Pain, and Why We Need It

Hueng-Chuen Fan, et al.

J Med Sci 2009;29(5):233-241 http://jms.ndmctsgh.edu.tw/2905233.pdf Copyright © 2009 JMS

Pain, and Why We Need It Hueng-Chuen Fan1,3, Yih-Jing Lee2,3, and Peter A. McNaughton3* 1

Department of Pediarics, Tri-Service General Hospital, National Defense Medical Center, Taipei; 2 School of Medicine, Fu-Jen Catholic University, Taipei, Taiwan, Republic of China; 3 Department of Pharmacology, University of Cambridge, Cambridge, UK

Pain is unique among sensations in that the perceived intensity increases, or sensitizes, during exposure to a strong stimulus. Efforts to determine how neurons sense pain-producing stimuli of a thermal, mechanical or chemical nature have revealed new signaling mechanisms and brought us closer to understanding the molecular events that facilitate transitions from acute to chronic pain. The thermo-gated transient receptor potential (TRP) channels play a critical role in the development of thermal hyperalgesia induced by a wide range of inflammatory mediators. Temperature sensing can be modulated by phosphorylation of intracellular residues by these protein kinases or by insertion of new channels into the cell membrane. Furthermore, the formation of a signaling complex is a final common element in heat hyperalgesia, on which the effects of multiple proinflammatory mediators converge. The integration of A kinase anchoring protein (AKAP) in inflammatory hyperalgesia may be a promising target for the development of novel analgesics. Key Words: pain, inflammation, protein kinases, TRP channel, AKAP

INTRODUCTION We all know about pain, or at least we think we do. Things that damage our bodies cause pain. We learn to avoid pain because the experience is nasty, and in the process we learn to avoid stimuli that might damage us. The more the damage, the greater the pain, and the greater we avoid it. Adaptation is different. Adaptation to a maintained stimulus is a general feature of sensory systems. A good example is light adaptation that allows us to operate over a wide range of ambient light intensities, a necessary property for any species that may at one moment be in bright sunlight and the next in a dark cave. Pain is unique among sensations in that the perceived intensity increases during exposure to a strong stimulus, a process that is referred to as sensitization or hyperalgesia. Pain is a complex experience that involves not only the transduction of noxious environmental stimuli, but also cognitive and emotional processing by the brain. For example, sometimes we fail to notice even quite severely damaging stimuli. Soldiers in battle similarly do not feel Received: May 6, 2009; Accepted: May 26, 2009 * Corresponding author: Peter A. McNaughton, Department of Pharmacology, University of Cambridge, The Old Schools, Trinity Lane, Cambridge CB2 1TN, UK. Tel:+44-1223-334012; Fax:+44-1223-334100; E-mail: [email protected]

severe injuries until after the battle is over. A letter written by a sailor at the battle of Trafalgar mentioned that he had been unaware at the time of the battle that a cannon ball had carried away three of his fingers. Although damaging stimuli do not necessarily hurt, however, we still can feel pain even without stimulus, or it can be caused by a stimulus that is clearly not damaging. One example is phantom limb pain. Patients with amputated limbs often complain of a feeling of pain in the ghost of the missing limb. A more day-to-day example that we are all familiar with occurs if the skin is burned, in getting a hot dish out of the oven. The burned area remains painful for days, and even a quite innocuous stimulus, such as a gentle touch or the warmth of contact with a hand, causes pain. So pain can be caused by a non-damaging stimulus, or even by no stimulus. However, if there is no direct or simple relationship between the strength of the stimulus and the magnitude of the pain, how can we sense it? Basically, pain-producing stimuli are detected in vivo by the nerve terminals of primary sensory neurons, whose cell bodies are found in sensory ganglia such as the dorsal root ganglia (DRG). These neurons, which can be activated by stimuli capable of causing tissue damages, are then transmitted along primary sensory nerve fibers to the dorsal horn of the spinal cord, and from there on to higher brain centers, where these are interpreted as pain1. The specialized primary sensory neurons involved in the transduction of painful stimuli into action poten233

