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Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, Pécs, HungaryJános Szentágothai Research Center, University of Pécs, Pécs, HungaryPharmInVivo Ltd., Pécs, HungaryMTA NAP B Pain Research Group University of Pécs, School of Medicine, Pécs, Hungary
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, Pécs, HungaryJános Szentágothai Research Center, University of Pécs, Pécs, HungaryMTA NAP B Pain Research Group University of Pécs, School of Medicine, Pécs, Hungary
DE-MTA Lendület Cellular Physiology Research Group, Departments of Physiology and Immunology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, Pécs, HungaryJános Szentágothai Research Center, University of Pécs, Pécs, HungaryPharmInVivo Ltd., Pécs, Hungary
János Szentágothai Research Center, University of Pécs, Pécs, HungaryDepartment of Experimental Zoology and Neurobiology, Faculty of Sciences, University of Pecs, Pecs, Hungary
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, Pécs, HungaryJános Szentágothai Research Center, University of Pécs, Pécs, Hungary
Laboratory of Molecular Neuropharmacology & iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, JapanUnited Graduate School of Child Development, Osaka University, Suita, Japan
DE-MTA Lendület Cellular Physiology Research Group, Departments of Physiology and Immunology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary
Although pituitary adenylate cyclase-activating polypeptide (PACAP) was described as a key vasoregulator in human skin, little is known about its expression in mouse skin. As it is important to investigate PACAP signaling in translational mouse dermatitis models, we determined its presence, regulation, and role in neurogenic and non-neurogenic cutaneous inflammatory mechanisms. The mRNA of PACAP and its specific receptor PAC1 was detected with real-time PCR in several skin regions at comparable levels. PACAP-38-immunoreactivity measured with radioimmunoassay was similar in plantar and dorsal paw skin and the ear but significantly smaller in the back skin. PACAP and PAC1 mRNA, as well as PACAP-38 and PAC1 protein expression, significantly increased in the plantar skin after intraplantar administration of capsaicin (50 μl, 100 μg ml−1), an agonist of the transient receptor potential vanilloid 1 (TRPV1) receptor, evoking chiefly neurogenic inflammation without inflammatory cell accumulation. Intraplantar complete Freund’s adjuvant (CFA; 50 μl, 1 mg ml−1) also increased PACAP/PAC1 mRNA but not the PACAP peptide. Capsaicin-induced neurogenic paw edema, but not CFA-evoked non-neurogenic swelling, was significantly smaller in PACAP-deficient mice throughout a 24-hour period. To our knowledge, we provide previously unreported evidence for PACAP and PAC1 expression upregulation during skin inflammation of different mechanisms and for its pro-inflammatory function in neurogenic edema formation.
Capsaicin (N-vanillyl-nonenamide), the pungent principle of chili peppers, is a pharmacological tool to activate the transient receptor potential vanilloid 1 (TRPV1) nonselective cation channel located on 50–70% of sensory nerves. TRPV1 has a variety of endogenous activators, such as protons, bradykinin, prostanoids, tumor necrosis factor-α, nerve growth factor, gasotransmitters, or lipid peroxidase products (
). Many of these are crucial participants of skin inflammatory processes. Capsaicin-sensitive sensory nerves, which innervate multiple skin layers, exhibit “triple” cutaneous functions. First, they mediate signal processing of various sensory modalities including pain, itch, and thermal sensation (afferent function). Second, in response to their activation, they release a multitude of (mostly) pro-inflammatory sensory neuropeptides (local efferent function) (
), which, among others, elicit arteriolar vasodilatation, promote plasma protein leakage from venules locally in the innervated area, and eventually induce acute neurogenic inflammation (
. Third, peptides with systemic anti-inflammatory and anti-nociceptive actions (such as somatostatin and endomorphins) are also derived from these fibers (systemic effect “sensocrine” function) (
Antiinflammatory and analgesic effects of somatostatin released from capsaicin-sensitive sensory nerve terminals in a Freund's adjuvant–induced chronic arthritis model in the rat.
