Genetic alteration of phospholipase C β3 expression modulates behavioral and cellular responses to μ opioids

Abstract

Morphine and other opioids regulate a number of intracellular signaling pathways, including the one mediated by phospholipase C (PLC). By studying PLC ␤3-deficient mice, we have established a strong link between PLC and opioid-mediated responses at both the behavioral and cellular levels. Mice lacking PLC ␤3, when compared with the wild type, exhibited up to a 10-fold decrease in the ED 50 value for morphine in producing antinociception. The reduced ED 50 value was unlikely a result of changes in opioid receptor number or affinity because no differences were found in whole-brain B max and K d values for , , and ␦ opioid receptors between wild-type and PLC ␤3-null mice. We also found that opioid regulation of voltage-sensitive Ca 2؉ channels in primary sensory neurons (dorsal root ganglion) was different between the two genotypes. Consistent with the behavioral findings, the specific agonist [D-Ala 2 ,(Me)Phe 4 , Gly(ol) 5 ]enkephalin (DAMGO) induced a greater whole-cell current reduction in a greater proportion of neurons isolated from the PLC ␤3-null mice than from the wild type. In addition, reconstitution of recombinant PLC protein back into PLC ␤3-deficient dorsal root ganglion neurons reduced DAMGO responses to those of wild-type neurons. In neurons of both genotypes, activation of protein kinase C with phorbol esters markedly reduced DAMGO-mediated Ca 2؉ current reduction. These data demonstrate that PLC ␤3 constitutes a significant pathway involved in negative modulation of opioid responses, perhaps via protein kinase C, and suggests the possibility that differences in opioid sensitivity among individuals could be, in part, because of genetic factors.Morphine and other opioids are widely used analgesics. Three opioid receptors, , ␦, and , have been cloned and characterized both functionally and by radioligand binding (1-4). These receptors couple to G i and G o proteins and, through ␣ and ␤␥ subunits, regulate a number of signaling pathways. These regulated pathways include inhibition of adenylyl cyclase activity (5, 6), activation of inwardly rectifying K ϩ channels (7,8), and inhibition of voltage-activated Ca 2ϩ channels, predominantly of the N and P͞Q types (9, 10). Recent work with cell lines has demonstrated that opioid receptors also activate phospholipase C (PLC; 11-17). Moreover, several physiological studies have implicated PLC-linked pathways in a diverse range of opioid-modulated events, such as pain regulation (18), response to brain injury (19), and opioid withdrawal (20). However, despite these data suggesting an important role for PLC-mediated pathways in opioid signal transduction, there is little direct evidence supporting a role of PLC in opioid responses.PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to produce two important second messengers, inositol triphosphate and diacylglycerol (21). The former increases release of Ca 2ϩ from intracellular stores, and the latter activates protein kinase C (PKC). Molecular cloning and biochemical characterizations have revealed four ...

Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

“…Mice and Culture of BMDMs-PLC␤3-deficient mice were on a C57BL/6 background after 7 generations of backcrossing (22), and PLC␤2-deficient mice were on 129SvEV background (23). For mice doubly deficient in PLC␤3 and PLC␤2, PLC␤2 Ϫ/Ϫ mice were crossed twice to C57BL/6 mice and a PLC␤2 ϩ/Ϫ mouse was crossed to PLC␤3 Ϫ/Ϫ mice.…”

Mammalian Fertilization: From Sperm Factor to Phospholipase Cζ

“…Gene targeting studies have been performed to generate PLCb1-and b3-null mice (Kim et al, 1997;Wang et al, 1998;Xie et al, 1999), and the results obtained with b3-null mice have proven intriguing. PLCb3-null mice have been produced in two different laboratories by targeted disruption of different exons (Wang et al, 1998;Xie et al, 1999).…”

“…PLCb3-null mice have been produced in two different laboratories by targeted disruption of different exons (Wang et al, 1998;Xie et al, 1999). In one case, disruption of PLCb3 did not have abnormal developmental consequences (Xie et al, 1999), whereas in the other case, PLCb3-deficient embryos died before the 4-cell stage, suggesting involvement of this enzyme in egg activation or in early embryonic development (Wang et al, 1998). However, determination of the precise time of the developmental arrest and whether or not [Ca 2+ ] i responses were present at fertili-zation were not assessed in the null embryos.…”

Exogenous nerve growth factor attenuates opioid-induced inhibition of voltage-activated Ba2+ currents in rat sensory neurons

“…One of these effects, mediated directly by G protein subunit interactions, is a modulation of VGCC in sensory neurons which leads to marked decreases in currents through VGCC. Opioid-mediated inhibition of VGCC is regulated by several kinases, including protein kinase C and PLC (Swartz, 1993;Zhu and Ikeda, 1994;Zamponi et al, 1997;Zhu and Yakel, 1997;King et al, 1999;Xie et al, 1999;Barrett and Rittenhouse, 2000). In addition, PI3 kinase and MAP kinases may be involved in opioid receptor desensitization (Schmidt et al, 2000;Tan et al, 2003).…”