Protein kinase Cδ mediates histamine-evoked itch and responses in pruriceptors
Molecular Pain volume 11, Article number: 1 (2015)
Itch-producing compounds stimulate receptors expressed on small diameter fibers that innervate the skin. Many of the currently known pruritogen receptors are Gq Protein-Coupled Receptors (GqPCR), which activate Protein Kinase C (PKC). Specific isoforms of PKC have been previously shown to perform selective functions; however, the roles of PKC isoforms in regulating itch remain unclear. In this study, we investigated the novel PKC isoform PKCδ as an intracellular modulator of itch signaling in response to histamine and the non-histaminergic pruritogens chloroquine and β-alanine.
Behavioral experiments indicate that PKCδ knock-out (KO) mice have a 40% reduction in histamine-induced scratching when compared to their wild type littermates. On the other hand, there were no differences between the two groups in scratching induced by the MRGPR agonists chloroquine or β-alanine. PKCδ was present in small diameter dorsal root ganglion (DRG) neurons. Of PKCδ-expressing neurons, 55% also stained for the non-peptidergic marker IB4, while a smaller percentage (15%) expressed the peptidergic marker CGRP. Twenty-nine percent of PKCδ-expressing neurons also expressed TRPV1. Calcium imaging studies of acutely dissociated DRG neurons from PKCδ-KO mice show a 40% reduction in the total number of neurons responsive to histamine. In contrast, there was no difference in the number of capsaicin-responsive neurons between KO and WT animals. Acute pharmacological inhibition of PKCδ with an isoform-specific peptide inhibitor (δV1-1) also significantly reduced the number of histamine-responsive sensory neurons.
Our findings indicate that PKCδ plays a role in mediating histamine-induced itch, but may be dispensable for chloroquine- and β-alanine-induced itch.
Itch, clinically known as pruritus, is an unpleasant sensory and emotional experience that leads to the desire to scratch [1, 2]. Chronic itch can result in severe anxiety, self-mutilation, and impaired overall quality of life that is comparable to chronic pain [3–5]. Several histamine-dependent and histamine-independent itch receptors have been recently identified; however, few of the intracellular mediators downstream of these receptors have been characterized. Elucidating the intracellular mediators that activate pruriceptors may provide a new set of targets to aid in the generation of more specific and efficacious treatments.
Intradermal histamine induces itch via direct activation of the H1 histamine receptor, which is expressed in sensory neurons [6–10]. Additionally, several subtypes of the recently characterized class of Mas-related G protein-coupled receptors (MRGPR) have been shown to respond selectively to a variety of non-histaminergic, itch-producing compounds. For example, MRGPRA3 is activated by the anti-malarial drug chloroquine (CQ) [11, 12], and β-alanine induces itch by activating a subset of nonpeptidergic MRGPRD-expressing sensory neurons [13, 14].
A common property of many of the identified pruritogen receptors, including the H1 histamine receptor (H1R), MRGPRA3, and MRGPRD, is that they are Gq protein-coupled receptors (GqPCRs) [10–12, 14]. Canonically, GqPCRs activate phospholipase C (PLC), which cleaves phosphatidylinositol (PIP2) into inositoltriphosphate (IP3) and diacylglycerol (DAG), resulting in release of intracellular calcium stores and activation of downstream targets. However, the itch-mediating factors downstream of PLC are largely unknown [10, 15]. Protein kinase C (PKC) is coupled to the canonical GqPCR/PLC pathway via activation by DAG and/or calcium and therefore may play a role in the signaling of itch.
A number of PKC isoforms are expressed in sensory neurons [16–19]. One of these isoforms is PKCδ, a member of the “novel” PKC isozymes, which depends on DAG but not calcium for its activation. Previously, we demonstrated that PKCδ is dispensable for withdrawal responses to acute noxious mechanical and thermal stimuli . However, studies of H1R signaling in human aortic endothelial cells and HeLa cells showed that PKCδ is phosphorylated in response to histamine [21, 22]. PKCδ also mediated histamine-induced H1R mRNA upregulation and downstream activation of ERK1/2 and p38 [21, 22]. These lines of evidence suggest that PKCδ could play a specific role as an intracellular modulator of itch in sensory neurons.
In this study, we tested the hypothesis that PKCδ contributes to pruritogen-induced itch. We determined the role of PKCδ in histaminergic and non-histaminergic itch by examining scratching responses to histamine and the non-histaminergic pruritogens chloroquine and β-alanine, which activate separate pruriceptor subpopulations. We characterized the distribution of PKCδ in sensory neurons and show that both genetic deletion and pharmacological inhibition of PKCδ significantly decrease the proportion of histamine-responsive neurons.
PKCδ mediates histamine-induced itch
To determine if PKCδ plays a role in behavioral responses to itch, we assessed scratching responses to histamine and non-histaminergic pruritogens in PKCδ knock-out mice (PKCδ-KO) and their wild type littermates. Mice were injected intradermally at the nape of the neck with one of three pruritogens: histamine (1 mg), chloroquine (CQ) (200 μg), or β-alanine (223 μg). PKCδ-KO mice scratched significantly less than their wild type littermates when injected with histamine (Figure 1A, p < 0.05). On the other hand, chloroquine-induced scratching was not significantly different between WT and PKCδ KO mice (Figure 1B, p = 0.129). There was also no difference in the number of scratch bouts induced by β-alanine (Figure 1C, p = 0.61). These results indicate that PKCδ mediates histaminergic itch, but is not necessary for non-histaminergic itch induced by CQ and β-alanine.