). Among the neuropeptides expressed by capsaicin-sensitive sensory fibers (e.g. calcitonin gene-related peptide, substance P, other tachykinins), we were particularly interested in pituitary adenylate cyclase-activating polypeptide (PACAP). PACAP exists in two forms, PACAP-27 and PACAP-38, the latter being predominant in mammals. It is the most highly conserved member of the vasoactive intestinal peptide/secretin/glucagon peptide superfamily (
). The specific PACAP receptor is PAC1 coupled to both Gs and Gq proteins activating adenylate-cyclase and phospholipase C signal transduction pathways. VPAC1 and VPAC2 receptors have similar binding abilities for both PACAP and vasoactive intestinal peptide, and they predominantly activate adenylate-cyclase leading to cAMP increase. PACAP and its receptors are widely distributed (
); therefore, PACAP-coupled signaling mechanisms are implicated in physiological and pathological processes, including vascular integrity, neuronal development and regeneration, circadian rhythm, and reproduction (
Capsaicin-evoked release of pituitary adenylate cyclase activating peptide (PACAP) and calcitonin gene-related peptide (CGRP) from rat spinal cord in vivo.
Concerning its localization in the human skin, PACAP and PAC1 have been described in nerve fibers close to the dermal–epidermal border, hair follicles, blood vessels, and sweat glands. Their marked upregulation was observed in psoriasis patients, indicating a role of PACAP in cutaneous inflammation (Steinhoff et al., 1999).
Despite numerous studies showing an inhibitory effect of PACAP on cellular inflammatory and immune processes (
), little is known about its actions on neurogenic inflammatory components. We have shown that exogenous PACAP inhibits sensory neuropeptide release and acute neurogenic inflammation (
). Studies with PACAP-deficient mice are useful in elucidating the function of endogenous PACAP in inflammatory reactions in vivo. Increased inflammatory reactions have been demonstrated in PACAP knockout mice in colitis, autoimmune encephalomyelitis, airway models, neuroinflammation, and allergic contact dermatitis models (
Targeted gene deletion reveals that pituitary adenylyl cyclase-activating polypeptide is an intrinsic regulator of Treg abundance in mice and plays a protective role in experimental autoimmune encephalomyelitis.
Pituitary adenylate cyclase-activating polypeptide plays an anti-inflammatory role in endotoxin-induced airway inflammation: in vivo study with gene-deleted mice.
As there is no information on the expression, inflammation-induced alterations, and functions of PACAP in mouse skin, our aim was to provide data for these points with radioimmunoassay and molecular biological techniques. In order to investigate a direct relationship between TRPV1 and PACAP signaling, functional tests were performed in two acute in vivo cutaneous inflammation models of distinct neurogenic and non-neurogenic mechanisms using gene-deficient mice.
Results
PACAP and PAC1 are expressed in the mouse skin
Specific PACAP mRNA transcripts were detected in different skin regions. Its relative mRNA expression compared with GAPDH gene was similar in the examined regions, albeit PACAP mRNA measured in the back skin was slightly, yet not significantly, smaller than in other areas. As anticipated, levels of PACAP-38 mRNA were about 4–5-fold higher in the hypothalamus and the pituitary gland, used as positive control tissues (Figure 1a). Similarly to the peptide itself, we could also identify the specific PACAP receptor, PAC1, in the mouse skin. Its specific mRNA also showed a relatively stable expression in different skin areas (Figure 1b).
Figure 1Specific pituitary adenylate cyclase-activating polypeptide (PACAP) and pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) mRNA transcripts are expressed in different mouse skin regions. RT-PCR determinations of PACAP and PAC1-specific mRNA transcripts, as detailed in the Methods section. The pituitary gland (PG) and the hypothalamus (HT) served as positive controls. N, non-template control. (a) Expression of molecules in n=4–5 mice in each group. (b) Averaged (means±SEM) relative expression values normalized to the housekeeping gene GAPDH.
PACAP-38 immunoreactivity (IR) was reliably measured in the homogenates of different mouse skin samples. Its concentration was relatively similar (20–25 fmol mg−g wet tissue) in the plantar and dorsal paw skin, as well as the ear. However, the amount of PACAP-IR was significantly smaller, 7–8 fmol mg−1, in the back skin (Figure 2a).
