, either isolated nerve terminals (synaptosomes) or neurons in primary culture. In rat striatal synaptosomes H₃R activation induced inositol 1,4,5-trisphosphate (IP₃) formation but failed to increase the intracellular calcium concentration ([Ca²⁺]_i). In striatal primary cultures H₃R activation resulted in IP₃ formation and increased the [Ca²⁺]_i in 18 out of 70 cells that responded with an elevation in the [Ca²⁺]_i to membrane depolarization with KCl (100 mM) as evaluated by microfluorometry. Confocal microscopy studies corroborated the increase in [Ca²⁺]i induced by H₃R activation in a fraction of those cells that were responsive to membrane depolarization. These results indicate that H₃R activation stimulates the PLC/IP₃/Ca²⁺ pathway but only in a subpopulation of striatal neurons.
Phospholipase C mediates (±)-1-(2,5-dimethoxy-4-iodophenyl)-2- aminopropane (DOI)-, but not lysergic acid diethylamide (LSD)-elicited head bobs in rabbit medial prefrontal cortex
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Paradoxically, U73122 also produced a small, but non-significant, PI hydrolysis signal in the current study, a phenomenon described elsewhere (Tyeryar et al., 2008). The authors also recognize that the specificity of U73122 is not limited to the PLC enzyme, though it is an accepted PLC inhibitor, especially when used in conjunction with its analog, U73343, which does not inhibit PLC activity (Bleasdale and Fisher, 1993). As is reported in the literature, the maximal PI hydrolysis signal stimulated by DOI in the present study was lower than the maximal signal stimulated by 5-HT, but greater than that stimulated by LSD (Berg et al., 1998; Edwards et al., 1992; Pierce and Peroutka, 1988).
The phenethylamine and indoleamine classes of hallucinogens demonstrate distinct pharmacological properties, although they share a serotonin_2A (5-HT_2A) receptor mechanism of action (MOA). The 5-HT_2A receptor signals through phosphatidylinositol (PI) hydrolysis, which is initiated upon activation of phospholipase C (PLC). The role of PI hydrolysis in the effects of hallucinogens remains unclear. In order to better understand the role of PI hydrolysis in the MOA of hallucinogens, the PLC inhibitor, 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U73122), was used to study the effects of two hallucinogens, the phenethylamine, (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and the indoleamine, lysergic acid diethylamide (LSD). PI hydrolysis was quantified through release of [3H]inositol-4-phosphate from living rabbit frontocortical tissue prisms. Head bobs were counted after hallucinogens were infused into the medial prefrontal cortex (mPFC) of rabbits. Both DOI and LSD stimulated PI hydrolysis in frontocortical tissue through activation of PLC. DOI-stimulated PI hydrolysis was blocked by 5-HT_2A/2C receptor antagonist, ketanserin, whereas the LSD signal was blocked by 5-HT_2B/2C receptor antagonist, SB206553. When infused into the mPFC, both DOI- and LSD-elicited head bobs. Pretreatment with U73122 blocked DOI-, but not LSD-elicited head bobs. The two hallucinogens investigated were distinct in their activation of the PI hydrolysis signaling pathway. The serotonergic receptors involved with DOI and LSD signals in frontocortical tissue were different. Furthermore, PLC activation in mPFC was necessary for DOI-elicited head bobs, whereas LSD-elicited head bobs were independent of this pathway. These novel findings urge closer investigation into the intracellular mechanism of action of these unique compounds.
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The involvement of specific phospholipase C isozymes in catecholamine release from digitonin permeabilized bovine adrenal medullary chromaffin cells
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The role of phospholipase C (PLC) in exocytosis has been investigated using digitonin permeabilized, [³H]noradrenaline ([³H]NA) loaded, bovine adrenal medullary chromaffin cells. The PLC inhibitor U-73122 caused a concentration-dependent suppression of Ca²⁺-evoked [³H]NA release but increased basal release (that occurring in the absence of Ca²⁺). Preincubation with antibodies against PLCγ1 or PLCβ3 (but not PLCδ1, δ2, β1 and β2) also inhibited [³H]NA release evoked by Ca²⁺ and increased basal release, indicating that only specific PLC isozymes are involved in these actions. Interestingly, PLCγ1 (but not PLCβ3) antibodies inhibited the ability of Ca²⁺ to increase PLC activity in these permeabilized cells. These data therefore suggest that PLCγ1 activity may have a specific role in regulating the exocytotic response from the adrenal chromaffin cell.
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Regular ArticleUse of U-73122 as an Inhibitor of Phospholipase C-Dependent Processes

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Use of U-73122 as an Inhibitor of Phospholipase C-Dependent Processes