PKCδ is preferentially expressed in small diameter DRG neurons
PKCδ is expressed in a variety of tissues, including the brain and peripheral nervous system [19, 21, 23–30]. To assess whether PKCδ is localized to potential pruriceptive sensory neurons, immunohistochemistry (IHC) was used to characterize the distribution of PKCδ in dorsal root ganglion (DRG) neurons. Antibody specificity was confirmed via western blot using PKCδ-KO DRG and spinal cord tissue. No antibody staining was found corresponding to the 78kD PKCδ band in PKCδ-KO DRG tissue (Figure 2A). This was further confirmed by IHC of knock-out and wild type DRG (Figure 2B). In wild type lumbar DRG, PKCδ was expressed in 43.2% of total neurons labeled with βIII tubulin (567/1314 cells, n = 3 animals) and PKCδ expression was predominantly restricted to small diameter neurons (average diameter 23.3 ± 0.19 μm, min 11.6 μm, max 36.2 μm) (Figure 2C).
PKCδ is expressed in peptidergic and non-peptidergic DRG neurons
We further characterized PKCδ expression in small diameter DRG neurons by immunohistochemical analysis of peptidergic and non-peptidergic markers. Of peptidergic neurons identified by anti-Calcitonin Gene Related Peptide (CGRP+) immunoreactivity, 26.8% expressed PKCδ, while 14.7% of PKCδ-expressing neurons were CGRP-positive (Table 1, Figure 3A-B). PKCδ was also expressed in non-peptidergic neurons identified by isolectin B4 (IB4) binding. Of IB4+ DRG neurons, 61.6% expressed PKCδ and 55.0% of PKCδ+ neurons exhibited IB4 binding (Table 1, Figure 3C-D). These findings indicate that PKCδ is expressed in both peptidergic and non-peptidergic sensory neurons, with greater expression overlap found with non-peptidergic IB4+ neurons.
Behavioral and physiological studies have shown that itch produced by histamine is largely dependent on the non-specific cation channel transient receptor potential vanilloid receptor 1 (TRPV1) [31–33]. Because PKC has previously been demonstrated to modulate TRPV1 function and may present one potential mechanism by which PKCδ regulates itch, we determined the degree of overlap between PKCδ and TRPV1 expression [34, 35]. We first confirmed the specificity of our antibody directed against TRPV1 using TRPV1 knockout mice (Figure 3E, inset). Our data indicate that 29.1% of PKCδ-positive neurons were also TRPV1-positive, and 48.9% of TRPV1-positive neurons also expressed PKCδ, suggesting a potential functional relationship between PKCδ and TRPV1 (Figure 3E-F; Table 1).
PKCδ-KO sensory neurons exhibit diminished responses to histamine
PKCδ is expressed in the brain, spinal cord, and the peripheral nervous system [30, 36–39]. Therefore, global genetic deletion of PKCδ in our knockout mice makes it difficult to pinpoint where PKCδ functions to modulate histamine-induced scratching. The expression of PKCδ in small diameter sensory neurons suggests that it may mediate histamine-evoked itch by signaling in nociceptive neurons responsive to pruritic agents (i.e., pruriceptors). To determine if PKCδ directly modulates neuronal responses to histamine, calcium imaging was performed on acutely dissociated adult mouse DRG neurons (Figure 4A-B). Of the total sensory neurons treated with histamine, 11.1% of wild type neurons responded to bath application of 100 μM histamine (126/1137 total WT neurons, N = 5 animals), but only 6.7% of PKCδ-KO neurons responded to histamine (47/706 total KO neurons, N = 3 animals), indicating a significant reduction of 39.6% in the proportion of histamine responsive neurons (p < 0.01, χ2 test) (Figure 4C). No significant difference in peak calcium responses to histamine was detected between knock-out and wild type cells (WT 33.6 ± 2.8% increase from baseline, n = 126 cells; KO 39.9 ± 6.6% increase from baseline, n = 47 cells, unpaired t-test, p = 0.304) (Figure 4D).
We hypothesized that PKCδ could contribute to neuronal responses to histamine by mediating histamine receptor coupling to TRPV1, or by regulating the normal expression or function of TRPV1. To investigate whether the absence of PKCδ affects the activation of TRPV1 within histamine-responsive neurons, we applied the TRPV1-specific agonist capsaicin after the histamine response. We found that 58.7% of WT histamine-responsive neurons subsequently responded to capsaicin (74/126 total His+ neurons), while only 40.4% of KO histamine-sensitive neurons responded to capsaicin (19/47 total His+ neurons; p < 0.05, χ2 test) (Figure 4E). To determine whether the functional expression of TRPV1 is altered in PKCδ-KO neurons, we tested WT and KO sensory neurons for responses to capsaicin. There were no differences in the total number of capsaicin-responsive neurons between KO and WT groups (117/194 (60.3%) WT neurons, 144/259 (55.6%) KO neurons, p = 0.315, χ2 test, Figure 4F). Together, these data indicate that the reduction in histamine responses is not due to altered levels of TRPV1 receptors in the KO, and support the idea that PKCδ could modulate TRPV1 downstream of histamine receptor activation.
To further control for the possibility of developmental effects or compensatory mechanisms that may occur with congenital genetic deletion of PKCδ, we performed calcium imaging experiments with the same experimental design using acute pharmacological inhibition in wild type DRG cultures. The δV1-1 peptide inhibitor has been shown to inhibit PKCδ activation in vitro and in vivo by competitively binding to receptor for activated C-kinase (RACK) proteins, which confer PKC isoform substrate specificity [28, 40–43]. DRG neurons were incubated with the peptide inhibitor δV1-1 for 30 minutes prior to recording. Consistent with our findings in PKCδ-KO neurons, there was a significant reduction in the total number of histamine-responsive neurons treated with peptide inhibitor when compared to scramble peptide-treated control neurons (18.5% (75/405) of scramble-treated neurons vs. 13.1% (69/527) of peptide-treated neurons, N = 4 animals, p < 0.05, χ2 test) (Figure 4G). Peak calcium responses induced by histamine were not different between inhibitor- and scramble-treated groups (inhibitor 33.23 ± 2.7% change from baseline, n = 69 cells; scramble 41.0 ± 3.7% change from baseline, n = 75 cells, p = 0.095, unpaired t test) (Figure 4H).