Figure 2PACAP-38 and pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) receptor protein are expressed in the mouse. (a) PACAP-immunoreactivity (PACAP-IR) was determined by radioimmunoassay in different mouse skin regions. Columns represent means±SEM of n=5–6 samples obtained from different animals (the Mann–Whitney U test vs. all other skin regions in panel a. (b) Upper panel: PAC1 protein is expressed in the basal and polygonal layers of the epidermis (arrowheads) and in the granular layer (arrows). The papillary layer of the dermis was positive for PAC1 protein as well. The signal specificity is supported by the peptide pre-absorption control experiment represented by the image taken from the identical area of the consecutive section from the same tissue block seen in the insert (scale bar=250 μm). Lower panel: depicts areas in higher magnification marked by the boxes in the upper panel (scale bar=60 μm).
As the plantar skin showed considerable PACAP expression and this is a suitable region for mouse inflammation models, we analyzed this area for specific PAC1-IR. PAC1 was detected in the papillary layer of the plantar skin (Figure 2b), where the superficial vessels and sensory nerve endings for sensing thermal, pain, and tactile stimuli are localized (
Expression levels of PACAP and PAC1 are differentially modulated by capsaicin and CFA
Next, we assessed whether a predominantly neurogenic inflammation induced by intraplantar injection of capsaicin or a chiefly non-neurogenic cutaneous inflammation evoked by complete Freund’s adjuvant (CFA) affects the expression levels of PACAP and/or PAC1. PACAP mRNA expression in the inflamed plantar skin samples slightly increased 4 hours after capsaicin administration, which reached a significant, 2.1-fold elevation at 24 hours. Meanwhile, PACAP mRNA increase was significant (1.6-fold) already 4 hours following CFA injection, and it was upregulated (4.6-fold) at the 24-h time point (Figure 3a).
Figure 3Capsaicin and complete Freund’s adjuvant (CFA) upregulate pituitary adenylate cyclase-activating polypeptide (PACAP) and pituitary adenylate cyclaseactivating polypeptide receptor type 1 (PAC1) mRNA expressions in the inflamed plantar skin. Expression of (a) PACAP and (b) PAC1 detected by qRT-PCR in capsaicin (Caps)- and CFA-treated plantar skin samples taken 4 and 24 hours after the induction of the inflammation relative to the respective vehicle-injected contralateral tissues. (c) The widely expressed inflammatory cytokine TNF-α mRNA was also determined from the same samples to compare the peptide and the receptor expression with the cellular components of the inflammation. Columns represent means±SEM of n=6 per group. Statistical analysis was performed by the Mann–Whitney test versus contralateral vehicle-injected tissue. TNF-α, tumor necrosis factor-α.
PAC1 mRNA similarly increased in response to both capsaicin and CFA at the 4 hours time point by 1.6- and 2-fold, respectively. This moderate, but statistically significant, expressional elevation remained stable (1.7-fold) 24 hours after capsaicin but increased further (5.4-fold) in case of CFA (Figure 3b). In order to compare PACAP and PAC1 mRNA alterations with the cellular component of the inflammatory reaction, we in parallel determined the widely expressed inflammatory cytokine tumor necrosis factor (TNF)-α mRNA in the respective skin samples. Expression of TNF-α was not significantly altered in case of the capsaicin-induced neurogenic inflammation, but it showed a significant 2.5-fold increase 24 hours after CFA administration (Figure 3c). It should be noted that both expressions of PACAP and PAC1 mRNA, referring to their local synthesis, as well as that of TNF-α were significantly greater in the case of the non-neurogenic CFA-induced inflammation compared with the “purely” neurogenic capsaicin-evoked reaction (Figure 3a–c).
At the peptide level, capsaicin elicited a significant, more than 2-fold increase in PACAP-IR in the plantar skin 24 hours post injection compared with the contralateral non-inflamed, vehicle-treated side. Unexpectedly, CFA injection into the plantar surface of the paw did not influence the total PACAP peptide quantity in the skin samples (Figure 4a). In agreement with the quantitative real-time PCR (qRT-PCR) results, immunohistochemistry revealed that the effect of capsaicin to evoke swelling of the plantar skin was accompanied by significantly increased PAC1-IR. Similarly, CFA treatment also upregulated the protein level of PAC1 when compared with non-inflamed vehicle-treated tissues (Figure 4b).
