A novel conformation‐sensitive antibody‐based strategy to explore PKC signaling induced by opiate activation of μ‐opioid receptors

Abstract

Opiates such as morphine and fentanyl are the most widely used analgesics for the treatment of pain; however, prolonged treatment with opiates decreases the responsiveness of opioid receptors, leading to the development of tolerance and opiate addiction. Recent alarming reports of the widespread misuse of fentanyl leading to the ‘Opioid Epidemic” have begun to refocus scientific investigations into the mechanisms of action of opioid receptors. Despite intense research efforts, the molecular mechanisms underlying opioid addiction remain unknown contributing to a public health challenge. Activation of opioid receptors leads to the initiation of a number of signaling cascades, resulting in enhanced protein phosphorylation. Among the various kinases, protein kinase C (PKC) has been reported to play a crucial role in the desensitization of opioid receptors. To date, the majority of studies with PKC have used genetic and/or pharmacological manipulation of PKC to explore the dynamics of PKC activation. In order to investigate the activation of endogenous PKC in native tissue, we developed conformational‐sensitive antibodies that are able to detect time‐ and ligand‐mediated activation of native PKC. We also generated antibodies to several substrates of PKC including pPP2A (regulatory subunit phosphoS90 PPP2R5D), phosphoSer143 MARCKS, and phosphoS375/T376 MOR. We used a high‐throughput microscopy approach to explore the temporal dynamics of opiate‐mediated PKC signaling and track the temporal dynamics of PKC activation in cells treated with PMA, morphine or fentanyl. Interestingly, in contrast to the PKC activation induced by morphine that is rapid but transient the activation by fentanyl is slow and sustained for a longer period of time. We are able to detect rapid activation of PKC in the striatum of animals acutely treated with morphine suggesting that the conformation‐sensitive activation, state sensitive anti‐PKC antibodies serve as hitherto unavailable, critical tools to investigate the role of PKC in opioid receptor signaling in particular and PKC‐mediated signaling in general.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.