Serotonin and noradrenaline modulate chronic itch processing in mice.

The roles of serotonin and noradrenaline in the modulation of chronic pruriceptive processing currently remain unclear. To clarify the contribution of serotonin and noradrenaline to chronic itch, the effects of the administration of antidepressants or noradrenaline reuptake inhibitors were evaluated in the present study. A pretreatment with milnacipran, a serotonin and noradrenaline reuptake inhibitor, and mirtazapine, a noradrenergic and specific serotonergic antidepressant, attenuated the induction of spontaneous scratching behavior in mice with chronic itch. The administration of a serotonin reuptake inhibitor, such as fluvoxamine and paroxetine, but not escitalopram, or a noradrenaline reuptake inhibitor, such as atomoxetine and nisoxetine, ameliorated the induction of spontaneous scratching behavior in mice with chronic itch. Furthermore, this attenuation was reversed by the administration of yohimbine, a selective α2-adrenoceptor antagonist, or methysergide, a non-selective serotonin receptor antagonist. These results suggest that elevated serotonin and noradrenaline levels are involved in the attenuation of scratching behavior induced by chronic itch, and serotonin receptors and an α2-adrenoceptor play a crucial role in chronic pruriceptive processing.

Pharmacological characteristics of hemokinin-1-derived peptides in rat pruriceptive processing.

Hemokinin-1 (HK-1) is a member of mammalian tachykinin peptide family, and [Leu11]-HK-1 has an antagonistic effect on HK-1. The attenuation of pruritogen-induced scratching behavior by pretreatment with [Leu11]-HK-1 indicates the involvement of HK-1 in pruriceptive processing. However, it remains unclear whether the intrathecal or intranasal administration of HK-1-derived peptides, such as [D-Trp7,9]-[Leu11]-HK-1 or [D-Trp7]-[Leu11]-HK-1, elicits the effects different from [Leu11]-HK-1. The induction of scratching by intrathecal administration of HK-1 was attenuated 30 min, 4 h and 24 h after pretreatment with [Leu11]-HK-1, [D-Trp7,9]-[Leu11]-HK-1 and [D-Trp7]-[Leu11]-HK-1 or [D-Trp9]-[Leu11]-HK-1, respectively. Similarly, the scratching induced by subcutaneous injection of pruritogens as chloroquine and histamine was ameliorated 30 min and 24 h after pretreatment with [Leu11]-HK-1 and these three HK-1-derived peptides, respectively. Moreover, the effective minimum concentrations of intrathecal administrations of [D-Trp9]-[Leu11]-HK-1 on scratching induced by chloroquine and histamine were 10-6 M, while the effective minimum concentrations of intranasal administration of this peptide on scratching induced by chloroquine and histamine were 10-5 M and 10-4 M, respectively. Thus, the present results indicate that the intrathecal administration of HK-1-derived peptides with D-Trp extends its effective time on scratching induced by intrathecal administration of HK-1 and pruritogens such as chloroquine and histamine. Similarly, the induction of scratching by pruritogens was attenuated by intranasal administration of HK-1-derived peptide, although the effective minimum concentration of this peptide was slightly lower than that of intrathecal administration, indicating that intranasal administration is an effective tool for carrying peptides into the brain.

Expression Analysis of Serotonin Receptors, Serotonin Transporter and l-Amino Acid Decarboxylase in the Mouse Sphenopalatine Ganglion by RT-PCR, Northern Blot Analysis and In Situ Hybridization.