Pain, and why we need it

tials are called nociceptors, a term coined by Sherrington 100 years ago. A century later, the isolated nerve cells cultured in a dish are found to respond in many ways like pain-sensitive nerve in a real animal2. Furthermore, temperature at which the isolated DRGs responds to heat is very similar to the temperature at which a human subject reports that the sensation from a warmed object changes from a pleasant feeling of warmth to a sensation of painful heat2. In fact, heat is found to directly stimulate the nerve endings and the process is caused by cations flowing in through the membrane of the cell2. To date, all such temperature-sensitive ion channels are members of the extensive transient receptor potential (TRP) family. The origin of the discovery of the TRP channels can be traced back to the 1960s, when a Drosophila mutant was found to show a transient response to prolonged bright light3. The trp gene was cloned in 1989, and was shown to encode a light-activated Ca2+ channel in Drosophila4. The mammalian TRP channel family contains more than 30 members, which can be divided into at least seven subfamilies, including TRPA (ankyrin), TRPC (canonical), TRPM (melastatin), TRPML (mucolipin), TRPN (NOMPC), TRPP (polycystin) and TRPV (vanilloid)5. Thermo-TRPs currently constains 9 members, including TRPV (TRPV1–4), TRPM (TRPM2, M4, M5 and M8) and TRPA (TRPA1). Each thermo-TRP is activated over a specific temperature range, and when working together in vivo they cover a wide cumulative temperature range from noxious heat (>52oC) to cold (42oC)2, acidic condition (pH 42oC) and various endogenous lipids. Residues reported to be involved in capsaicin and lipids binding are shown in green. In addition, residues which are critical for proton- and heatbinding are shown in purple and red, respectively.

inflammatory mediators, such as bradykinin2, prostaglandins35, anandamide36 and NGF37. The effects of these inflammatory mediators on nociceptors mainly activate kinases, including PKC2, PKA38, and Src39, which in turn sensitizes the heat-activated current in neurons, In fact, TRPV1 is one of the most important targets for these kinases in inflammatory hyperalgesia. Bradykinin Bradykinin is generated in tissue injury and noxious stimulation40. Injection of bradykinin into human skin produces a dose-dependent pain and a heat hyperalgesia41, suggesting that bradykinin is capable of exciting nociceptors as well as sensitizing their response to heat. The evidence that action potentials evoked by bradykinin are dramatically reduced by the TRPV1 antagonist capsazepine42,43, and that the response of C-fibers to bradykinin is much more decreased in TRPV1 knock-out mice than that in wild type mice23 supports the sensitization of TRPV1 is mainly by bradykinin. The biological effects of bradykinin in vivo are mediated through two transmembrane G-protein-coupled receptors, the B1 and B2 receptors44,45. B1 receptor, is mainly absent under normal non-inflamed conditions, but can be induced and overexpress in chronic inflammation46,47. B2 receptors are widely expressed in the nervous system. After activation of B2 receptor, PLCβ is triggered to breakdown PIP2 into

Fig. 2 Inflammatory mediators and intracellular signaling pathways modulating TRPV1. NGF (brown color) activates TrkA receptor, which then promotes the insertion of TRPV1 into a membrane, from a pool located in subcellular vesicles, via a pathway of PI3K-PKCδ-Src. Then it activates trafficking to the membrane by phosphorylating TRPV1 (blue). G-protein coupled receptors (purple colour) are activated by a range of inflammatory mediators, such as bradykinin. These receptors activate PLCβ, and consequently activate PKCε, which enhances the activity of TRPV1 through a phosphorylation manner. PGE2 and PGI2 activate EP and IP receptors (pink color), which couple to PKA and PKCε to activate TRPV1.

IP3, causing a burst of calcium in the cell, and the release of diacylglycerol (DAG), which in turn activates PKC. In addition, there are far more complicated molecular cascades looming after activation of B2 receptor, such as phospholipases A2, phospholipases D, tyrosine kinases, phosphatases and MAP kinases46. Functionally, bradykinin can amplify the current activated by heat in isolated DRG neurons2. This effect is similar to the result of direct activation of PKC. Moreover, PKCε is found to rapidly move to the cell membrane after exposure to bradykinin48, indicating that PKCε may connect to the thermal hyperalgesia evoked by bradykinin. The enhancement of TRPV1 activity through the PLCβ/PKCε pathway is a major molecular mechanism for bradykinin sensitizing nociceptors49. Furthermore, activation of PKCε by bradykinin via the B2 receptor leads to phosphorylation of TRPV1 at two serine residues, S502 and S800, which in turn potentiates the gating of TRPV1 by noxious stimuli50-53 (Fig. 2). 235