Figure 4Capsaicin and complete Freund’s adjuvant (CFA) differentially upregulate PACAP-38 and pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) receptor protein in the inflamed plantar skin. (a) PACAP-immunoreactivity (PACAP-IR) was determined by radioimmunoassay in homogenates of different mouse skin regions. Columns represent mean±SEM of n=5–6 samples obtained from different animals; two-way ANOVA followed by Bonferroni’s post-tests was used to determine the statistical differences. (b) Neurogenic inflammation was induced by capsaicin (Caps), whereas predominantly non-neurogenic inflammation was evoked by CFA. The contralateral sides treated with the respective solvents served as non-inflamed controls. Column graphs represent statistical analysis of the semi-quantitative PAC1 immunofluorescence evaluation performed on multiple sections from n=6 animals in each group, and the mean±SEM values are shown. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s post-test. ANOVA, analysis of variance.
PACAP and TRPV1 gene-deficient mice exhibit smaller neurogenic but not non-neurogenic edema response
We then intended to assess the putative mechanism of action of capsaicin with the help of PACAP and TRPV1 gene-deficient mice. In both wild-type CD1 (background for PACAP−/−) and C57BL/6 (background for TRPV1−/−) wild-type mice, the potent and selective TRPV1 receptor agonist capsaicin evoked paw swelling, which reached its maximum of 25–30% 2 hours after the intraplantar injection, then it gradually decreased to 5–10% during the 24 hours of the study (Figure 5a and b). Notably, this acute capsaicin-induced neurogenic edema formation was significantly smaller during the total duration of the experiment in PACAP gene-deficient mice (Figure 5a). As expected, capsaicin did not evoke swelling in TRPV1−/− mice (Figure 5b).
Figure 5The edema-inducing effect of capsaicin, unlike that of complete Freund’s adjuvant (CFA), depends on pituitary adenylate cyclase-activating polypeptide (PACAP) and transient receptor potential vanilloid 1. Inflammation was induced by intraplantar injection of capsaicin (a and b) or CFA (c and d) in gene-deficient (PACAP−/−, TRPV1−/−) mice, as well as in their respective wild-type littermates (PACAP+/+, C57Bl/6 WT); the dynamics of paw swelling was monitored at the time points. Symbols indicate % increase in the volume of the hindpaws compared with the initial values, measured with plethysmometry before the induction of the inflammation. Each data point represents the mean±SEM of n=5–6 mice per group, **P<0.01 *P<0.001 versus wild-type (one-way ANOVA followed by Bonferroni’s modified t-test). ANOVA, analysis of variance.
Intraplantar CFA administration also resulted in paw edema, but this swelling had different kinetics compared with the capsaicin effect. It reached a significant level (25–35% over baseline) 2 hour post injection, remained stable for 6 hours, and then increased again to 45–55% at 24 hours in both wild-type strains. In contrast to what was observed in the case of capsaicin, there was no measurable difference between the extent of the CFA-evoked edema in either PACAP or TRPV1 gene-deficient mice when compared with their respective wild types (Figure 5c and d).
The histological analysis revealed negligible dermal edema formation and inflammatory cell infiltration in either the capsaicin- or the CFA-treated group 4 hours after the treatment (Supplementary Figure S1 online). Immune cells were also absent 24 hours following capsaicin injection; however, a significant inflammatory cell accumulation (predominantly neutrophils and macrophages) was observed after CFA application at this time point compared with its vehicle paraffin oil. Although reliable morphological quantitative evaluation was not possible on the plantar skin slides owing to different sectioning angles and regions examined, differences were not observed in the number of infiltrating cells between the wild type and the PACAP−/− groups (Supplementary Figure S2 online).
Identification of pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP type 1 receptor in human skin: expression of PACAP-38 is increased in patients with psoriasis.
), the role of PACAP and its receptor in skin inflammation and sensory-immune communication is still poorly understood. As PACAP is vaso- and immune-regulator, and a variety of mechanisms and translational disease models are widely used in mice for dermatological investigations, it is important to map its expression in different mouse skin regions. To our knowledge, the results demonstrate previously unreported evidence that PACAP and its specific receptor, PAC1, can be detected in murine skin from various regions. They are also synthesized locally besides being present on sensory nerves, as they are detected at the mRNA level. Their relative expressions are almost the same in dorsal and plantar paw skin and the ear. PACAP mRNA is slightly but not significantly smaller in the back skin. The concentration of the PACAP-38 peptide is similar in paw skin samples and the ear but smaller in the back skin. This might be due to a less dense sensory innervation with less PACAP of neural origin but also to the thicker skin and the different cutaneous structure. The presence of PACAP has been shown in capsaicin-sensitive sensory nerves (
Identification of pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP type 1 receptor in human skin: expression of PACAP-38 is increased in patients with psoriasis.