The sphenopalatine ganglion (SPG) is a gathering of the cell bodies of parasympathetic fibers that dominate the nasal gland, lacrimal gland and cerebral blood vessels. The SPG controls nasal secretions, tears, and the dilation of cerebral blood vessels. However, it is unclear how serotonin regulates SPG functions. In this study, we investigated the expression of genes involved in the serotonergic system in the mouse SPG. We examined the mRNA expression levels of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3A, 5-HT3B, 5-HT4, 5-HT5A, 5-HT5B, 5-HT6 and 5-HT7 receptors, as well as serotonin transporter, tryptophan hydroxylases 1 and 2, and L-amino acid decarboxylase (AADC) by RT-PCR. It revealed that the 5-HT3A and 5-HT3B ionotropic receptors and AADC were likely to be highly expressed in the SPG, as measured by RT-PCR. We next performed in situ hybridization on the SPG to examine the expression of these three genes at the cellular level after validating the specificity of each cRNA probe by northern blotting. The 5-HT3A receptor, 5-HT3B receptor, and AADC were expressed in 96.5% ±â€¯1.0%, 29.7% ±â€¯10.7%, and 57.4% ±â€¯2.9% of neuronal cell bodies in the SPG, respectively, indicating that the 5-HT3A receptor was virtually expressed in all SPG neurons. Our results on the expression of these critical serotonin system genes in the parasympathetic SPG provide insight into the pathogenetics of rhinitis, conjunctivitis and headache. Furthermore, our findings suggest that targeting the 5-HT3A receptor might have therapeutic potential in the treatment of these ailments.

Role of serotonin and noradrenaline in the acute itch processing in mice.

The contribution of serotonin and noradrenaline to the modulation of pruriceptive processing was evaluated by administrating antidepressants or noradrenaline reuptake inhibitors. The pretreatment with milnacipran, a serotonin and noradrenaline reuptake inhibitor, and mirtazapine, a noradrenergic and specific serotonergic antidepressant, attenuated the induction of scratching behavior by chloroquine, a representative pruritogen, indicating the involvement of serotonin and/or noradrenaline in the modulation of pruriceptive processing. By contrast, the single administration of noradrenaline reuptake inhibitor such as atomoxetine and nisoxetine or serotonin reuptake inhibitor such as fluvoxamine and escitalopram had little effect on chloroquine-induced scratching, whereas the induction of scratching behavior by chloroquine was significantly ameliorated by co-administration of serotonin reuptake inhibitors and noradrenaline reuptake inhibitors. These results indicate that the simultaneous increases of serotonin and noradrenaline elicit the attenuating effect on pruriceptive processing induced by acute itch, and may also play a crucial role in the descending itch inhibitory system.

Distribution of hemokinin-1 in the rat trigeminal ganglion and trigeminal sensory nuclear complex.

OBJECTIVE: A new mammalian tachykinin peptide encoded in a TAC4 gene was identified and designated as hemokinin-1 (HK-1). A representative of the tachykinin peptide family is substance P (SP), and the function of SP has been well characterized as a pain transmitter or modulator, while it is possible that HK-1 is involved in pruriceptive processing, but, as yet, the distribution of HK-1 peptide in the trigeminal sensory system is still unknown. Thus, the aim of the present study was to elucidate the distribution of HK-1, while comparing the expression of SP, in the trigeminal ganglion and trigeminal sensory nuclear complex. DESIGN: The trigeminal ganglion and the brain stem of male SD rats were used in the immunohistochemical study. Since the amino acid sequence in the carboxyl-terminal regions of HK-1 and SP is common, polyclonal antibodies of HK-1 and SP derived from 6 amino acids consisting of amino-terminal regions of these peptides were produced in guinea pig and rabbit, respectively. The immunohistochemical staining of HK-1 and SP was conducted using frozen sections of the trigeminal ganglion and brain stem in rats. RESULTS: Immunohistochemical studies revealed the expression of HK-1 in small- and medium-sized trigeminal ganglion neurons, in the paratrigeminal nucleus, and in lamina I of the trigeminal nucleus caudalis, while there was no immunoreactivity of HK-1 in the trigeminal nucleus principalis, trigeminal nucleus oralis, and trigeminal nucleus interpolaris. CONCLUSION: These findings indicate that HK-1 is a target molecule for treatment of itch in the orofaicial regions.

