Results

Nociceptin-induced scratching

An intradermal injection of nociceptin (30 nmol per site) markedly elicited scratching in ICR mice. The onset of scratching was within 1 min after injection in all mice examined. The effect peaked during the first 10-min period and had almost completely subsided by 30 min (Figure 1a). The scratching was concentration dependently increased in the range of 1–100 nmol per site of nociceptin (Figure 1b). An intradermal injection of nociceptin (30 nmol per site) also elicited scratching in C57BL/6 mice, but it was without effect in ORL1^-/- mice (Figure 2).

Figure 1.

Scratching after intradermal injection of nociceptin in ICR mice. (a) Time-course of scratching after nociceptin (30 nmol per site) injection. (b) Dose–response curves for the scratch-inducing effect of nociceptin. Mice were given an intradermal injection of nociceptin or vehicle (VH: saline). Values represent the meansSEM for eight animals. ^*p<0.05 when compared with VH (Dunnett's multiple comparisons).

Full figure and legend (14K)

Figure 2.

ORL1 receptor deficiency abolishes scratching induced by intradermal injection of nociceptin in mice. Nociceptin (30 nmol per site) was injected intradermally into C57BL/6 (ORL1^+/+) and ORL1^-/- mice. Values represent the meansSEM for eight animals. ^*p<0.05 (Student's t test).

Full figure and legend (5K)

Effects of naloxone on nociceptin-induced scratching

Subcutaneous pre-treatment with naloxone (1 mg per kg) significantly inhibited scratching induced by nociceptin (30 nmol per site) (Figure 3a). On the other hand, an intradermal injection of naloxone (100 nmol per site) together with nociceptin (30 nmol per site) did not affect nociceptin-induced scratching (Figure 3b).

Figure 3.

Effect of naloxone on nociceptin-induced scratching in ICR mice. The mice were given an intradermal injection of nociceptin (30 nmol per site) and scratch bouts were counted for 1 h. (a) Naloxone (1 mg per kg) and vehicle (VH: saline) were injected subcutaneously 15 min before nociception. (b) Naloxone (100 nmol per site) was injected intradermally together with nociceptin (30 nmol per site). Values represent the meansSEM for eight animals. ^*p< 0.05 (Student's t test).

Full figure and legend (11K)

Effects of anti-allergic agents on nociceptin-induced scratching

Scratching induced by an intradermal injection of nociceptin (30 nmol per site) was significantly inhibited by oral pre-treatment with azelastine (10 mg per kg), but not with terfenadine (30 mg per kg) (Figure 4a). The LTB₄ receptor antagonist ONO-4057 (30 and100 mg per kg) concentration dependently inhibited the nociceptin-induced scratching (Figure 4b).

Figure 4.

Effects of anti-allergic agents on nociceptin-induced scratching in ICR mice. The mice were given an intradermal injection of nociceptin (30 nmol per site) and scratch bouts were counted for 1 h. (a) Azelastine (AZL; 10 mg per kg) and terfenadine (TRF; 30 mg per kg) were administered orally 1 h before nociceptin. (b) ONO-4057 (30–100 mg per kg) was administered orally 1 h before nociceptin. Values represent the meansSEM for eight animals. ^*p<0.05 when compared with vehicle (VH) (Dunnett's multiple comparisons).

Full figure and legend (14K)

Prepronociceptin and ORL1 receptor mRNAs in the dorsal root ganglia and skin

Prepronociceptin mRNA was substantially expressed in the skin, but, on the other hand, the expression level was very low in the dorsal root ganglia in ICR mice (Figure 5a). ORL1 receptor mRNA was also substantially expressed in the skin, whereas the expression level was very low in the dorsal root ganglia (Figure 5b).

Figure 5.

Expression of prepronociceptin (PPN) and ORL1 receptor mRNAs in the mouse skin and dorsal root ganglia (DRG). The expression levels of mRNAs of prepronociceptin and ORL1 receptor were determined with RT-PCR method (for detailed procedures see Materials and Methods). The expression level was normalized to the level of -actin mRNA. Typical examples of the bands of PPN and -actin mRNAs are shown above the corresponding columns. Values represent the meansSEM for three animals.