As a second step, we described the upregulation of locally produced PACAP in mouse plantar skin in inflammatory conditions of distinct mechanisms. Capsaicin evokes an acute neurogenic inflammation by selectively activating a subpopulation of nociceptive sensory nerves via TRPV1 and consequently releasing several vasoactive neuropeptides (
Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides.
) including PACAP. Meanwhile, CFA elicits a chronic, predominantly non-neurogenic inflammatory response by triggering the innate immune system through stimulating macrophages and T lymphocytes and producing cellular infiltration with sensory neuronal elements involved only at a later stage (
). Significant upregulation of PACAP was observed in the plantar skin at both mRNA and the peptide levels 24 hours after capsaicin injection. Capsaicin selectively activates the TRPV1 channel, which has been described not only on sensory nerves but also on keratinocytes, dendritic cells, and sebocytes in the skin (
). Increased PACAP expression in response to TRPV1 activation can be explained either by its direct effect at extraneural TRPV1 receptors or an indirect action via the release of sensory nerve–derived inflammatory mediators. Substance P and calcitonin gene-related peptide are able to stimulate epithelial and inflammatory cells; therefore, they might increase PACAP synthesis in these structures. We have previously provided in vivo evidence that PACAP-38 is released from capsaicin-sensitive fibers of the skin in response to systemic TRPV1 receptor stimulation (
). As capsaicin elevated both PACAP mRNA and peptide in the skin, its local upregulation and release from the stimulated sensory fibers are suggested. Meanwhile, CFA increased the PACAP mRNA level significantly more than capsaicin did at the 24 hours time point, but interestingly it did not alter the peptide-IR. This virtual contradiction can be explained by the fact that, at the mRNA level, we only measure the locally synthesized non-neural PACAP but not that of sensory neural origin (the mRNA of which is located in the dorsal root ganglia). This represents only a small fraction of the total peptide amount in the tissue, and the much higher neural PACAP expression is also supported by our findings. We propose that, in the CFA model, the infiltrating and accumulating inflammatory cells are the major local sources of PACAP mRNA increase by two mechanisms: (i) increased number and/or (ii) enhanced PACAP synthesis. However, this might not be sufficient to considerably elevate the peptide concentration compared with the total amount measured in the skin. Another explanation is that there was a 50% paw swelling in the CFA model at the 24 hours time point. As the peptide IR is normalized to the wet tissue weight, the edema-induced greater weight might result in a smaller relative peptide concentration.
Upregulation of PACAP has been shown in various types of neuronal damage, such as motor nerve injury, sciatic nerve transection and neuronal inflammation, and sympathetic chain lesion, which might indicate endogenous adaptation to injuries and its possible role in regeneration. Upregulated trophic factors and neuropeptides may be important for post-axotomy/post-inflammation survival and restoration (
PAC1 expression also increased in both types of inflammation at the mRNA and protein levels, suggesting that this target is likely to mediate the effects of PACAP in the skin. PAC1 was shown to be upregulated after some injuries depending on the local microenvironment; PACAP can also induce the upregulation of its receptor. Our results suggest that PACAP signaling (both PACAP and PAC1 receptor) is stronger in both types of inflammation (
The role of PACAP can only be determined under in vivo experimental conditions with gene-deficient mice, as stable, selective pharmacological tools are not available. Our results with PACAP-deficient mice also revealed the in vivo functional relevance of the locally increased PACAP in acute vascular inflammatory responses. PACAP proved to be a potent pro-inflammatory mediator, which increases edema due to short-lasting neurogenic but not non-neurogenic mechanisms. Intradermally injected PACAP induces plasma extravasation in the hindpaw (
). In contrast, we have shown earlier that a low dose of intraperitoneal PACAP-38 inhibits acute neurogenic plasma leakage in the rat plantar skin evoked by TRPV1 or TRPA1 agonists and also decreased carrageenan-induced “mixed-type” inflammation (
) and other inflammatory mediators from sympathetic fibers or cellular sources. Furthermore, its direct inhibitory effect on plasma extravasation and edema at the level of the vascular endothelium can also be assumed. This is supported by the presence of PAC-1 on endothelial cells (
Pituitary adenyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) in the cochlea: evidence for specific transcript expression of PAC1-R splice variants in rat microdissected cochlear subfractions.