The role of spinal serotonin receptor and alpha adrenoceptor on the antiallodynic effects induced by intrathecal milnacipran in chronic constriction injury rats.

Milnacipran, a reuptake inhibitor of noradrenaline (NA) and serotonin (5-HT), elicits an antiallodynic effect in rats with neuropathic pain; however, the role of NA and 5-HT receptors in the induction of the antiallodynic effect of milnacipran remains unclear. Thus, we examined the effects of prazosin as an α1 adrenoceptor antagonist, yohimbine as an α2 adrenoceptor antagonist, metergoline as a 5-HT1, 5-HT2 and 5-HT7 receptor antagonist, cyanopindolol as a 5-HT1A/1B receptor antagonist, ketanserin as a 5-HT2 receptor antagonist, and ondansetoron as a 5-HT3 receptor antagonist on the antiallodynic effect of milnacipran in neuropathic rats with chronic constriction injury (CCI). The CCI rats expressed mechanical and thermal allodynia, which was attenuated by intrathecal injection of milnacipran. Yohimbine, but not prazosin, reversed the milnacipran-induced antiallodynic effect. The antiallodynic effect of milnacipran was also reversed by metergoline, ketanserin and ondansetron, while cyanopindolol reversed the antiallodynic effect on mechanical, but not thermal stimulation. Furthermore, c-Fos expression in lamina I/II of the spinal dorsal horn was enhanced by thermal stimulation and the enhanced expression of c-Fos was suppressed by milnacipran. This effect of milnacipran was reversed by yohimbine, metergoline, katanserin and ondansetron, but not prazosin. These results indicate that the effect of milnacipran on mechanical and thermal allodynia and c-Fos expression is elicited through the α2 adrenoceptor, but not α1 adrenoceptor, and 5-HT2 and 5-HT3 receptors; furthermore, the 5-HT1A/1B receptor is involved in mechanical allodynia, but not thermal allodynia.

Pharmacological characteristics of endokinin C/D-derived peptides in nociceptive and inflammatory processing in rats.

Endokinins designated from the human TAC4 gene consist of endokinin A, endokinin B, endokinin C (EKC) and endokinin D (EKD). EKC/D is a peptide using the common carboxyl-terminal in EKC and EKD and consists of 12 amino acids, and exerts antagonistic effects on the induction of scratching behavior by substance P (SP). Some of SP-preferring receptor antagonists have several d-tryptophan (d-Trp); however, the pharmacological effect of EKC/D-derived peptides with d-Trp remains to be solved. Therefore, to clarify the pharmacological characteristics of EKC/D-derived peptides, effects of pretreatment with these peptides on SP-induced scratching and thermal hyperalgesia, formalin-induced flinching and carrageenan-induced inflammation were evaluated. Intrathecal administration of [d-Trp(8)]-EKC/D and [d-Trp(10)]-EKC/D showed a markedly long inhibitory effect, at least 14 h, whereas the antagonistic effects of [d-Trp(8,10)]-EKC/D and EKC/D without d-Trp disappeared after 1h. Furthermore, the inhibitory effect of [d-Trp(10)]-EKC/D-derived peptides was dependent on the number of amino acids from the amino-terminus, and the more numerous the amino acids, the more marked the antagonistic effect. Thus, these results indicate that the effective duration of EKC/D-derived peptides is dependent on the number of d-Trp in the carboxyl-terminal region and the amino-terminal region regulates the antagonistic effect of EKC/D.

Knockdown of the tachykinin neurokinin 1 receptor by intrathecal administration of small interfering RNA in rats.

