Abstract
Chronic morphine causes the mu opioid receptor (MOR) to switch its coupling from Gi/o to Gs, resulting in excitatory signaling via both Gαs and its Gβγ dimer. Ultra-low-dose naloxone (NLX) prevents this switch and attenuates opioid tolerance and dependence. This protective effect is mediated via a high-affinity interaction of NLX to a pentapeptide region in c-terminal filamin A (FLNA), a scaffolding protein interacting with MOR. In organotypic striatal slice cultures, we now show that acute morphine induces a dose-dependent Go-to-Gs coupling switch at 5 and 15 min that resolves by 1 hr. The acute Gs coupling induced by 100 µM morphine was completely prevented by co-treatment with 100 pM NLX, (+)NLX, or naltrexone (NTX), or their pentapeptide binding site (FLNA2561–2565), which we show can act as a decoy for MOR or bind to FLNA itself. All of these co-treatments presumably prevent the MOR–FLNA interaction. Since ultra-low-dose NTX also attenuates the addictive properties of opioids, we assessed striatal cAMP production and CREB phosphorylation at S133. Correlating with the Gs coupling, acute morphine induced elevated cAMP levels and a several-fold increase in pS133CREB that were also completely blocked by NLX, NTX or the FLNA pentapeptide. We propose that acute, robust stimulation of MOR causes an interaction with FLNA that allows an initially transient MOR–Gs coupling, which recovers with receptor recycling but persists when MOR stimulation is repeated or prolonged. The complete prevention of this acute, morphine-induced MOR–Gs coupling by 100 pM NLX/NTX or 10 µM pentapeptide segment of FLNA further elucidates both MOR signaling and the mechanism of action of ultra-low-dose NLX or NTX in attenuating opioid tolerance, dependence and addictive potential.
Highlights
Ultra-low-dose opioid antagonists have been shown to enhance opioid analgesia, minimize opioid tolerance and dependence [1,2] and attenuate the addictive properties of opioids [3,4]
Acute morphine-induced G protein coupling switch blocked by 100 pM NLX/NTX or 10 mM filamin A (FLNA) pentapeptide
Coupling to pertussis toxin-sensitive G proteins Gi and Go to inhibit the adenylyl cyclase/cAMP pathway (Laugwitz et al, 1993;Connor and Christie, 1999), mu opioid receptor (MOR) switches to Gs coupling after chronic opioid administration, resulting in excitatory signaling by both Gas and Gbc subunits [5,6,8]
Summary
Ultra-low-dose opioid antagonists have been shown to enhance opioid analgesia, minimize opioid tolerance and dependence [1,2] and attenuate the addictive properties of opioids [3,4]. Electrophysiology data suggested that ultra-low-dose opioid antagonists block excitatory signaling of opioid receptors [1]. Ultra-low-dose NLX co-treatment suppresses opioid tolerance and dependence by preventing these MOR signaling alterations [5], and we recently identified the NLX binding site that mediates its protective effects as a pentapeptide segment in c-terminal FLNA [9]. Implicating FLNA in desensitization, our recent organotypic striatal slice culture data showed that potentially disrupting the MOR–FLNA interaction via NLX’s high-affinity binding to FLNA blocks the chronic morphine-induced G protein coupling switch by MOR [9]. FLNA peptide fragments containing the NLX binding site blocked the protective effect of NLX on both the MOR–Gs coupling and downstream cAMP accumulation induced by chronic morphine (twice daily 1-hr exposures for 7 days), presumably by interfering with NLX’s binding to FLNA in the tissues
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