Full figure and legend (14K)

Distribution of nociceptin and ORL1 receptor in the skin

ORL1 receptor-like immunoreactivity was localized in the epidermis, especially its superficial layers, of the back skin and absent there in ORL1^-/- mouse (Figure 6, upper panels). Nociceptin-like immunoreactivity was also localized in the superficial layer of epidermis, which was not affected by the deficiency of ORL1 receptors (Figure 6, lower panels). Similar results were obtained in other four C57BL/6 mice and four ORL1^-/- mice.

Figure 6.

Typical examples of distribution of nociceptin and ORL1 receptor in the mouse skin. Nociceptin and ORL1 receptor were immunostained in the back skin of C57BL/6 (ORL1^+/+) and ORL1^-/- mice. Scale bar=50 m.

Full figure and legend (179K)

Production of LTB₄ in keratinocytes

In the above experiments, ORL1 receptor-like immunoreactivity was localized in the epidermis, the major cell of which is keratinocytes. Since LTB₄ is produced by keratinocytes (^{Andoh et al, 2001}) and elicits scratching at relatively low intradermal doses (^{Andoh and Kuraishi, 1998}), we examined whether nociceptin would increase the production of LTB₄ in the keratinocytes in vitro. A bath application of nociceptin (10 and 100 M) increased the production of LTB₄ in a concentration-dependent manner in primary cultures of keratinocytes from ICR and C57BL/6 mice (Figure 7). The effect of nociceptin (100 M) was abolished by zileuton (10 M) and deficiency in ORL1 receptors (Figure 7).

Figure 7.

Nociceptin-induced production of LTB₄ in mouse keratinocytes. Keratinocytes were prepared from the epidermis of the back skin of ICR, C57BL/6, and ORL1^-/- mice. Nociceptin (10 and 100 M) was administered to primary cultures of the mouse keratinocytes, and 5 min later the supernatant was taken for the assay. Zileuton (10 M) was administered 1 h before nociceptin. Values represent the means SEM for six animals, ^* and ^#p<0.05 when compared with the group without nociception and nociceptin (100 M) alone (Dunnett's multiple comparisons).

Full figure and legend (11K)

Discussion

Nociceptin-induced scratching

One aim of this study was to test whether nociceptin would elicit itch. An intradermal injection of nociceptin elicited scratching in mice. The effective dose range (1–100 nmol per site) of nociceptin wassimilar to those of substance P (^{Andoh et al, 1998}), histamine (^{Maekawa et al, 2000}), and serotonin (^{Yamaguchi et al, 1999}). Systemic administration of the opioid antagonist naloxone markedly inhibited nociceptin-induced scratching, whereas a local injection of naloxone was without effect. Scratching induced by substance P and serotonin in healthy mice and the spontaneous scratching of mice with chronic dermatitis or xerosis are inhibited by systemic injections of opioid antagonists (^{Andoh et al, 1998;Yamaguchi et al, 1999,2001;Miyamoto et al, 2002}). In mice with chronic dermatitis, systemic administration of the opioid antagonist naltrexone suppresses the spontaneous scratching without effect on the increased firing of cutaneous branch of sensory nerve (^{Maekawa et al, 2002}). Thus, the results suggest that opioid receptors in the skin (for example, on the peripheral terminals of sensory neurons) are not involved in the nociceptin action and that naloxone inhibits nociceptin-induces scratching through central action. This idea is consistent with the findings that nociceptin does not show any activity at the opioid receptors (^{Mahis et al, 1997}). Opioid antagonists reduce experimentally elicited itch (^{Bernstein et al, 1982}) and itching of patients with pruritic diseases such as cholestasis, chronic urticaria, and atopic dermatitis (^{Monroe 1989;Bergasa et al, 1995}). Opioids may induce pruritus through mu-opioid receptors in the central nervous system (^{Szarvas et al, 2003}). Thus, the inhibition of nociceptin-induced scratching by systemic naloxone suggests that the scratching is an itch-associated response.