Pruritic stimuli can activate sensory neurons via specific intracellular signaling cascades, which represent potential targets for anti-pruritics, but these cascades remain poorly understood. Characterizing signaling components is a significant challenge, in part because of the great diversity of recently identified pruritic receptors. Some receptors involved in pruritus are coupled to Gi/o cascades such as the H4 histamine receptor, while others utilize kinase signaling pathways like the TSLP/IL-7 receptors [6, 7, 44, 45]. However, the majority of identified pruritic receptors, including the H1 receptor and the “orphan” family of MRGPR receptors, are linked by a common Gαq signaling mechanism [10, 46–49]. In this study we focused on a component downstream of the canonical Gαq signaling pathway, the serine/threonine kinase PKC.
PKC isozymes are divided into three groups: classic (activated by DAG and Ca2+), novel (activated by DAG but not Ca2+), and atypical (activated by neither DAG nor Ca2+) [50, 51]. Specific PKC isoforms have been shown to selectively regulate nociceptive behavior and nociceptor physiology [16, 17, 19, 52–58]. We previously showed that the novel isozyme PKCδ is dispensable for acute mechanical and thermal nociceptive behaviors . Previous reports have implicated PKCδ in H1R signaling in non-neuronal cells [21, 22], but the specific role of PKCδ in pruriceptor signaling and itch had not been explored. In this study we found that PKCδ was necessary for the full expression of histamine-induced itch, but it did not have significant effects on histamine-independent itch produced by the MRGPR ligands chloroquine or β-alanine.
To determine whether the scratching deficit we observed in PKCδ null mice could be attributed to loss of function within sensory neurons, we examined anti-PKCδ staining in mouse lumbar DRG. Previous studies indicated that PKCδ is expressed in murine spinal cord and DRG, but the precise subset of PKCδ-positive neurons had not been characterized [30, 36–39]. We found that PKCδ expression was restricted to small diameter dorsal root ganglion neurons. Furthermore, although PKCδ was expressed in both peptidergic and non-peptidergic sensory neurons, it was greatly enriched in the non-peptidergic subset. Both peptidergic and nonpeptidergic fibers have been shown to play a role in pruritus. Histaminergic itch is largely dependent on CGRPα-positive neurons , however, a subset of histamine-responsive neurons also express the nonpeptidergic marker IB4 [11, 60].
Calcium imaging studies of dissociated DRG neurons demonstrated that genetic deletion of PKCδ resulted in a significant reduction of the proportion of adult sensory neurons that were histamine-responsive. We further confirmed these results using acute pharmacological inhibition, supporting the hypothesis that PKCδ functions within normal wild type sensory neurons to mediate acute histaminergic signaling. The expression of PKCδ in small diameter sensory neurons, along with the reduction of histamine-responsive sensory neurons, suggest a peripheral mechanism for the behavioral effects of global PKCδ deletion on histamine-induced scratching.
Following histamine release, sensory neuron signaling to produce itch is thought to depend on functional coupling of H1R to TRPV1. Supporting this idea, mice lacking TRPV1 exhibit greatly reduced scratching behavior and cellular responses to histamine, and blocking TRPV1 channel function likewise abolishes the response of sensory neurons to histamine [31, 33, 61]. The mechanisms by which H1R recruits TRPV1 are complex and several different signaling pathways have been implicated [32, 33, 62, 63]. One possible mechanism by which histamine could couple to TRPV1 in sensory neurons is through PLC-induced PKC activation. This is further supported by an expanding body of literature indicating that PKC directly modulates TRPV1 function [34, 35]. Indeed, inhibitors for PLC and PKC prevent histamine-induced TRPV1-potentiation . We previously found that acute mechanical and thermal pain were independent of PKCδ. In contrast, PKCδ was necessary for the full expression of thermal hyperalgesia during Complete Freund’s Adjuvant-induced inflammation, which is a TRPV1 dependent process .
In this study, 49% of TRPV1-expressing neurons were PKCδ-positive and we showed a significant reduction in the proportion of capsaicin-responsive neurons within the subset of neurons responsive to histamine. Additionally, sensory neurons from PKCδ-KO mice not previously treated with histamine responded similarly to wild type neurons when challenged with capsaicin. These observations indicate that acute detection of heat stimuli by TRPV1 is not dependent on PKCδ, but suggest that PKCδ could function downstream of the histamine receptor to modulate TRPV1 function. In further support of this idea, several other inflammatory mediators including PGE2, NGF, and IL-6 have been shown to activate PKCδ [39, 64–66]. It is possible that pruritic dermatoses marked by inflammation may recruit PKCδ, resulting in sensory neuron modulation that could potentiate itch.
In contrast to histaminergic signaling, the MRGPR receptors appear to produce their pruritic effects through an alternative, PKCδ-independent pathway. Chloroquine activates MRGRPA3 which couples to the irritant receptor TRPA1 to produce itch. Neuronal responses to chloroquine were prevented by inhibiting Gβγ subunit activity, suggesting that the Gαq pathway is not necessary for chloroquine-induced itch . However, MRGPRA3 was also recently shown to sensitize TRPV1 via a PKC mechanism likely dependent on Gαq signaling, suggesting a possible mechanism for thermal sensitization . This suggests that MRGPRA3 may have biased signaling mechanisms that lead to itch and/or TRPV1-related sensitization. Thus, the histamine receptor and MRGPRs may share a pathway leading to sensitization of TRPV1 via PKC signaling, despite an alternative Gβγ mechanism for MRGPRA3 signaling of itch [46, 68].
