Morphine-Mediated Signal Transduction Pathways and Receptor Desensitization
Treatment of chronic pain has been a challenge as the most effective treatment that uses opiates has many unwanted side effects; for example, chronic exposure leads to desensitization of opioid receptors, development of tolerance, and addiction [1
]. One of the alarming effects, reported in 2016, is that more than 100 people die daily due to opioid-related overdose (CDC/NCHS, National Vital Statistics System, Mortality, CDC Wonder, Atlanta, GA: US Department of Health and Human Services, CDC; 2017).
Opiates, such as morphine and heroin, interact with opioid receptors and it is generally thought that they function primarily via the activation of the μ opioid receptor (MOR), although, at high concentrations, they can activate δ and κ opioid receptors [2
]. Opioid receptors are located both pre-and-post-synaptically and are coupled to the Gi/Go proteins. Upon ligand binding, Gi/Go-coupled receptors, acutely inhibit adenylyl cyclase (AC) activity, decreasing the levels of the cyclic AMP (cAMP) and decreasing the activity of the protein kinase A (PKA) [3
], or of the exchange protein directly activated by cAMP (EPAC) [4
]. Opioid receptor activation also leads to the stimulation of inward rectifying potassium channels and the inhibition of voltage-gated calcium channels, causing a decreased neurotransmitter release from the pre-synaptic nerve terminal. Thus, the net effect of acute opiate administration is to inhibit neuronal transmission, and this is thought to lead to analgesia [5
Chronic opiate administration, on the other hand, has been shown to upregulate the activity of AC and PKA [6
]. This upregulation of the cAMP pathway has been reported to occur in several regions of the brain, reduce analgesia, and is thought to contribute to opiate addiction [7
In addition to PKA, opioid receptors have been shown to regulate a number of other kinases. Activation of opioid receptors leads to the activation of G protein-coupled receptor kinases (GRK), mitogen-activated protein kinase (MAPK), protein kinase B (PKB/AKT), calcium/calmodulin-dependent kinase II (CAMKII), and protein kinase C (PKC) [2
]. Some of these kinases are thought to play a role in opiate-mediated tolerance, dependence [14
], and addiction [15
]. In the case of PKC, studies show that PKC inhibitors decrease receptor desensitization, development of opiate tolerance, and opiate addiction [16
], (reviewed in [18
PKC is a family of serine/threonine kinases, composed of eleven different isoenzymes, divided into three sub-families. These include, (i) classical PKCs (cPKCs) including α, Βi, βII, and γ, which are calcium-dependent and are activated by phosphatidyl serine (PS) and diacylglycerol (DAG), (ii) novel PKCs including δ, ε, η, and θ, which are calcium-independent, but depend on PS and DAG for their activation, and (iii) atypical PKCs including ζ and λ/ι, which are calcium-independent [19
] and are thought to be activated by protein–protein interactions [20
]. Different PKC isoenzymes are expressed at different subcellular locations. For example, PKCα is found in both pre-and-post-synaptic sites, at the outer surface of synaptic vessels. However, PKCγ in adult rats is only expressed in postsynaptic dendrites, perikaryal cytoplasm, and postsynaptic densities. On the other hand, PKCε is found only in small and medium-sized dorsal root ganglion (DRG) neuronal soma, and presynaptic terminals of nociceptive neurons in the dorsal spinal horn (reviewed in [18
]). It has not yet been determined if these PKCs are active, and what proteins they are interacting with, or are being phosphorylated by them.
Distinct PKC isoenzymes have been implicated in opioid receptor desensitization and addiction. Protein levels and activity of PKCα and γ are increased in the dorsal spinal cord, during chronic exposure to morphine [21
]. Selective inhibitors for PKCα, γ, and ε completely reverse morphine-tolerance [18
]. In particular, PKCγ has been suggested to play a central role in morphine tolerance, both in the spinal cord and the nucleus accumbens (NAc), having a role in sensory signal processing [23
]. Determining the exact role of PKC, in addiction, has been difficult, due to the fact that PKC also plays a critical role in the formation and maintenance of memory [24
], including drug-induced memory [26
]. However, it is not clear how PKC is activated, following the activation of MOR, by morphine. Following are the possible mechanisms that follow from the MOR activation:
MOR activation enables the Gβγ subunit to activate PLC which then would lead to PKC activation [27
MOR activation leads to an activation of a Gq-coupled receptor that, in turn, leads to PLCβ activation, as seen in the case of M3 muscarinic receptor activation-mediated increase in the MOR desensitization [28
MOR activation leads to activation of the receptor-coupled and non-coupled tyrosine kinases, which in turn lead to PLCγ activation.
MOR activation leads to the activation of a small G protein which would then activate PLCε and subtypes of PLCβ and γ [29
MOR activation leads to activation of PI3K which then activates PKC.