On the basis of these data, the role of endogenous PACAP in acute neurogenic edema formation in the skin is pro-inflammatory, whereas the effect of the exogenous peripherally administered peptide is inhibitory on sensory neuropeptide release (
). This latter action is not mediated by the known PACAP receptors, as their signaling mechanisms are all stimulatory on the nerve terminals. This might be explained by distinct inhibitory mechanisms and targets via different, currently not identified, receptors or splice variants on the sensory nerves. The role of PACAP in inflammation is complex. It increases the early vascular phase by inducing vasodilatation, plasma protein leakage from the fenestrated veins, inflammatory cell extravasation, and consequent immune cell accumulation induced both by neurogenic and non-neurogenic mechanisms. In contrast, it inhibits the later cellular responses by decreasing the activation and further accumulation of immune cells (
In summary, in this study, we provide evidence for the expression and capsaicin-induced upregulation of PACAP in murine skin both at the mRNA and peptide levels. Furthermore, using PACAP and TRPV1 gene-deficient mice, we demonstrate that PACAP is a regulator of TRPV1-mediated acute neurogenic edema formation. The role of PACAP in chronic inflammatory skin diseases and its human relevance awaits further clarification.
Materials and Methods
Animals
Male CD1 wild-type and PACAP gene-deleted mice (25–35 g) (
) were bred and kept in the Animal House of the Department of Pharmacology and Pharmacotherapy, University of Pécs, at 24–25 °C and provided with standard mouse chow and water ad libitum. The breeding pairs of the TRPV1 receptor gene-deleted mice (TRPV1−/−) and C57Bl/6 wild-types were from Jackson Laboratories (Bar Harbor, ME).
Animals were killed by cervical dislocation under ketamine-xylazine (100 mg kg−1-5 mg kg−1 intraperitoneal) anesthesia; the skin samples were excised and cut into three parts: (i) stored in RNA later for molecular biological studies, (ii) frozen at -20 °C for radioimmunoassay, and (iii) put into 4% paraformaldehyde for immunohistochemistry.
Experiments were carried out according to the 1998/XXVIII Act of the Hungarian Parliament on Animal Protection and Consideration Decree of Scientific Procedures of Animal Experiments (243/1988) and complied with the recommendations of the International Association for the Study of Pain. The experiments were approved by the Ethics Committee on Animal Research of the University of Pécs (license number: BA02/2000-2/2012).
Measurement of PACAP, PAC1, and TNF-α mRNA in the skin
Expressions of PACAP (Adcyap1, GenBank Accession: NM_009625) and PAC1 (Adcyap1r1, NM_007407, NM_001025372)-specific mRNA were determined by semi-quantitative RT-PCR in different mouse skin samples (
). Total RNA was isolated with TRIzol. One μg of total RNA was then reverse transcribed into cDNA by using 15U of AMV reverse transcriptase and 0.025 μg μl−1 random primers. Subsequent PCR amplification (95 °C for 2 minutes, 35 cycles of 94 °C for 1 minute; 61.5 °C for PACAP for 1 minute, 62.5 °C for PAC1 for 90 seconds, 55.5 °C for GAPDH for 60 seconds, 72 °C for 1 minute; 72 °C for 2 minutes) was performed on the GeneAmp PCR System 2400 DNA Thermal Cycler (Applied Biosystems, now Thermo Fisher Scientific, Waltham, MA). Primers were synthesized by Invitrogen (now Thermo Fisher Scientific) (PACAP, forward: 5′-GGGGCAAGTCTAGCTCCTCT-3′, PACAP, reverse: 5′-GGGTCTCCAGAAAATCCACA-3′, product: 225 bp; PAC1, forward: 5′-AGGCAATGAGTCGAGCATCT-3′, reverse: 5′-GTCTTTCCCTCTTGCTGACG-3′, product: 220 bp; Glyceraldehyde 3-phosphate dehydrogenase (Gapdh, NM_001289726, NM_008084), forward: 5′-ATGGTGAAGGTCGGTGTGAAC-3′; reverse: 5′-GCTGACAATCTTGAGGGAGT-3′, product: 459 bp). PCR products were visualized on a 1.5 agarose gel with ethidium bromide (0.5 mg ml−1) under UV, and the photographed bands were quantified by an Image Pro Plus 4.5.0 software (Media Cybernetics, Rockville, MD).