In summary, we found that PKCδ is a mediator of histaminergic itch signaling in sensory neurons. Although we specifically investigated PKCδ in this study, other PKC isoforms may also be involved in modulating the response to itch. For example, another novel PKC isozyme, PKCϵ, is expressed in largely IB4+ neurons, and has been shown to also modulate TRPV1 responses [16, 57, 58, 69]. The sensory neuron responses involved in itch are complex, involving multiple molecular cascades which may be differentially mediated by specific PKC isoforms. Future studies that investigate the roles of PKC isozymes in itch may contribute to better therapeutic specificity for the treatment of acute and chronic pruritus.
Our studies indicate that PKCδ significantly contributes to histamine-induced scratching behavior, but may be dispensable for non-histaminergic itch induced by the pruritogens chloroquine and β-alanine. In the peripheral nervous system, PKCδ expression is restricted to small diameter sensory neurons, and is found in both peptidergic and nonpeptidergic neurons. Physiological studies of cultured adult DRG demonstrate that PKCδ mediates histamine-induced responses of sensory neurons using genetic and pharmacological tools. In addition, PKCδ may act downstream of the histamine receptor to modulate TRPV1 activity. We conclude that PKCδ regulates sensory neuron responses necessary for acute histaminergic itch, and future studies should address the role of PKCδ in persistent and inflammatory pruritic conditions. Because PKCδ shows no effects on acute pain , but contributes to histaminergic itch, PKCδ inhibition may be a potential therapeutic target to selectively control pruritus.
Subjects and ethical approval
All experiments were conducted in accordance with the National Institute of Health guidelines and received the approval of the Animal Care and Use Committee of Washington University School of Medicine. 8–12 week old male littermate mice were housed on a 12 hour light–dark cycle and allowed ad libitum access to food and water.
PKCδ-KO mice were obtained from Dr. Michael Leitges . These mice were generated using a standard gene targeting approach to insert a LacZ/neo cassette in the first transcribed exon of the PKCδ gene to abolish transcription, resulting in a global knock-out . PKCδ-KO mice were backcrossed on a C57BL/6 background for at least 6 generations prior to use. PKCδ-KO mice were then crossed with wild type C57BL/6 mice to generate heterozygous mice, which were used to generate wild type and KO littermates.
Pruritogen-induced scratching behavior
The nape of the neck and upper back were shaved with electric clippers one day prior to behavioral experiments. On the day of experiment, mice were placed in individual plexiglass observation boxes and allowed to acclimate in the presence of white noise for 2 hours. Using gentle restraint, 50 μl consisting of pruritogen dissolved in 0.9% normal saline was injected intradermally at the nape of the neck using a 29½ gauge insulin syringe. The following pruritogen amounts were used: 1 mg histamine (Sigma Aldrich, St. Louis, MO), 200 μg chloroquine (Sigma Aldrich, St. Louis, MO), and 223 μg β-alanine (Sigma Aldrich, St. Louis, MO). A single scratch bout was defined as one or more rapid back-and-forth motions of the hindpaw directed at the injection site, ending with either a pause, licking, or biting of the toes or placing of the hindpaw on the floor. Scratch bouts by the hind-paw directed at the injection site were counted over a period of 30 minutes. Experimenters were blinded to mouse genotype.
PKCδ immunohistochemistry/Western Blotting protocol
For Western blotting, mice were euthanized by swift decapitation and lumbar spinal cord and lumbar DRG were removed. Tissue samples were homogenized in homogenization buffer (20 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1 mM sodium pyrophosphate, 25 μg/ml aprotinin, 25 μg/ml leupeptin and 100 μM phenylmethylsufonyl fluoride) on ice. 7 μg of spinal cord and DRG protein were separated using 4-12% SDS-PAGE, then transferred to nitrocellulose membrane. Membrane was blocked in Odyssey blocking buffer for 1 hour, then incubated in rabbit anti-PKCδ (1:1000, Santa Cruz) and mouse anti-β-Tubulin (1:1000, Sigma-Aldrich) primary antibodies in Odyssey buffer with 0.1% Tween-20 at 4°C overnight. Blots were then washed in TBS-0.1% Tween-20, and incubated for 1 hour at room temperature in secondary antibodies (goat anti-rabbit Alexa Fluor 680 (1:20,000, Sigma Aldrich); goat anti-mouse IR800 (1:20,000, Sigma Aldrich)). Blots were washed and scanned using an Odyssey infrared scanner.
For immunohistochemistry (IHC), mice were deeply anesthetized with a ketamine, xylazine, and acepromazine cocktail, then perfused intracardially with cold PBS followed by 4% paraformaldehyde in PBS. Lumbar DRG were removed and cryoprotected in 30% sucrose. Transverse sections were cut at 18 μm thickness on a cryostat and collected on slides. To determine the percentage of total neurons that express PKCδ, dual labeling was performed with rabbit anti-PKCδ (1:50, Santa Cruz) and mouse anti-β-tubulin (1:1000, Sigma Aldrich) primary antibodies. Briefly, sections were blocked in 2% BSA, 0.1% Milk powder, 0.05% Tween-20 TBS for 1 hr, then incubated in primary antibodies overnight at 4°C. On day 2, slides were washed and incubated in secondary antibodies for 2–4 hours at 4°C (Alexa Fluor 488 Donkey anti-rabbit 1:200, Alexa Fluor 555 donkey anti-mouse 1:200, Invitrogen). Images were obtained using an upright epifluorescent microscope (Nikon 80i, CoolSnapES camera). Labeled neurons were counted in at least 3 randomly selected sections separated by >50 μm per animal. The size distribution of PKCδ+ neurons was determined using ImageJ software to measure cell diameter. The percentage of PKCδ+ neurons that also expressed CGRP or IB4 was determined using dual labeling for PKCδ and CGRP (goat anti-CGRP 1:400, Serotec) or Alexa-568 conjugated to IB4 (1:400, Invitrogen) using the above-described procedures. PKCδ-TRPV1 coexpression was determined using a goat anti-PKCδ antibody (1:50, Santa Cruz) and a rabbit antibody directed against the TRPV1 C-terminus peptide (1:500) .
