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|Title:||The molecular mechanism of peripheral antinociceptive action of morphine: activation of PI3Kγ/AKT/nitric oxide signaling pathway|
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|Authors/Affiliations:||Thiago M. Cunha1, Celina M. Lotufo1, Mani I. Funez1, Andressa C. Domingues1, Waldiceu A. Verri, Jr1, Mauro M. Teixeira2, John S. Hothersall1, Fernando Q. Cunha1 & Sergio H. Ferreira1|
1Department of Pharmacology, Faculty of Medicine of Ribeirão Preto University of Sao Paulo, Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brazil. 2Department of Biochemistry and Imunology, Institute of Bilogical Science (ICB), Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil.
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|Content:||Objectives: The increased intensity of nociception during inflammation (hypernociception) is primary due to the sensitization of specific classes of nociceptive neurons. Morphine directly blocks hypernociception via the activation of nitric oxide/cGMP/PKG/K(ATP) channels signaling pathway. In the present study we tested whether the initiation of this signaling cascade depends on the stimulation of the PI3Kγ/AKT. |
Materials and Methods: Mechanical hypernociception was evaluated in PGE2 sensitized rats and mice paws using a modification of Randall-Sellito test and an electronic version of the von Frey test, respectively. The expression of m-opioid receptor, PI3Kγ and TRPV1 in dorsal root ganglion (DRG) neurons was detected by immunofluorescense (confocal microcopy). Phosphorylated AKT expression was analyzed by western blot of DRG culture neurons. Nitric oxide production by DRG neurons was evaluated using fluorescent DAF indicator (confocal).
Results: Confocal analysis showed that PI3Kγ is expressed in DRG neurons, mainly in neurons of small size, which also express TRPV1 and m-opioid receptor. PI3Kγ is also expressed in IB4 positive neurons. Non-selective (wortmannin, 1-10 μg/paw) or selective (AS 605240, 10-90 μg/paw) pharmacological inhibition of PI3Kγ prevented in rats the peripheral anti-hypernociceptive action of morphine (6 μg/paw) or DAMGO (1 μg/paw). In PI3Kγ deficient mice (-/-) mice morphine as well DAMGO did not show peripheral anti-hypernociceptive effect. Further investigating the PI3Kγ downstream signaling pathway it was shown that the AKT selective inhibitor (3-30 mg/paw) also prevents morphine and DAMGO anti-hypernociceptive effects. Corroborating with this in vivo observation the incubation of DRG culture neurons with morphine (10 μM) or DAMGO (1 μM) produce an increase in the phosphorylation of AKT. This activation was prevented by naloxone (10 μM), PI3Kγ inhibitor (AS 605240; 1 μM) and found reduced in cultured neurons of PI3Kγ (-/-). Incubation of DGR neurons with morphine induced an increase of nitric oxide production which was inhibited by PI3Kγ and AKT inhibitors.
Conclusion: The present results suggest that the peripheral blockade of hypernociception by morphine seems to be dependent on m-opioid receptor, with an initial activation of PI3Kγ/AKT signaling pathway, which is ultimately responsible for the activation of nitric oxide/cGMP/PKG/K+(ATP) channels pathway.
This work was supported by grants from FAPESP and CNPq. We thank Ieda Regina dos Santos Schivo and Sergio Roberto Rosa for excellent technical support.
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