The expression of PACAP (Adcyap1), PAC1 (Adcyap1r1), and TNF-α (Tnf, NM_013693, NM_001278601) mRNA was determined by quantitative real-time PCR in capsaicin and CFA-treated plantar skin samples. PCR amplification (two-step protocol: 95 °C for 10 minutes; 40 cycles of 95 °C for 30 seconds and 60 °C for 1 minute) was performed on the Stratagene MX3000P system (Agilent Technologies, Santa Clara, CA). Primers: PACAP, forward: 5′-GACCAGAAGACGAGGCTTACG-3′, reverse: 5′-GTCCGCTGGATAGTAAAGGGC-3′, product: 128 bp; PAC1, forward: 5′-GAGAACGTCAGCAAGAGGGA-3′, reverse: 5′-GCCTGTACCTCCCCATTCAG-3′, product: 107 bp; TNFα, forward: 5′- CAGGCGGTGCCTATGTCTC-3′, reverse: 5′-CGATCACCCCGAAGTTCAGTAG-3′, product: 89 bp; Gapdh, forward: 5′-AATGGTGAAGGTCGGTGTG-3′, reverse: 5′-GTGGAGTCATACTGGAACATGTAG-3′, product: 150 bp. The reference gene Gapdh was used as an internal control. Samples were measured in duplicates, and Ct values were averaged to reduce technical variability. Fold changes were calculated by the ΔΔCt method and standardized as described by
). In brief: Antiserum: “88111-3” (working dilution 1:10,000). Tracer: mono-125I-labeled ovine PACAP 24–38 prepared in our laboratory (5,000 cpm per tube). Standard: ovine PACAP-38 was used as a radioimmunoassay standard ranging from 0 to 1,000 fmol ml−1. Buffer: the assay was prepared in 1 ml 0.05 mol l−1 (pH 7.4) phosphate buffer containing 0.1 mol l−1 sodium chloride, 0.25 w/v % BSA, and 0.05 w/v % sodium azide. Incubation time: 48–72 h incubation at 4 °C. Separation solution: charcoal/dextran/milk powder (10:1:0.5 g in 100 ml distilled water). PACAP-38 concentrations of the unknown samples were read from a calibration curve; detection limit of the assay was 2 fmol ml−1.
Histology, immunohistochemistry
Plantar skin samples were fixed in 4% paraformaldehyde for 1 hour at room temperature, and serial histological sections were prepared. The specimens were rinsed in phosphate-buffered saline, permeabilized in 0.1% Triton X-100 for 5 minutes, and incubated with 0.1% BSA, 1% normal goat serum, and 0.1% Na-azide for 1 hour to minimize nonspecific labeling. Sections were incubated overnight at room temperature with anti-PAC1 antibody raised in rabbit (1:100, given by Seiji Shioda). For semi-quantitative purposes, sections were incubated for 2 hours in the dark with the corresponding Alexa Fluor “568” secondary antibody, raised in goat (1:1,000), and mounted with Fluoromount-G. Digital photographs were taken with a Nikon Eclipse 80i microscope (Tokyo, Japan). PAC1 intensity was measured with the ImageJ 1.440 software; expression levels were corrected with the tissue background (standard duration, settings, unit range: 250 × 250 pixels, 10 measurements/slide averaged).