Scratching behavior was evoked with pruritic stimuli applied to the back skin where site directed scratching occurs. We expanded our functional analyses of neuronal physiology to include both thoracic and lumbar DRG. Mice were euthanized rapidly by decapitation and DRG removed and acutely dissociated using previously described methods . Briefly, DRG were incubated in 45U papain/L-cysteine in Hank’s buffered saline solution (HBSS) without Ca2+ or Mg2+ and with 10 mM HEPES for 20 minutes at 37°C and 5% CO2. Ganglia were then washed, followed by 20 minute incubation in 1.5 mg/ml collagenase in HBSS + HEPES. Ganglia were then triturated with fire-polished Pasteur pipettes, the dissociated cells were filtered through a 40 μm cell strainer, and were plated on poly-D-lysine and collagen-coated glass coverslips. Cells were incubated overnight at 37°C in 5% CO2 humidified air in culture medium (Neurobasal A with B27, pen/strep, 2 mM glutamax, 5% fetal bovine serum (Gibco)). All experiments were performed within 24 hours of plating.
Cells were incubated in 3 μg/ml Fura-2 AM (Molecular Probes) for 30 minutes and then incubated for 30 minutes in external solution (in mM): 130 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 30 Glucose, 10 HEPES. For each recording, a coverslip was placed in a perfusion chamber and perfused with external solution at room temperature. Cells were viewed under an inverted microscope (Olympus Optical, Tokyo, Japan) and images were captured with a Hamamatsu Orca camera. SimplePCI Software was used to draw regions of interest (ROI) around Fura-loaded cells prior to recording. The ratio of fluorescence emission at an excitation wavelength of 357 and 380 nm was measured for each ROI. The experimental protocol consisted of a 2 minute baseline followed by 30 second bath application of histamine (100 μM in external solution), >8 minutes of external solution wash, 10 second application of capsaicin (200 nM), >8 minutes of external solution wash, and 10 seconds of KCl (50 mM) followed by wash (<2 minutes) to determine live neurons. A 10% or greater change from baseline 357 nm/380 nm ratio was considered a response to histamine. Capsaicin experiments were performed similarly except with 10 second capsaicin application (200nM). For experiments using PKCδ peptide inhibitor and scrambled peptide, cells were incubated in 100 μM peptide solution (dissolved in external solution) for 30 minutes prior to recording (δV1-1 peptide inhibitor (Myr-SFNSYELGSL-NH2), peptide inhibitor scramble (Myr-GLSFSEYLSN-NH2), Biomatik).
Protein kinase C
Gq Protein-coupled receptor
Mas-related gene protein coupled receptor
Dorsal root ganglion
- PIP2 :
- IP3 :
Histamine receptor 1
Extracellular signal regulated kinase 1/2
Calcitonin gene-related peptide
Hank’s buffered saline solution
Transient receptor potential vanilloid receptor 1
Transient receptor potential cation channel, subfamily A, member 1
- PGE2 :
Nerve growth factor
Ikoma A, Steinhoff M, Ständer S, Yosipovitch G, Schmelz M: The neurobiology of itch. Nat Rev Neurosci 2006,7(7):535–47. 10.1038/nrn1950
Davidson S, Giesler GJ: The multiple pathways for itch and their interactions with pain. Trends Neurosci 2010,33(12):550–8. 10.1016/j.tins.2010.09.002
Armstrong AW, Schupp C, Wu J, Bebo B: Quality of Life and Work Productivity Impairment among Psoriasis Patients: Findings from the National Psoriasis Foundation Survey Data 2003–2011. PLoS ONE 2012,7(12):e52935. 10.1371/journal.pone.0052935
Yosipovitch G, Bernhard JD: Clinical practice, Chronic pruritus. N Engl J Med 2013,368(17):1625–34. 10.1056/NEJMcp1208814
Weisshaar E, Dalgard F: Epidemiology of itch: adding to the burden of skin morbidity. Acta Derm Venereol 2009,89(4):339–50. 10.2340/00015555-0662
Rossbach K, Nassenstein C, Gschwandtner M, Schnell D, Sander K, Seifert R, et al.: Histamine H1, H3 and H4 receptors are involved in pruritus. Neuroscience 2011, 190:89–102.
Bell JK, McQueen DS, Rees JL: Involvement of histamine H4 and H1 receptors in scratching induced by histamine receptor agonists in Balb C mice. Br J Pharmacol 2004,142(2):374–80. 10.1038/sj.bjp.0705754
Rimmer SJ, Church MK: The pharmacology and mechanisms of action of histamine H1-antagonists. Clin Exp Allergy 1990,20(Suppl 2):3–17.
Davies MG, Greaves MW: Sensory responses of human skin to synthetic histamine analogues and histamine. Br J Clin Pharmacol 1980,9(5):461–5.