Pain, and why we need it

Prostaglandins and anandamide Prostaglandins are generated in response to noxious stimuli and inflammatory insults54. Several prostanoid receptor subtypes are found in DRG neurons, including the PGE2 receptors EP1-455,56 and the PGI2 receptor IP57. Behavioral studies of mice lacking IP, EP1 or EP3 receptors show a significant reduction in pain perception and thermal hyperalgesia, suggesting that EP and IP receptors play an important role in inflammation and pain58-61. Thermal hyperalgesia induced by PGI2 is found to abrogate in mice without TRPV1, indicating that TRPV1 is an essential contributor to thermal hyperaligesia induced by prostaglandin. So far, there are at least two secondmessenger pathways involved in potentiation of TRPV1 by PGE2 and PGI2: PGE2 activates both PGE receptor 1 (EP1) and PGE receptor 4 (EP4) in sensory neurons. The EP1 receptor is Gq-coupled and therefore activates PKC60, and the EP4 receptor is Gs-coupled and therefore activates PKA. PKA has also been shown to potentiate the activation of TRPV138,62 (Fig. 2). They sensitize TRPV1 by phosphorylating specific sites on the channel63,64. Anandamide (N-arachidonoyl-ethanolamide), which can activate the cannabinoid 1 (CB1) receptor, is found to be involved in inflammatory hyperalgesia31,65. Interestingly, TRPV1 and the cannabinoid 1 (CB1) receptor not only show a high degree of colocalisation66,67, structure analysis also shows a similarity to some extent between anandamide and olvanil, a synthetic vanilloids. All these findings suggests that anandamide may be able to activate TRPV168. In fact, the concentration of anandamide is found to be increasing in neurons with TRPV1 expression in inflammatory and pathological conditions. Moreover, bradykinin and PGE2 are found to activate pathways that convert anandamide to a potent TRPV1 activator in inflammation69. No doubt, anandamide indeed participates in inflammatory hyperalgesia. Althongh PKA is known to be able to sensitize TRPV1, the effect of CB1 receptor to TRPV1, in the presence of forskolin (an adenylate cyclase activator) is not like that. On the contrary, when exposure to forskolin, the activation of CB1 receptor attenuates TRPV1 activity70. Thus, the effect of CB1 activation on TRPV1 may be yin and yang: positive in the presence of anandamide and negative in the presence of forskolin. Nerve growth factor (NGF) NGF, a member of the neurotrophin family, interacts with two types of cell surface receptors, the tyrosine kinase receptor A (TrkA) and the pan-neurotrophin p75NTR receptor71,72. NGF is a potent growth factor, 236

which promotes the targeting of outgrowing nerve fibers in the developing organism, and it seems to play a role in the adult organism, as a proinflammatory mediator amongst its other roles73. Experiments show that injection of NGF into adult rat paw produced a rapid and prolonged hypersensitivity to noxious thermal stimulation73, and the pain became noticeable within a few minutes and maintained for several days74. In addition, the thermal hyperalgesia induced by injection of NGF is largely absent in TRPV1 knock-out mice75. Therefore, thermal hyperalgesia induced by NGF is mainly caused by the sensitization of TRPV1. In vitro study shows that administration of NGF (~ 10 minutes) enhances the response of DRG neurons to heat and capsaicin37,76,77, while long term (4~6 days) application of NGF increases the number of capsaicin-sensitive DRG neurons in culture78,79, suggesting that NGF may increase the activity of TRPV1 and the expression level of TRPV1 in DRG neurons. However, NGF does not increase TRPV1 mRNA levels in DRG neurons80, nor does affect the TRPV1 mRNA level in PC12 cells81. These data indicate that NGF regulates TRPV1 expression by increasing translation and transport of the channel without involvement of gene transcription. More and more molecular investigations conclude that there are at least two pathways for NGF-induced hyperalgesia. One is the PLCγ/ PKCε signaling pathway, which causes phosphorylation of TRPV1 at the S502 and S801. The other is the TrkA-dependent MAPK pathways, which are involved in NGF up-regulating TRPV1 expression82. In addition, Erk and PI3K are also involved in this process82. By site-directed mutagenesis, our lab shows that the activation of Y760 site on TrkA stimulates PI3 kinase, which then triggers PKCδto activate Src. Src kinase phosphorylates TRPV1 at a single tyrosine residue, Y200, leading to trafficking and insertion of the new generation, channels into the surface membrane and thus amplifying membrane ionic currents (Fig. 2). How are diverse signals to be integrated? At a given time point, there are various extracellular signals across the cell membrane to intracellular targets. Signaling kinases are widely distributed, and often have rather broad substrate selectivity. It is still unclear how these distinct activators converge upon the opening of the channel. Diverse studies on the various inflammatory mediators report multiple and sometimes contradictory effects on TRPs that occur via different signaling pathways. Even until now, the precise regulation of these polymodal sensory channels has not been completely understood. So far, we only know some intracellular sig-