For qualitative purposes, the 3,3’-diaminobezidine technique was preferred. The same protocol was used, but after the same primary antibody, the sections were incubated in biotinylated goat anti-rabbit serum (1:200) for 1 hour at room temperature and transferred into the avidin-biotin complex for 1 hour. The immunoreaction was developed in 0.02% 3,3’-diaminobezidine in Tris buffer with 0.00003% H2O2, for 10 minutes. Sections were mounted on gelatin-coated slides, treated with for 2 × 10 minutes with xylene, and covered with DePex. To test the specificity of the antibodies, some sections were treated by non-immune goat serum instead of the primary or secondary antibody. In addition, in three separate vials, the 1:100 diluted primary antiserum was pre-incubated for 2 hours at room temperature with 0.1, 1, or 10 μg of synthetic (blocking) peptide, to which the PAC1 antibody was raised. After development, immunoreaction signal was not observed in either cases, and the pre-absorption peptide completely abolished the immunolabeling in all three cases.
Induction and assessment of acute paw inflammation
Neurogenic and mixed-type inflammatory reactions were evoked by intraplantar injection of 50 μl capsaicin (100 μg ml−1) or complete Freund’s adjuvant (CFA, killed Mycobacteria tuberculosis suspended in paraffin oil; 50 μl, 1 mg ml−1) into the left hindpaw, respectively. The respective solvents were injected in the same volume into the contralateral paw. Mice were killed in deep anesthesia 4 or 24 hours after capsaicin or CFA. The plantar skin of the hindpaws was removed and cut into half for further PCR and radioimmunoassay processing, as described.
To assess the role of PACAP and TRPV1 in paw swelling induced by inflammatory stimuli, such as capsaicin and CFA activating distinct inflammatory mechanisms, the paw volume was measured with plethysmometry (Ugo Basile, Gemonio, Italy) before and repeatedly after the induction of the inflammation. Edema was expressed as % of initial control (n=6–8/group).
Statistical analysis
Statistical evaluation of plasma PACAP-IR and mRNA data was performed with the Mann–Whitney U-test. For analyzing the results in the inflammation experiments (edema, PACAP/PAC1 immunoreactivities), analysis of variance+Bonferroni’s t-test was used (P<0.05 was considered significant).
Drugs and chemicals
PACAP-38, capsaicin (8-methyl-N-vanillyl-6-nonenamide), CFA, and ethidium bromide were obtained from Sigma (St Louis, MO), ethanol and Tween 80 from Reanal (Budapest, Hungary), ketamine from Richter Gedeon (Budapest, Hungary) and xylazine from Eurovet Animal Health BV (Bladel, The Netherlands), RNA-later from Qiagen (Hilden, Germany), TRIzol and primers from Invitrogen (now part of Thermo Fisher Scientific, Renfrew, UK), AMV reverse transcriptase from Promega (Madison, WI), and Thermal Cycler from Applied Biosystems (now part of Thermo Fisher Scientific). Alexa Fluor “568” secondary antibody and Fluoromont-G were purchased from Southern Biotech (Birmingham, AL), biotinylated goat anti-rabbit serum and the avidin-biotin complex (Vectastain Elite ABC Kit) from Vector Laboratories (Burlingame, CA), and DePex from Fluka (Germany). Capsaicin was dissolved in 10% ethanol, 10% Tween 80, and 80% saline (0.9% NaCl) to a 10 mg ml−1 stock solution and further diluted with saline. PACAP-38 antiserum was provided by Prof. A. Arimura (Tulane University, New Orleans, LA).
ACKNOWLEDGMENTS
This work was sponsored by Hungarian Grants OTKA 104984, OTKA PD100706, SROP-4.2.2.B-15/KONV-2015-0011 Supporting Scientific Training of Talented Youth at the University of Pécs 2015, Arimura Foundation, and Hungarian Brain Research Program (grant KTIA_13_NAP-A-III/5). DR and TB are supported by “Lendület” Program of the Hungarian Academy of Sciences, whereas TB received further support from OTKA 101761, OTKA 105369, and TÁMOP-4.2.2./A-11/1/KONV-2012-0025. JK and EP were supported in the form of a scholarship by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/ 2-11/1-2012-0001 “National Excellence Program.” HH was supported in part by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research, KAKENHI, Grant Numbers 26293020 and 26670122, the Funding Program for Next Generation World-Leading Researchers, Grant number LS081, and by a grant for research from Uehara Memorial Foundation, Japan. The project was subsidized by the European Union and co-financed by the European Social Fund. We thank Dr Agnes Kemény for editing the figures.
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