Han SK, Mancino V, Simon MI: Phospholipase Cbeta 3 mediates the scratching response activated by the histamine H1 receptor on C-fiber nociceptive neurons. Neuron 2006,52(4):691–703. 10.1016/j.neuron.2006.09.036
Liu Q, Tang Z, Surdenikova L, Kim S, Patel KN, Kim A, et al.: Sensory neuron-specific GPCR Mrgprs are itch receptors mediating chloroquine-induced pruritus. Cell 2009,139(7):1353–65. 10.1016/j.cell.2009.11.034
Liu Q, Weng HJ, Patel KN, Tang Z, Bai H, Steinhoff M, et al.: The distinct roles of two GPCRs, MrgprC11 and PAR2, in itch and hyperalgesia. Sci Signal 2011,4(181):ra45.
Decombaz J, Beaumont M, Vuichoud J, Bouisset F, Stellingwerff T: Effect of slow-release beta-alanine tablets on absorption kinetics and paresthesia. Amino Acids 2012,43(1):67–76. 10.1007/s00726-011-1169-7
Liu Q, Sikand P, Ma C, Tang Z, Han L, Li Z, et al.: Mechanisms of itch evoked by beta-alanine. J Neurosci 2012,32(42):14532–7. 10.1523/JNEUROSCI.3509-12.2012
Wilson SR, Gerhold KA, Bifolck-Fisher A, Liu Q, Patel KN, Dong X, et al.: TRPA1 is required for histamine-independent, Mas-related G protein-coupled receptor-mediated itch. Nat Neurosci 2011,14(5):595–602. 10.1038/nn.2789
Cesare P, Dekker LV, Sardini A, Parker PJ, McNaughton PA: Specific involvement of PKC-epsilon in sensitization of the neuronal response to painful heat. Neuron 1999,23(3):617–24. 10.1016/S0896-6273(00)80813-2
Aley KO, Messing RO, Mochly-Rosen D, Levine JD: Chronic hypersensitivity for inflammatory nociceptor sensitization mediated by the epsilon isozyme of protein kinase C. J Neurosci 2000,20(12):4680–5.
Velazquez KT, Mohammad H, Sweitzer SM: Protein kinase C in pain: involvement of multiple isoforms. Pharmacol Res 2007,55(6):578–89. 10.1016/j.phrs.2007.04.006
Mandadi S, Armati PJ, Roufogalis BD: Protein kinase C modulation of thermo-sensitive transient receptor potential channels: Implications for pain signaling. J Nat Sci Biol Med 2011,2(1):13–25. 10.4103/0976-9668.82311
Zhao C, Leitges M, Gereau RW: Isozyme-specific effects of protein kinase C in pain modulation. Anesthesiology 2011,115(6):1261–70.
Hao F, Tan M, Xu X, Cui MZ: Histamine induces Egr-1 expression in human aortic endothelial cells via the H1 receptor-mediated protein kinase Cdelta-dependent ERK activation pathway. J Biol Chem 2008,283(40):26928–36. 10.1074/jbc.M803071200
Mizuguchi H, Terao T, Kitai M, Ikeda M, Yoshimura Y, Das AK, et al.: Involvement of protein kinase Cdelta/extracellular signal-regulated kinase/poly(ADP-ribose) polymerase-1 (PARP-1) signaling pathway in histamine-induced up-regulation of histamine H1 receptor gene expression in HeLa cells. J Biol Chem 2011,286(35):30542–51. 10.1074/jbc.M111.253104
Haubensak W, Kunwar PS, Cai H, Ciocchi S, Wall NR, Ponnusamy R, et al.: Genetic dissection of an amygdala microcircuit that gates conditioned fear. Nature 2010,468(7321):270–6. 10.1038/nature09553
Amano T, Amir A, Goswami S, Paré D: Morphology, PKCdelta expression, and synaptic responsiveness of different types of rat central lateral amygdala neurons. J Neurophysiol 2012,108(12):3196–205. 10.1152/jn.00514.2012
Baines CP, Molkentin JD: STRESS signaling pathways that modulate cardiac myocyte apoptosis. J Mol Cell Cardiol 2005,38(1):47–62. 10.1016/j.yjmcc.2004.11.004
Pan TT, Neo KL, Hu LF, Yong QC, Bian JS: H2S preconditioning-induced PKC activation regulates intracellular calcium handling in rat cardiomyocytes. Am J Physiol Cell Physiol 2008,294(1):C169–77. 10.1152/ajpcell.00282.2007
Castaneda D, Zhao H, Mochly-Rosen D, Steinberg GK: Activating deltaPKC antagonizes the protective effect of ERK1/2 inhibition against stroke in rats. Brain Res 2009, 1251:256–61.
Bright R, Raval AP, Dembner JM, Pérez-Pinzón MA, Steinberg GK, Yenari MA, et al.: Protein kinase C delta mediates cerebral reperfusion injury in vivo. J Neurosci 2004,24(31):6880–8. 10.1523/JNEUROSCI.4474-03.2004
Minami H, Owada Y, Suzuki R, Handa Y, Kondo H: Localization of mRNAs for novel, atypical as well as conventional protein kinase C (PKC) isoforms in the brain of developing and mature rats. J Mol Neurosci 2000,15(2):121–35. 10.1385/JMN:15:2:121
Sakaue Y, Sanada M, Sasaki T, Kashiwagi A, Yasuda H: Amelioration of retarded neurite outgrowth of dorsal root ganglion neurons by overexpression of PKCdelta in diabetic rats. Neuroreport 2003,14(3):431–6.