Hueng-Chuen Fan, et al.

naling pathways involved in regulating the activity and expressions of TPRV1, including PKC-, PKA-, and tyrosine phosphorylation, and the temperature threshold of TRPV1 is lowered by a variety of pro-inflammatory mediators such as bradykinin, prostaglandin E2 (PGE2) and nerve growth factor (NGF). With these diverse signaling pathways acting on TRPV1 to potentiate or desensitize the channel, cells are specialized and equipped with “integrators” to organize various information, to prevent signals from diffusing, and to ensure a specific outcome to a particular ligand exposed at a particular time to a particular site. Hence, subcellular targeting complexes through association with anchoring proteins has emerged as an important mechanism by which cells localize signaling molecules to sites where they can be accessed optimally by specific activators83. The A kinase anchoring protein (AKAP) family of scaffolding proteins were originally named for their ability to target PKA to appropriate substrates, but are known to assemble a wide range of kinases and phosphatases into signaling complexes with appropriate targets84. A number of ion channels are subject to modulation by AKAPs, including glutamate receptors, calcium channels and the M-type potassium channels85-88. The process of the potentiation of TRPV1 by PKA is found to be augmented by the presence of AKAP89. Work in our lab has shown that AKAP150 (rat) is found to co-express TRPV1 in rat trigeminal ganglia (TG) neurons. Functionally, knockdown AKAP150 attenuates the sensitization of TRPV1 by PKA, and administration of an AKAP antagonist significantly reduces sensitization to thermal stimuli by PGE2. Furthermore, the phosphorylation level of TRPV1 by PKA is significantly reduced when AKAP 150 is knock-downed by siRNA. PKA and PKC are anchored adjacent to TRPV1 through AKAP79 (human), and that the binding site of this complex has been identified. Therefore, not only can AKAPs assemble a wide range of kinases and phosphatases into signaling complexes with appropriate targets, but also fine-tune the effects of PKC and PKA-mediated phosphorylation/sensitization on the TRPV190. It is AKAP79 that orchestras distinct signaling molecules, such as PKA and PKC, and controls their phosphorylation leading to modulating their target proteins (Fig. 3).

CONCLUSION TRP channels are known to be molecular gateways in sensory systems, and interfaces between the environment and the nervous system. TRPV1 is able to trans-

Fig. 3 AKAP79/150 Forms a Signaling Complex with TRPV1 The scaffolding protein AKAP79/150 binds to the C-terminal domain of TRPV1 (orange cylinder). The formation of this scaffolding complex aligns PKCε, the phosphatase calcineurin (PP2B) and PKA so as to control phosphorylation of key sites on TRPV1. The principal site involved in control of trafficking of TRPV1 to the membrane is S502. The tyrosine kinase Src can also promote trafficking to the membrane by phosphorylating Y200 but does so independently of AKAP79/150.

duce thermal, chemical, and mechanical stimuli into inward currents, an essential and first step for eliciting thermal and pain sensations91. On nociceptive terminals, phosphorylation of TRPV1 results in the sensitization to many different stimuli, contributing to the development of hyperalgesia. The phosphorylation is mediated by a number of messengers, such as PKC and/or PKA. During inflammation, the activation of the Gq-coupled B1 and B2 receptors by bradykinin causes the temperature threshold of TRPV1 to be lowered by PKCε-mediated phosphorylation48. Prostaglandins, are a group of inflammatory mediators, activate PKA38 and PKC53 to phosphorylate TRPV1. NGF potentiates TRPV1 via a pathway involving PI3K and the tyrosine kinase Src, triggering an insertion of TRPV1 into the neuronal cell membrane39. Taken together, the ways these inflammatory mediators delicately and precisely regulate the sensitivity of TRPV1 are incredibly manipulated by a diverse and complex network. Among them, the scaffolding protein A-kinase anchoring protein (AKAP) is the key to orchestrate and integrate complicated signaling molecules to modulate this sensory gateway. The integration of these scaffolding proteins in inflammatory hyperalgesia may be a promising target for the development of novel analgesics. 237

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ACKNOWLEDGMENTS Professor Peter McNaughton would like to express his deepest gratitude to the Tri-Service General Hospital and National Defense Medical Center and the grant NSC 098-2912-i-016-006 from National Science Council for the support of this work.

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