In these experiments, an injection of nociceptin into the rostral back elicited scratching. On the other hand, an injection of nociceptin into the hind paw was shown to elicit withdrawal response in mice (^{Inoue et al, 1998}). In their experiments, mice were suspended and could not lick and bite the hind paw, although painful and pruritic stimulation of the hind paw induces licking (or flinching) and biting behaviors, respectively, in mice (^{Hagiwara et al, 1999}). Thus, it is possible that the withdrawal-like response of suspended animal is related to itch. It is also possible that nociceptin is more effective in exciting nociceptors in the plantar skin than in the back skin, and a subcutaneous injection of nociceptin into the plantar skin elicits pain in mice. To determine whether intraplantar nociceptin elicits itch and/or pain, this peptide should be injected into the plantar region of freely moving animals.

Origin and target cells of nociceptin

Nociceptin-induced scratching was abolished by deficiency of ORL1 receptors, suggesting that the action is mediated by ORL1 receptors. It is suggested that keratinocytes are the main origin cells for nociceptin in the skin. The expression level of prepronociceptin mRNA was relatively high in the skin and nociceptin-like immunoreactivity was localized in the superficial layers of the epidermis. These results strongly suggest that keratinocytes in the superficial layers synthesize and contain nociceptin. On the other hand, there were no nociceptin-immunoreactive nerve fibers in the skin. The expression level of prepronociceptin mRNA was low in the dorsal root ganglia. The result is consistent with the finding that the level of prepronociceptin mRNA is very low in the dorsal root ganglia of normal rats (^{Andoh et al, 1997}).

These results strongly suggest that keratinocytes are the main target cells for intradermal nociceptin, and co-localization of nociceptin and its receptor in the keratinocytes suggest the autocrine-like action of nociceptin. The expression level of ORL1 receptor mRNA was relatively high in the skin and ORL1 receptor-like immunoreactivity was localized in the superficial layers of the epidermis. The immunoreactivity was absent in the skin of ORL1^-/- mice. On the other hand, the expression level of ORL1 receptor mRNA was very low in the dorsal root ganglia and there were no nerve fibers immunoreactive for ORL1 receptors. But we do not rule out the possibility of nociceptin action on primary afferents. The splice variants of ORL1 receptor were reported to be present in the dorsal root ganglia (^{Xie et al, 1999}), and nociceptin was suggested to act on primary afferents (^{Inoue et al, 1998}).

LTB₄ involvement in nociceptin-induced scratching

Nociceptin-induced scratching was inhibited by the LTB₄ antagonist ONO-4057, which inhibits LTB₄-induced scratching in mice (^{Andoh et al, 2001}). An intradermal injection ofLTB₄ at relatively low doses elicits scratching in mice (^{Andoh and Kuraishi, 1998}). Therefore, LTB₄ may play an important role in the nociceptin-induced scratching. Azelastine (10 mg per kg) suppressed nociceptin-induced scratching. This dosage inhibits LTB₄ production in the skin and LTB₄-induced scratching in mice (^{Andoh and Kuraishi, 2002b}). In addition, azelastine suppresses depolarization-induced increase in intracellular Ca²⁺ concentration in cultured dorsal root ganglion neurons (^{Ohtsuka et al, 2003}). These actions may be involved in the inhibition by azelastine of nociceptin-induced scratching.

Nociceptin induced the production of LTB₄ in primary cultures of keratinocytes. This effect was abolished by the deficiency of ORL1 receptors, suggesting the involvement of ORL1 receptors. The mechanism of the nociceptin-induced production of LTB₄ is not clear; however, ORL1 receptor is a pertussis toxin-sensitive-G-protein (Gi)-coupled receptor (^{Hawes et al, 1998}) and Gi activates phospholipase C (^{Ueda et al, 1995}). Activated phospholipase C increases intracellular Ca²⁺ concentration. In our preliminary experiments, nociceptin increased intracellular Ca²⁺ concentration in mouse keratinocytes (Andoh and Kuraishi: unpublished observation). An increase in intracellular Ca²⁺ activates the secretory- and cytosolic-type phospholipase A₂ (^{Clark et al, 1990}). Such processes may lead to the production of LTB₄ in keratinocytes.