Imamachi N, Park GH, Lee H, Anderson DJ, Simon MI, Basbaum AI, et al.: TRPV1-expressing primary afferents generate behavioral responses to pruritogens via multiple mechanisms. Proc Natl Acad Sci U S A 2009,106(27):11330–5. 10.1073/pnas.0905605106
Shim WS, Tak MH, Lee MH, Kim M, Koo JY, Lee CH, et al.: TRPV1 mediates histamine-induced itching via the activation of phospholipase A2 and 12-lipoxygenase. J Neurosci 2007,27(9):2331–7. 10.1523/JNEUROSCI.4643-06.2007
Zygmunt PM, Ermund A, Movahed P, Andersson DA, Simonsen C, Jönsson BA, et al.: Monoacylglycerols activate TRPV1–a link between phospholipase C and TRPV1. PLoS ONE 2013,8(12):e81618. 10.1371/journal.pone.0081618
Bhave G, Hu HJ, Glauner KS, Zhu W, Wang H, Brasier DJ, et al.: Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). Proc Natl Acad Sci U S A 2003,100(21):12480–5. 10.1073/pnas.2032100100
Bhave G, Gereau RW: Posttranslational mechanisms of peripheral sensitization. J Neurobiol 2004,61(1):88–106. 10.1002/neu.20083
Brenner GJ, Ji RR, Shaffer S, Woolf CJ: Peripheral noxious stimulation induces phosphorylation of the NMDA receptor NR1 subunit at the PKC-dependent site, serine-896, in spinal cord dorsal horn neurons. Eur J Neurosci 2004,20(2):375–84. 10.1111/j.1460-9568.2004.03506.x
Kupcova Skalnikova H, Navarro R, Marsala S, Hrabakova R, Vodicka P, Gadher SJ, et al.: Signaling proteins in spinal parenchyma and dorsal root ganglion in rat with spinal injury-induced spasticity. J Proteomics 2013, 91:41–57.
Morioka N, Yoshida Y, Nakamura Y, Hidaka N, Hisaoka-Nakashima K, Nakata Y: The regulation of exon-specific brain-derived neurotrophic factor mRNA expression by protein kinase C in rat cultured dorsal root ganglion neurons. Brain Res 2013, 1509:20–31.
Andratsch M, Mair N, Constantin CE, Scherbakov N, Benetti C, Quarta S, et al.: A key role for gp130 expressed on peripheral sensory nerves in pathological pain. J Neurosci 2009,29(43):13473–83. 10.1523/JNEUROSCI.1822-09.2009
Chen L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, et al.: Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC. Proc Natl Acad Sci U S A 2001,98(20):11114–9. 10.1073/pnas.191369098
Pabla N, Dong G, Jiang M, Huang S, Kumar MV, Messing RO, et al.: Inhibition of PKCdelta reduces cisplatin-induced nephrotoxicity without blocking chemotherapeutic efficacy in mouse models of cancer. J Clin Invest 2011,121(7):2709–22. 10.1172/JCI45586
Qi X, Inagaki K, Sobel RA, Mochly-Rosen D: Sustained pharmacological inhibition of deltaPKC protects against hypertensive encephalopathy through prevention of blood–brain barrier breakdown in rats. J Clin Invest 2008,118(1):173–82.
Lin HW, Defazio RA, Della-Morte D, Thompson JW, Narayanan SV, Raval AP, et al.: Derangements of post-ischemic cerebral blood flow by protein kinase C delta. Neuroscience 2010,171(2):566–76. 10.1016/j.neuroscience.2010.08.058
Oda T, Morikawa N, Saito Y, Masuho Y, Matsumoto S: Molecular cloning and characterization of a novel type of histamine receptor preferentially expressed in leukocytes. J Biol Chem 2000,275(47):36781–6. 10.1074/jbc.M006480200
Wilson SR, Thé L, Batia LM, Beattie K, Katibah GE, McClain SP, et al.: The epithelial cell-derived atopic dermatitis cytokine TSLP activates neurons to induce itch. Cell 2013,155(2):285–95. 10.1016/j.cell.2013.08.057
Solinski HJ, Gudermann T, Breit A: Pharmacology and signaling of MAS-related G protein-coupled receptors. Pharmacol Rev 2014,66(3):570–97. 10.1124/pr.113.008425
Hill SJ, Ganellin CR, Timmerman H, Schwartz JC, Shankley NP, Young JM, et al.: International Union of Pharmacology, XIII. Classification of histamine receptors. Pharmacol Rev 1997,49(3):253–78.
Grazzini E, Puma C, Roy MO, Yu XH, O′Donnell D, Schmidt R, et al.: Sensory neuron-specific receptor activation elicits central and peripheral nociceptive effects in rats. Proc Natl Acad Sci U S A 2004,101(18):7175–80. 10.1073/pnas.0307185101
Leurs R, Smit MJ, Timmerman H: Molecular pharmacological aspects of histamine receptors. Pharmacol Ther 1995,66(3):413–63. 10.1016/0163-7258(95)00006-3
Steinberg SF: Structural basis of protein kinase C isoform function. Physiol Rev 2008,88(4):1341–78. 10.1152/physrev.00034.2007
Wu-Zhang AX, Newton AC: Protein kinase C pharmacology: refining the toolbox. Biochem J 2013,452(2):195–209. 10.1042/BJ20130220
Miyano K, Tang HB, Nakamura Y, Morioka N, Inoue A, Nakata Y: Paclitaxel and vinorelbine, evoked the release of substance P from cultured rat dorsal root ganglion cells through different PKC isoform-sensitive ion channels. Neuropharmacology 2009,57(1):25–32. 10.1016/j.neuropharm.2009.04.001
Parada CA, Reichling DB, Levine JD: Chronic hyperalgesic priming in the rat involves a novel interaction between cAMP and PKCepsilon second messenger pathways. Pain 2005,113(1–2):185–90.
