There is a largely arbitrary transition between ‘acute’ and ‘chronic’ pain, with temporal cut-offs after which point acute pain becomes chronic pain. Thus, an understanding of the cellular mechanisms that underlie chronic pain states is essential for the development of new long-term therapeutic strategies. At multiple sites within the pain pathway, neuronal plasticity is now widely accepted as critical in the maintenance of chronic pain.
Cellular mechanisms underlying this plasticity are not well known, although they are likely to involve changes in gene expression and regulation of protein synthesis. Furthermore, in the nociceptor, changes in synaptic strength and efficacy have the potential to trigger increases in the transduction of pain signals.
At peripheral terminals of nociceptors, there is the potential for plasticity to occur, with changes in receptor and channel expression, distribution, and activation thresholds being able to generate hypersensitivity. These changes occur through multiple intracellular pathways. For instance, protein kinase A (PKA), PKC, and PKG cascades can generate posttranslational modifications on target proteins – which in turn affect their activation and trafficking.
Figure 1. Intracellular signaling in chronic pain states. MAPK and PI 3-K/mTOR signaling pathways are thought to be the primary pathways involved in chronic pain and the regulation of gene transcription and translation in nociceptors. PKC, PKA, and PKG pathways control posttranslational regulation of receptor and channel proteins, and also have influences on gene expression. Long term modulation of nociceptor plasticity in this way can lead to hyperalgesia and persistent pain states.
Regulating gene transcription and translation that controls the expression of proteins has been thought to occur primarily through the ERK and PI 3-K/mTOR signaling cascades (Figure 1), which have been identified and subsequently highlighted as potential areas that can produce future therapeutic pain targets.
Protein Kinase A
Protein Kinase A (also known as PKA) exerts a profound modulatory role in sensory nociceptor physiology. The activation of PKA by cAMP is enough to create hyperalgesia in nociceptors. This activation may mediate some of these effects through direct phosphorylation of TTx-R sodium channels – for instance, such as NaV1.8 or TRPV1 channels. Interestingly, in a neuropathic pain model, both of these have been shown to induce pain.
Further, PKA also interacts with MEK, which is a component of the MAPK pathway. This, in turn, leads to the activation of further downstream targets. Such a scenario is capable of regulating gene expression, thus affecting neuronal plasticity. Meanwhile, opioid receptors can produce analgesia by inhibiting adenylyl cyclase, thus blocking PKA activation. Hence, this makes it a key therapeutic target in pain.
Before a painful stimulus, the injection of a PKA inhibitor, cAMPS-Rp has been known to inhibit hyperalgesia. Also, H 89, which is also a PKA inhibitor, has displayed an ability to block the nociceptive response to an inflammatory agent when it comes to sensory pain fibers. Finally, the activation of PKA signaling pathways implicated in pain is also possible through the use of drugs such as 8-BromocAMP or cAMPS-Sp, which, in effect, are membrane-permeable cAMP analogs with the capacity to stimulate PKA phosphorylation.
Protein Kinase C
PKC consists of 15 isozymes that can be further subdivided into three groups: a conventional, novel, and atypical. The activation of conventional isozymes is brought about by a process that requires phospholipase C (PLC), diacylglycerol (DAG) and calcium. In contrast, the novel forms do not need calcium, while atypical forms do not require either DAG or calcium.
The activation of PKC enhances the TTx-R sodium currents and TRPV1 channel currents. In response to inflammatory mediators such as bradykinin and substance P, PKCε, in particular, can translocate to the plasma membrane of nociceptors.
The activity of PKCε also bears a link to the maintenance of ‘primed’ state, whereby nociceptors have increased sensitivity to noxious stimuli, particularly characteristic to chronic pain. Inhibitors of PKC like the GF 109203X and chelerythrine have displayed significant reductions in rat models of mechanical hyperalgesia. Thus, it will be interesting to see how PKC inhibitors and other isoforms advance in the current field of research.
Protein Kinase G and Nitric Oxide
In contrast to PKC, there is far less information about the role of cGMP/PKG signaling and nitric oxide (NO)-mediated modulation of pain. Guanylyl cyclase is stimulated by NO, which activates cGMP and PKG.