Role of histamine in nociceptin-induced scratching

Nociceptin-induced scratching was not affected by the H₁ histamine receptor antagonist terfenadine. Since the same dosage inhibits histamine-induced scratching in mice (^{Ohtsuka et al, 2001}), histamine may not play an important role in nociceptin-induced scratching. An intradermal injection of nociceptin increases vascular permeability through the release of histamine from mast cells in rats (^{Kimura et al, 2000}). But terfenadine suppresses plasma extravasation, but not scratching, induced by immediate allergy (^{Ohtsuka et al, 2001}), suggesting that mediator(s) involved in scratching is different from those of plasma extravasation.

Summary

Nociceptin released from epidermal keratinocytes may stimulate ORL1 receptors on the keratinocytes through autocrine mechanisms. Stimulated keratinocytes produce LTB₄, which acts on primary afferents to induce itch sensation. In our preliminary experiments, nociceptin was increased in the inflamed skin of itching mice (NC strain), a mouse model of atopic dermatitis (^{Yamaguchi et al,2001}). Although further experiments are needed, it is possible that nociceptin is involved in inflammatory itchy skin diseases. Nociceptin and ORL1 receptor in the skin may be possible target molecules for new antipruritic agents.

Materials and Methods

Animals

We used male ICR mice (4–5 weeks old; Japan SLC, Shizuoka, Japan), male C57BL/6 (ORL1^+/+; Japan SLC), and ORL1 receptor-deficient mice (ORL1^-/-) with C57BL/6 genetic background (9–10 weeks old). They were housed 7–8 per cage in a room under controlled temperature (221°C), humidity (5510%), and light (lights on from 07:00 to 19:00 h). Food and water were freely available. Procedures in the animal experiments were approved by the Committee for Animal Experiments at Toyama Medical and Pharmaceutical University.

Materials

Nociceptin (Peptide Institute, Minoh, Japan) was dissolved in physiological saline. Azelastine (Eizai, Tokyo, Japan), 5-[2-(2-carboxyethyl)-3-oxyphenoxy] valeric acid (ONO-4057; Ono Pharmaceutical, Osaka, Japan), and terfenadine (Sigma Chemical, St Louis, Missouri) were suspended in 0.5% (wt/vol) sodium carboxymethyl cellulose (Wako Pure Chemical, Osaka, Japan). In in vivo experiments, azelastine and terfenadine were administered orally 30 min before nociceptin injection, and ONO-4057 before 1h. Naloxone hydrochloride (Sigma Chemical) was dissolved in physiological saline and injected subcutaneously 15 min before nociceptin or intradermally together with nociceptin. Zileuton (Ono Pharmaceutical) was dissolved in dimethyl sulfoxide.

Behavioral experiments

The hair was clipped over the rostral part of the mouse back. The next day, nociceptin was injected intradermally in a volume of 50 L into the interscapular region using a 27-gauge needle connected to a microsyringe via PE-10 tubing. Before behavior observation, the mice (four animals per observation) were put into an acrylic cage composed of four cells (13 9 30 cm) for at least 1 h for acclimation. Immediately after intradermal injection, the animals were put back into the same cells and their behaviors were videotaped using an 8 mm video camera for 1 h with personnel kept out of the observation room. Playing back of the video served for counting scratching behavior. The mice stretched either hind paw toward the injection site, leaned the head toward the hind paw, and rapidly scratched several times for about 1 s. A series of these movements was counted as one bout of scratching (^{Kuraishi et al, 1995}); scratch bout was considered to end when the mouse lowered its hind paw.