The contribution of tachykinin neurokinin 1 (NK1) receptor to nociceptive processing in the dorsal horn has been evaluated by tachykinin NK1 receptor antagonism and knockout or knockdown of tachykinin NK1 receptor; however, these results have not always been consistent. Therefore, to reevaluate the role of tachykinin NK1 receptor in the dorsal horn, a solution of hemagglutinating virus of the Japan envelope (HVJ-E) with small interfering RNA (siRNA) against tachykinin NK1 receptor was administered intrathecally and then the effect of treatment on tachykinin NK1 receptor immunohistochemistry and on the induction of inflammation, thermal hyperalgesia and scratching behavior was evaluated. This treatment resulted in marked reduction of tachykinin NK1 receptor immunoreactivity through the spinal dorsal horn, and the induction of thermal hyperalgesia and scratching behavior by substance P was significantly attenuated in rats with tachykinin NK1 receptor siRNA. In addition, only one intrathecal injection of tachykinin NK1 receptor siRNA reduced carrageenan-induced inflammation and thermal hyperalgesia significantly and markedly attenuated the induction of flinching after formalin injection and c-Fos expression in the dorsal horn following formalin injection. The efficient down-regulation of tachykinin NK1 receptor by intrathecal administration tachykinin NK1 receptor siRNA suggests that this method may be a valuable tool for examining the function of genes expressed in the dorsal horn.

Subcutaneous injection of endokinin C/D attenuates carrageenan-induced inflammation.

Endokinins, encoded by the human preprotachykinin C (PPT-C)/TAC4 gene, are peptides that consist of endokinin A (EKA), B (EKB), C (EKC) and D (EKD) and belong to the tachykinin family. Intrathecal injection of EKC/D (using the common carboxyl-terminal duodecapeptide in EKC and EKD) markedly attenuated the induction of thermal hyperalgesia and scratching behavior by intrathecal administration of substance P (SP), indicating that EKC/D has an antagonistic effect on the neurokinin 1 receptor (NK1R), SP-preferring receptor, at the spinal level; however, the pharmacological function of EKC/D at the periphery is not yet understood. Therefore, to clarify the effect of EKC/D on the peripheral tissue, the effect of subcutaneous injection of EKC/D on carrageenan-induced inflammation was examined. Subcutaneous injection of EKC/D attenuated an increase in paw volume following carrageenan-induced inflammation in a dose-dependent manner. Indeed, the increased paw volume was significantly decreased 40 min after treatment with 10(-4) M (10 nmol) and 10(-3) M (100 nmol) EKC/D (100 microl/rat). Similarly, injection of NK1R antagonists such as L-703,606 and Spantide I (10(-3) M) attenuated the increased paw volume following inflammation. Furthermore, the reduced withdrawal latency evoked by inflammation following subcutaneous injection of carrageenan was also dose-dependently attenuated by EKC/D administration. These results indicate that subcutaneous injection of EKC/D elicits an anti-inflammatory effect on carrageenan-induced inflammation.

Differential effects of substance P or hemokinin-1 on transient receptor potential channels, TRPV1, TRPA1 and TRPM8, in the rat.

Two tachykinin peptides, substance P (SP) and hemokinin-1 (HK-1), and three transient receptor potential (TRP) channels, TRPV1, TRPA1 and TRPM8, are similarly localized in the spinal dorsal horn and dorsal root ganglion, suggesting that TRP channels may be related or modulated by these tachykinin peptides. Thus, to clarify whether the responses of TRP channels are modulated by SP or HK-1, the effects of pretreatment with SP or HK-1 on the induction of scratching behavior by TRP channel agonists were examined. Pretreatment with SP or HK-1 enhanced the induction of scratching behavior by resiniferatoxin, a TRPV1 agonist, whereas scratching behavior induced by menthol, a TRPM8 agonist, was suppressed by pretreatment with these peptides. On the other hand, pretreatment with SP, but not HK-1, suppressed the induction of scratching behavior by cinnamaldehyde, a TRPA1 agonist. Taken together, the present results indicate that SP or HK-1 differentially modulated the response of TRPV1, TRPA1 or TRPM8 channel.