Historically, studies have shown that intracutaneous injections of NO precursors evoke pain, thereby this implicates it as a pronociceptive mediator. However, NO is known to mediate the analgesic effects of opioids and other analgesic substances, hence suggesting that it plays a complex and diverse role in nociception.
To help define the role of NO in pain, classical inhibitors such as L-NAME can be harnessed. Furthermore, newer products, such as the soluble guanylyl cyclase inhibitor, NS 2028, have the potential to offer increased selectivity in targeting the cGMP/PKG pathway.
A critical kinase in cell signaling pathways is MAPK, which transduces extracellular stimuli into intracellular translational and transcriptional responses. The stimulation of nociceptive DRG neurons can increase phosphorylation of various types of MAPK, thus initiating changes in short-term acute pain or long-term transcription. Within the MAPK family, three major family members – ERK, p38, and c-Jun N-terminal kinase (JNK) – are three different types of MAPK signaling cascades. While the inhibition of all three MAPK pathways has the potential to attenuate persistent pain after nerve injury, a spike in ERK and/or p38 activity has been linked to pain plasticity upstream of ERK.
The ERK pathway involves a sequential cascade, including Ras, Raf, and MEK. Moreover, MEK inhibitors such as PD 98059 and U0126 have an inherent ability to block acute pain behavior post the injection of formalin, thus suggesting that ERK necessarily plays a role in short-term nociception, considering the short time involved. However, ERK produces not just short-term functional changes by non-transcriptional mechanisms, but also generates long-term adaptive changes by gene transcription modification and translation.
So far, the use of direct ERK inhibitors has been beneficial in blocking this pathway since the selective ERK inhibitor, FR 180204. p38 is typically activated by MKK4 protein kinases and can be inhibited by SB 203580. Thus, this demonstrates an ability to reduce mechanical allodynia and thereby reverse the pain associated with arthritis.
The activation of p38 MAPK has also been known to increase TRPV1 channel expression at the plasma membrane, which suggests that it contributes to pain hypersensitivity and the early development of mechanical allodynia. However, although less is known about the role of JNK in pain, SP 600125 – which is a JNK inhibitor – attenuates pain after repeated injection over several days, thus demonstrating an accumulated analgesic effect in a rat cancer-induced bone pain model.
PI 3-K/Akt/mTOR Pathway
When it comes to plasticity, the PI 3-K/mTOR pathway appears to play a key role. Since the PI 3-kinase (PI 3-K) is a lipid kinase, it tends to generate PIP3 from membrane phosphoinositides. In turn, this thereby activates the serine/threonine kinases Akt and mTOR, which are responsible for regulating gene expression. Following nerve injury, the increased activation of Akt and mTOR is observed in DRG and dorsal horn neurons. Thus, it implies that inhibition of this pathway is an important target in pain research.
Some selective PI 3-kinase inhibitors, such as the LY 294002 and 740 Y-P, have displayed an ability to block downstream phosphorylation of Akt and the initiation of hyperalgesia. In animal models of pain, the inhibition of mTOR activity in the spine by rapamycin shows antinociceptive effects. Furthermore, the systemic administration of Torin 1, which is also an mTOR inhibitor, has the effect of reducing the response to mechanical and cold stimuli in mice that experience neuropathic pain.
Also, KU 0063794 is a selective mTOR inhibitor that shows no activity at PI 3-kinase. Thus, this inhibitor may prove to be useful in the investigation of the physiological role of mTOR in nociception. Furthermore, it has been observed that mTOR plays a key role in phosphorylation of a protein named 4E-BP, which controls the initiation of protein translation.
The direct inhibition of protein translation can be achieved through the targeting of the downstream binding protein, eIF4E, with 4E1RCat, which is a small molecule inhibitor that prevents assembly of the regulatory protein complex. Thus, mTOR plays multiple roles in transcription, translation, and posttranslational modifications, which make it an important target in pain research, if specificity or direct targeting of peripheral nociceptors can be achieved.
To conclude, investigating these complex intracellular pathways using selective inhibitors and activators has the potential to help deepen scientific understanding of the genesis of both acute and long-term chronic pain, while also helping to identify new targets for pharmacological intervention.
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