RT-PCR

The dorsal root ganglia (C1–T13 levels) and skin were removed, immediately frozen in liquid nitrogen, and stored at -80°C until assay. Total RNA was extracted with RNAzol B (Cinna/Biotecx, Houston, Texas) and treated with DNase I (Takara, Shiga, Japan). Total RNA (1 g) was incubated at 37°C for 60 min with a mixture of 100 U of reverse transcriptase (Superscript II, Gibco BRL, Rockville, Maryland), 1 buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 20 mM dithiothreitol), 0.5 mM dNTP, and 50pmol oligo(dT)₁₆ primer. Thereafter, the reaction mixture was incubated at 99°C for 5 min and then chilled on ice for 5 min. An aliquot (1 L) of the RT product was mixed with PCR solution containing 1.5 mM MgCl₂, 1 buffer, 1.5 U Taq DNA polymerase (Applied Biosystems, Branchburg, New Jersey), and 50 pmol of sense and antisense primers pair. The sequences of primers were as follows: prepronociceptin (sense), 5'-gtgactctgagcagctcagc-3'; prepronociceptin (antisense), 5'-ttctggttggccaacttccg-3'; ORL1 (sense) 5'-gatcgagtgcctggtggagatcc-3'; ORL1 (antisense) 5'-catgggcaggtccacgcctagtc-3'; -actin (sense), 5'-tcagaaggactcctatgtgg-3'; and -actin (antisense), 5'-tctctttgatgtcacgcacg-3'. A program for prepronociceptin was 33 cycles (94°C for 1 min, 70°C for 1 min, 72°C for 1 min), that for ORL1 33 cycles (94°C for 1 min, 55°C for 1 min, 72°C for 1 min), and that for -actin 28 cycles (94°C for 1 min, 55°C for 1 min, 72°C for 1 min). After the separation of PCR product with gel electrophoresis and ethidium bromide staining, the density of the band of predicted size was analyzed using NIH Image software (National Institute of Health, Bethesda, Maryland).

Immunohistochemistry

The animals were anesthetized with diethyl ether and euthanized via cervical dislocation. After exsanguination, the skin was removed from the rostral back, frozen in liquid nitrogen, and cut into sections of 20 m thickness. The sections were fixed by incubation with acetone at 4°C for 5 min and washed 4 times for 5 min with phosphate-buffered saline (pH 7.4). For quenching of endogenous peroxidase, the samples were treated with 0.3% H₂O₂ in methanol for 20 min, washed 4 times with phosphate-buffered saline, and then treated with the blocking buffer (0.5% fetal bovine serum in phosphate-buffered saline containing 0.1% Tween 20) for 30 min. They were then incubated with goat anti-nociceptin or goat anti-ORL1 receptor antibody (diluted 1:500 with blocking buffer; Santa Cruz Biotechnology, Santa Cruz, California) at 4°C overnight. After being washed with phosphate-buffered saline, they were incubated with horseradish peroxidase-conjugated donkey anti-goat IgG (diluted 1:2000 with blocking buffer; Santa Cruz Biotechnology) at room temperature for 1.5 h. After being washed with phosphate-buffered saline, the section was re-washed four times with 0.05 M Tris-HCl buffer (pH 7.6) containing 0.9% NaCl. Finally, they were treated with 0.2 mg per ml 3,3-diaminobenzidine (Wako Pure Chemical) in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.9% NaCl and 0.05% H₂O₂ at room temperature for 30 min. The staining was observed using a light microscope (AX80, Olympus, Osaka, Japan) with a CCD camera (Axio Cam, Carl Zeiss, Jena, Germany).

Enzyme immunoassay for LTB₄

Keratinocytes were prepared from neonatal mice and cultured (^{Andoh et al, 2001}). Nociceptin and zileuton (1 h before nociceptin) were added to the medium of primary cultures of mouse keratinocytes (10⁶ cells per well). Samples were taken from the medium 5 min after nociceptin administration and diluted with enzyme immunoassay buffer (Cayman Chemical, Ann Arbor, Michigan) for the assay. The amount of LTB₄ was determined using an enzyme immunoassay kit (Cayman Chemical).

Data processing

All data are presented as means and SEM. Statistical significance was analyzed using Dunnett's multiple comparisons or Student's t test; p<0.05 was considered significant.

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