Marchand F, D′Mello R, Yip PK, Calvo M, Muller E, Pezet S, et al.: Specific involvement of atypical PKCzeta/PKMzeta in spinal persistent nociceptive processing following peripheral inflammation in rat. Mol Pain 2011, 7:86. 10.1186/1744-8069-7-86
Chen R, Furman CA, Zhang M, Kim MN, Gereau RW 4th, Leitges M, et al.: Protein kinase Cbeta is a critical regulator of dopamine transporter trafficking and regulates the behavioral response to amphetamine in mice. J Pharmacol Exp Ther 2009,328(3):912–20. 10.1124/jpet.108.147959
He YQ, Chen Q, Ji L, Wang ZG, Bai ZH, Stephens RL Jr, et al.: PKCgamma receptor mediates visceral nociception and hyperalgesia following exposure to PTSD-like stress in the spinal cord of rats. Mol Pain 2013,9(1):35. 10.1186/1744-8069-9-35
Pan HL, Zhang YQ, Zhao ZQ: Involvement of lysophosphatidic acid in bone cancer pain by potentiation of TRPV1 via PKCepsilon pathway in dorsal root ganglion neurons. Mol Pain 2010, 6:85. 10.1186/1744-8069-6-85
Cang CL, Zhang H, Zhang YQ, Zhao ZQ: PKCepsilon-dependent potentiation of TTX-resistant Nav1.8 current by neurokinin-1 receptor activation in rat dorsal root ganglion neurons. Mol Pain 2009, 5:33. 10.1186/1744-8069-5-33
McCoy ES, Taylor-Blake B, Street SE, Pribisko AL, Zheng J, Zylka MJ: Peptidergic CGRPalpha Primary Sensory Neurons Encode Heat and Itch and Tonically Suppress Sensitivity to Cold. Neuron 2013,78(1):138–51. 10.1016/j.neuron.2013.01.030
Han L, Ma C, Liu Q, Weng HJ, Cui Y, Tang Z, et al.: A subpopulation of nociceptors specifically linked to itch. Nat Neurosci 2013,16(2):174–82.
Shim WS, Oh U: Histamine-induced itch and its relationship with pain. Mol Pain 2008, 4:29. 10.1186/1744-8069-4-29
Kajihara Y, Murakami M, Imagawa T, Otsuguro K, Ito S, Ohta T: Histamine potentiates acid-induced responses mediating transient receptor potential V1 in mouse primary sensory neurons. Neuroscience 2010,166(1):292–304. 10.1016/j.neuroscience.2009.12.001
Kim BM, Lee SH, Shim WS, Oh U: Histamine-induced Ca(2+) influx via the PLA(2)/lipoxygenase/TRPV1 pathway in rat sensory neurons. Neurosci Lett 2004,361(1–3):159–62.
Ayabe S, Murata T, Maruyama T, Hori M, Ozaki H: Prostaglandin E2 induces contraction of liver myofibroblasts by activating EP3 and FP prostanoid receptors. Br J Pharmacol 2009,156(5):835–45. 10.1111/j.1476-5381.2008.00051.x
Kobayashi K, Murata T, Hori M, Ozaki H: Prostaglandin E2-prostanoid EP3 signal induces vascular contraction via nPKC and ROCK activation in rat mesenteric artery. Eur J Pharmacol 2011,660(2–3):375–80.
Corbit KC, Foster DA, Rosner MR: Protein kinase Cdelta mediates neurogenic but not mitogenic activation of mitogen-activated protein kinase in neuronal cells. Mol Cell Biol 1999,19(6):4209–18.
Than JY, Li L, Hasan R, Zhang X: Excitation and Modulation of TRPA1, TRPV1, and TRPM8 Channel-expressing Sensory Neurons by the Pruritogen Chloroquine. J Biol Chem 2013,288(18):12818–27. 10.1074/jbc.M113.450072
Hager UA, Hein A, Lennerz JK, Zimmermann K, Neuhuber WL, Reeh PW: Morphological characterization of rat Mas-related G-protein-coupled receptor C and functional analysis of agonists. Neuroscience 2008,151(1):242–54. 10.1016/j.neuroscience.2007.09.085
Loo L, Shepherd AJ, Mickle AD, Lorca RA, Shutov LP, Usachev YM, et al.: The C-type natriuretic peptide induces thermal hyperalgesia through a noncanonical Gbetagamma-dependent modulation of TRPV1 channel. J Neurosci 2012,32(35):11942–55. 10.1523/JNEUROSCI.1330-12.2012
Leitges M: Exacerbated vein graft arteriosclerosis in protein kinase Cdelta-null mice. J Clin Investig 2001,108(10):1505–12. 10.1172/JCI200112902
Yang D, Gereau RW: Peripheral group II metabotropic glutamate receptors (mGluR2/3) regulate prostaglandin E2-mediated sensitization of capsaicin responses and thermal nociception. J Neurosci 2002,22(15):6388–93.
This work was supported in part by NIH funding NS089130 & GM007200 (MVV); NS076324 (SD); NS48602 & NS42595 (RWG). The authors would like to thank Sherri Vogt for exceptional mouse colony care and biochemistry technical support; Dr. Judith Golden for helpful discussions and critical reading of the manuscript; Dr. Vijay Samineni for experiment support; and Gereau lab members for helpful discussions. We thank Dr. George Wilcox for the PKCδ inhibitor.
The authors declare that they have no competing interests.
MVV, SD, and CZ performed experiments and analyzed data. MVV, SD, CZ, and RWG conceived the study and contributed to writing the manuscript. ML provided PKCδ-KO mice. All authors read and agreed to the final version of the manuscript.
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Valtcheva, M.V., Davidson, S., Zhao, C. et al. Protein kinase Cδ mediates histamine-evoked itch and responses in pruriceptors. Mol Pain 11, 1 (2015). https://doi.org/10.1186/1744-8069-11-1
- PKC isoform
- Novel PKC
- Peptide inhibitor