
   1. Maria C. Olianas,
   2. Simona Dedoni,
   3. Alessandra Olianas and
   4. Pierluigi Onali

   1.
      Section of Biochemical Pharmacology, Department of Neuroscience (M.C.O., S.D., P.O.) and Department of Sciences Applied to Biosystems (A.O.), University of Cagliari, Cagliari, Italy

   1. Address correspondence to:
      Dr. Pierluigi Onali, Section of Biochemical Pharmacology, Department of Neuroscience, University of Cagliari, Cittadella Universitaria di Monserrato, 09042 Monserrato (Ca), Italy. E-mail: onali{at}unica.it

Abstract

AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controlling cell survival, energy metabolism, and food intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DORs stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators, and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase, and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase treatment, Gq/11 antagonism, and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca2+ concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate accumulation. In SH-SY5Y neuroblastoma cells and primary olfactory bulb neurons, DOR activation failed to affect AMPK phosphorylation per se but potentiated the stimulation by either muscarinic agonists or 2-methyl-thio-ADP. Sequestration of G protein βγ subunits (Gβγ) blocked the DOR potentiation of AMPK phosphorylation induced by oxotremorine-M. In CHO cells, the AMPK activator 5-aminoimidazole-4-carboxamide1-β-d-ribonucleoside stimulated AMPK phosphorylation and glucose uptake, whereas pharmacological inhibition of AMPK, expression of a dominant-negative mutant of AMPKα1, and P2Y receptor blockade reduced DOR-stimulated glucose uptake. The data indicate that in different cell systems, DOR activation up-regulates AMPK through a Gβγ-dependent synergistic interaction with Gq/11-coupled receptors, potentiating Ca2+ release and CaMKKβ-dependent AMPK phosphorylation. In CHO cells, this coincident signaling mechanism is involved in DOR-induced glucose uptake.
Footnotes

    *

      Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

      http://dx.doi.org/10.1124/mol.111.075267.
    *

      ABBREVIATIONS:

      ACC
          acetyl-coenzyme A carboxylase
      ADV
          adenovirus
      AICAR
          5-aminoimidazole-4-carboxamide1-β-d-ribonucleoside
      AMPK
          AMP-activated protein kinase
      ANOVA
          analysis of variance
      BSA
          bovine serum albumin
      CaMKK
          Ca2+/calmodulin-dependent protein kinase kinase
      CCh
          carbachol
      CHO
          Chinese hamster ovary
      CHO/DOR
          CHO cells stably expressing human DOR
      compound C
          6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine
      dBcAMP
          dibutyryl-cAMP
      DN
          dominant-negative
      DOR
          δ-opioid receptors
      DPDPE
          [d-Pen(2,5)]-enkephalin
      DTT
          dithiothreitol
      FCS
          fetal calf serum
      GLUT
          glucose transporter
      Go 6983
          3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione
      GRK2-CT
          C-terminal region of G protein-coupled receptor kinase 2
      HPLC
          high-performance liquid chromatography
      IGF-1
          insulin-like growth factor-1
      IP
          inositol phosphate
      LDH
          lactate dehydrogenase
      MAP
          mitogen-activated protein
      MeSADP
          2-methylthio ADP
      MRS 2179
          N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate
      naltrindole
          17-(cyclopropylmethyl)-6,7-dehydro-4,5α-epoxy-3,14-dihydroxy-6,7–2′,3′-indolomorphinan
      NTI
          naltrindole
      Oxo-M
          oxotremorine-M
      PBS
          phosphate-buffered saline
      PD 98059
          2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one
      PI3K
          phosphatidylinositol 3-kinase
      PKA
          protein kinase A
      PKC
          protein kinase C
      PLC
          phospholipase C
      PPADS
          pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid
      PTX
          Bordetella pertussis toxin
      Rp-cAMPS
          adenosine-3′,5′-cyclic monophosphorothioate, Rp-isomer
      SB203580
          4-[5-(4-fluorophenyl)-2-[4-(methylsulphonyl)phenyl]-1H-imidazol-4-yl]pyridine
      SAMS peptide
          HMRSAMSGLHLVKRR
      SNC 80
          (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide
      SP600125
          anthra[1–9-cd]pyrazol-6(2H)-one
      Sp-cAMPS
          adenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer
      STO-609
          7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid
      TAK1
          transforming growth factor-β-activated kinase 1.

    * Received August 11, 2011.
    * Accepted October 26, 2011.
