Reactive oxygen species are second messengers of neurokinin signaling in peripheral sensory neurons

John E. Linleya,1, Lezanne Ooia,b,1, Louisa Pettingera, Hannah Kirtona, John P. Boylec, Chris Peersc, and Nikita Gampera,2 aInstitute of Membrane and Systems Biology, Faculty of Biological Sciences, and cLeeds Institute for Genetics, Health and Therapeutics, Faculty of Medicine and Health, University of Leeds, LS2 9JT Leeds, United Kingdom; and bUniversity of Western Sydney, School of Medicine, Penrith, New South Wales 2751, Australia AUTHOR SUMMARY

Mammals are equipped with nerve endings causing acute several systems to sense the en- “spontaneous” pain (that is, vironment. A somatosensory LOCAL INFLAMMATION pain not induced by external system collects tactile infor- stimulation). Bradykinin also mation from the outside world, . . increases the sensitivity of pe- monitors the status of the body, Antinociception Hyperalgesia Spontaneous pain ripheral nerves to thermal and and also informs the brain about + SP + - + mechanical stimulation (hyper- BK the occurrence of injury, thus algesia). Previous research sug- 2 2 2 2 M M PM PIP PIP PIP PIP generating a sensation that we DAG DAG gests that the spontaneous

G G R 2 CaCC i/o PKCε TRPV1 PKCε q/11

know as pain. Physiological pain B pain induced by bradykinin is NK1 IP3 is crucial for survival; however, ROS PLC P PLCβ mediated by the IP -induced Ca2+ 3 disease or injury can result in release of Ca2+ from specific pathological pain (e.g., arthritis intracellular reservoirs of Ca2+ pain, migraine, neuropathic Mitochondria ions called the “endoplasmic pain) that has no benefit to the reticulum” (Fig. P1). IP3 opens organism but brings suffering Ca2+-permeable pores within and distress. Such pain is diffi- Fig. P1. Signaling cascades generated in nociceptive neurons by the endoplasmic reticulum, substance P (SP) and bradykinin (BK). B2R, bradykinin receptor subtype 2+ − cult to treat with conventional 2; CaCC, Ca2+-activated Cl channels; DAG, diacylglycerol; ER, allowing the release of Ca into analgesics and, despite much the cytosol. This Ca2+ release endoplasmic reticulum; Gi/o and Gq/11, G protein alpha subunit i/o effort, the success of new treat- or q/11 types, respectively; M, M-type K+ channel; NK1, neurokinin mediates several excitatory ac-

ment strategies is limited. The receptor subtype 1; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, tivities in nociceptors, including somatosensory system consists inositol 1, 4, 5-trisphosphate; PKC, protein kinase C; PLC, phospholipase inhibition of M-type K+ chan- of the peripheral sensory nerves, C; PM, plasma membrane; TRPV1, transient receptor potential cation nels (which are needed to which have highly sensitive channel, subfamily V, member 1. “+” indicates activation and “−” maintain nociceptive neurons in endings in the skin and viscera depicts inhibition. a low-excitability state) and ac- − from which they reach the spinal tivation of a Ca2+-activated Cl cord. However, peripheral nerves are not mere “receivers” of the channels (also called “TMEM16A” or “ANO1”), which make primary external stimuli; they also have the ability to respond nociceptive neurons more excitable (1) (Fig. P1). In contrast, independently to external stimulation by releasing chemical bradykinin-induced hyperalgesia is linked to the sensitization of factors such as neuropeptides into the surrounding tissues. Thus, sensory receptors, such as the heat sensor TRPV1 (Fig. P1), an pain-sensing (nociceptive) nerves produce several neuropeptides effect that makes nociceptive neurons “overreact” upon pre- and can release them at their peripheral endings and into the sentation with external thermal stimuli. Surprisingly, we found spinal cord. For example, the peripheral release of one such that although injection of bradykinin in the rat hind paw pro- neuropeptide, known as “substance P,” can produce local duces both spontaneous pain (i.e., the injection is painful) and (“neurogenic”)inflammation. There is growing evidence that the thermal hyperalgesia, injection of substance P produces only peripheral sensory nerves themselves also are equipped with hyperalgesia but is not painful. The present study explains the substance P receptors (e.g., specialized protein receptors, called difference in painful effects of substance P and bradykinin and “neurokinin receptors”), of which three (NK1, NK2, and NK3) allows us to understand the cellular basis of the separate have been identified to date. However, until now, the purposes mechanisms responsible for spontaneous inflammatory pain of these receptors in sensory nerves and the sequence of in- and hyperalgesia. tracellular events they trigger have not been elucidated. Electrophysiological measurements and imaging of cultured Neurokinin receptors belong to a group of plasma membrane dorsal root ganglia (DRG) sensory neurons established that receptors called “G protein-coupled receptors”; they signal through the intracellular signaling cascade involving G protein alpha subunits q or 11 (Gq/11) and phospholipase C. Phospholi- pase C hydrolyzes plasma membrane phospholipid phosphati- Author contributions: J.E.L., L.O., J.P.B., C.P., and N.G. designed research; J.E.L., L.O., L.P., H.K., J.P.B., and N.G. performed research; J.E.L., L.O., L.P., H.K., J.P.B., C.P., and N.G. dylinositol 4,5-bisphosphate (PIP2) to produce the plasma analyzed data; and J.E.L., L.O., L.P., J.P.B., C.P., and N.G. wrote the paper. membrane-bound second messenger diacylglycerol and a soluble The authors declare no conflict of interest. second messenger, inositol trisphosphate (IP3). This signaling cascade is responsible for the action of many inflammatory This Direct Submission article had a prearranged editor. mediators (chemical components of the tissue immune response Freely available online through the PNAS open access option. that serve as signaling molecules), which are able to excite no- 1J.E.L. and L.O. contributed equally to this work. ciceptive neurons and so cause inflammatory pain. A classical 2To whom correspondence should be addressed. E-mail: [email protected]. example of such inflammatory mediators is bradykinin, a peptide See full research article on page E1578 of www.pnas.org. that is released by damaged tissue; bradykinin excites nociceptive Cite this Author Summary as: PNAS 10.1073/pnas.1201544109.