However, this last scenario has been shown to occur only in the case of atypical PKCs that are insensitive to DAG [30
]. In intestinal epithelial cells, MOR has been shown to activate PI3K, via Gβγ, leading to a decrease in cell death [31
]). Not much is also known about which PKC isoenzymes are activated by morphine. A recent study with DRG neurons and HEK-293 cells that were overexpressing MOR, showed that both PKCα and ε were activated at the plasma membrane within the first minute of the receptor activation by morphine, and that this activation was sustained for at least 20 min. This was specific to morphine, since MOR activation by DAMGO did not activate PKC, in this time frame. The authors also demonstrated that PKCα was activated by Gβγ, and led to MOR phosphorylation at specific sites, that restricted the plasma membrane localization of MOR and inhibited subsequent nuclear activation of the extracellular signal-regulated kinase (ERK) [32
]. Sequential activation of PKCα and ε has been previously shown to be responsible for sustained ERK1/2 activation, upon mechanical stress [33
]. If this also happens in the case of MOR activation by morphine, or whether both PKCs are activated simultaneously, remains to be determined. Therefore, understanding the spatial and temporal dynamics of the PKC signaling can help us elucidate the mechanisms that lead to MOR desensitization, mediated by these kinases.
It is clear that PKC has an important role not only in receptor desensitization but also in inhibition of receptor recycling [18
]. One of the main features that distinguishes morphine from other potent MOR agonists, such as DAMGO and fentanyl, is that morphine activates PKC signaling (and minimally activates GRK), whereas, DAMGO robustly activates GRK which phosphorylates the receptor and recruits β-arrestin, leading to a receptor endocytosis and recycling [32
]. Even though PKC has been demonstrated to be a key target in morphine-mediated receptor desensitization, the mechanism by which PKC mediates this process is still not clear. Targeting PKC itself to decrease receptor desensitization could be problematic, as PKC is involved in several processes, including mediating immunological responses specifically against viral infections [34
]. Thus, identifying PKC targets can be useful in elucidating the signal transduction processes involved in MOR desensitization and opioid-tolerance. MOR itself is a PKC target [35
]. The carboxy-terminal tail of MOR contains 12 serine/threonine residues and two of them have the consensus sequence for phosphorylation by PKC. Mutations of eleven of these phosphorylation sites (including the two PKC sites) led to a functional receptor that was not desensitized or internalized, indicating that phosphorylation of MOR is important for receptor recycling [36
]. A possibility that MOR activation of PKC leads to the phosphorylation of proteins other than the receptor, and that these PKC targets participate in desensitization, has not been well explored [35
]. In order to address this issue, we developed a new strategy to identify the PKC interacting proteins/substrates within the context of an acute morphine treatment. For this, we used an antibody that specifically recognizes the active state of cPKCs (anti-C2Cat) [40
]. Using this anti-C2Cat antibody, we immunoprecipitated active PKC-associated proteins from Neuro-2A cells treated with acute morphine, and identified the associated proteins by mass spectrometry. A number of proteins were identified, including a few known PKC targets.
In this article, we describe these proteins, discussing them in the context of pain mediated by nerve growth factor (NGF) signaling. In nociceptive neurons, NGF has a central role in pain. Inflammation leads to the release of NGF and activation of tropomyosin receptor kinase A (TrkA), a tyrosine kinase coupled to the NGF receptor [41
]. NGF-binding leads to TrkA dimerization, auto-phosphorylation and subsequent binding and activation of PLCγ [42
]. Amongst the several pathways activated by TrkA, PLCγ activation causes DAG generation and opening of an ion channel, the transient receptor potential cation channel subfamily V member 1 (TRPV1), a nonselective cation channel involved in a variety of nociceptive processes and activated by several stimuli (including acidic pH, heat, endocannabinoids, endogenous lipids, and capsaicin). Activation of TRPV1 causes a cation influx followed by depolarization and pain [43
]. PKA and PKC bind to AKAP79/150, and this complex can then phosphorylate and activate TRPV1 [44
] (Figure 1
). One of morphine’s targets is TRPV1 (reviewed in [45
]). Blocking PKA-signaling by MOR, inhibits the TRPV1 channel activity and the TRPV1 active multimer-translocation to the membrane [46
]. Moreover, a cAMP analog, 8Br-cAMP, can reverse the opioid-mediated inhibition of TRPV1, in DRG neurons [47
]. Furthermore, blocking TRPV1 decreases morphine tolerance [48
]. In DRG neurons and the spinal cord, TRPV1 and MOR are co-localized and their expression increases, upon inflammation [48
]. These observations suggest that TRPV1 [50
] and TrkA [51
] could be drug targets for the development of non-opioid analgesics. Therefore, understanding the interaction between morphine-mediated analgesia and TrkA mediated pain could lead to the development of analgesics with lesser side-effects than the currently used drugs.