9246–9247 | PNAS | June 12, 2012 | vol. 109 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.1201544109 Downloaded by guest on September 28, 2021 PNAS PLUS (i) substance P can induce phospholipase C activity in sensory receptors signal through the Gi/o-coupled pathway. Interestingly, neurons; (ii) substance P increases neuronal responses to the overexpression of cloned NK1 in the immortalized cell line TRPV1 agonist capsaicin, suggesting sensitization of TRPV1; or in cultured DRG neurons resulted in reconstitution of iii 2+ ( ) in contrast to bradykinin, substance P usually does not the classical Gq/11 cascade featuring robust release of Ca 2+ induce release of Ca from the endoplasmic reticulum; and from the endoplasmic reticulum and inhibition of M current. (iv), intriguingly, substance P not only failed to inhibit M current This observation strongly suggests that endogenous and overex- + (a current conducted by the M-type K channels) in sensory pressed neurokinin receptors in neurons couple to different neurons but instead caused marked augmentation of M current, signaling pathways, probably because of the association of en- an effect that was mirrored by decreased excitability. Thus, dogenous neurokinin receptors with another, yet to be identified, substance P signaling in sensory neurons displayed marked de- Gi/o-coupled receptor. viation from that expected by the Gq/11-mediated pathway. Our present study provides a reconstruction of the molecular The M current-augmenting effect of substance P was entirely events triggered in the peripheral nociceptive neuron by sub- unexpected. Therefore, we investigated it further. The effect of stance P. We report a signaling cascade in which endogenous substance P resembled that of the augmentation of M current reactive oxygen species are used as second messengers to aug- by reactive oxygen species that we reported earlier (2). Reactive ment M channel activity acutely, reducing the excitability of oxygen species are highly reactive oxygen-containing small nociceptive neurons. Moreover, we provide clear evidence that molecules such as hydrogen peroxide, which are produced by fl various enzymes of cellular metabolism and which increasingly spontaneous in ammatory pain and hyperalgesia can be induced by distinct underlying mechanisms within a single nociceptive are recognized as intracellular signaling molecules. The effect of fi substance P on M current was reversed by the application of neuron (Fig. P1). The latter nding is of particular importance a reducing agent, DTT, suggesting that, like reactive oxygen for designing future strategies for analgesic drug discovery, be- species, substance P induces oxidative modification of the M- cause spontaneous pain and hyperalgesia are very poorly dis- channel protein. Therefore we hypothesized that substance P can tinguished conceptually at present. In fact, most animal models induce the production and release of endogenous reactive oxy- currently used for testing analgesic drug efficacy are based on gen species in DRG neurons. Indeed, imaging of cytosolic con- hyperalgesia measurements (4) even though most patients with centrations of reactive oxygen species with a specific dye chronic pain suffer from spontaneous pain rather than from indicated that substance P does induce elevated levels of cyto- hyperalgesia. Accordingly, despite the rapid progress in our un- solic reactive oxygen species. Moreover, genetically introducing derstanding of the mechanisms of hyperalgesia, there has been a molecular sensor of reactive oxygen species into cultured DRG rather limited achievement in treatment of pain in humans (5). neurons revealed “sparks” of mitochondrial release of reactive Our study provides a mechanistic concept for the difference oxygen species in response to substance P. We then used a res- between these two types of pain and highlights the need to pirometry technique that allows measurement of the activity of change current approaches to animal pain models and the mitochondrial electron-transport chain, which is a main drug testing. source of intracellular reactive oxygen species. Substance P in- duced marked inhibition of electron-transport chain activity, 1. Liu B, et al. (2010) The acute nociceptive signals induced by bradykinin in rat sensory possibly at a complex III (one of the four major electron-trans- neurons are mediated by inhibition of M-type K+ channels and activation of Ca2+- porting complexes of electron-transport chain), an observation activated Cl- channels. J Clin Invest 120:1240–1252. fi + that is consistent with the reported release of reactive oxygen 2. Gamper N, et al. (2006) Oxidative modi cation of M-type K channels as a mechanism of cytoprotective neuronal silencing. EMBO J 25:4996–5004. species induced by the inhibition of the electron-transport chain 3. Zorov DB, Filburn CR, Klotz LO, Zweier JL, Sollott SJ (2000) Reactive oxygen species complex III (3). Consistently, inhibition of complex III mimicked (ROS)-induced ROS release: A new phenomenon accompanying induction of the the substance P-induced augmentation of M current. mitochondrial permeability transition in cardiac myocytes. J Exp Med 192:1001–1014. Because substance P signaling in sensory neurons deviated 4. Vierck CJ, Jr. (2006) Animal models of pain. Wall and Melzack’s Textbook of Pain, eds McMahon SB, Koltzenburg M (Elsevier Churchill Livingstone, Edinburgh), 5th Ed, pp from the Gq/11 pathway, we investigated if this signaling is me- 175–185. diated by another G protein alpha subunit. Indeed, the phar- 5. Hruby VJ, et al. (2006) New paradigms and tools in drug design for pain and addiction. macological evidence suggested that endogenous neurokinin AAPS J 8:E450–E460. NEUROSCIENCE

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