Induction by carrageenan inflammation of prepronociceptin mRNA in VR1-immunoreactive neurons in rat dorsal root ganglia
Introduction
Nociceptin, also known as orphanin FQ, is peptide which was discovered as an endogenous ligand for opioid receptor-like 1 (ORL1) receptor (Meunier et al., 1995, Reinscheid et al., 1995). In these reports, an intracerebroventricular injection of nociceptin was shown to induce hyperalgesia by hot plate and tail-flick tests in mice (Meunier et al., 1995, Reinscheid et al., 1995). Thereafter, some reports showed that an intrathecal injection of nociceptin induced both thermal hyperalgesia and mechanical allodynia (Hara et al., 1997, Sakurada et al., 1999, Minami et al., 2000). On the contrary, other reports showed that an intrathecal injection of nociceptin induced thermal analgesia (King et al., 1997, Tian et al., 1997). Thus, although the precise role of nociceptin is unknown, this peptide may be involved in the regulation of nociceptive information in the spinal dorsal horn.
When examined with reverse transcription-polymerase chain reaction (RT-PCR), the highest level of prepronociceptin (PPN) mRNA expression is localized in the cerebral cortex, pons/medulla, and midbrain in rats; a moderate level in the cerebellum, hypothalamus, and striatum; a low level in the thalamus, hypothalamus, and spinal cord (Andoh et al., 1997). Although there is little or no expression of PPN mRNA in the dorsal root ganglia (DRG) of the naive rats, a peripheral inflammation caused by carrageenan was found to produce a rapid induction of PPN mRNA there (Andoh et al., 1997). The induction peaked 30 min after carrageenan injection; it followed peripheral inflammation and preceded thermal hyperalgesia, suggesting that nociceptin induced in primary sensory neurons by peripheral inflammation is involved in thermal hyperalgesia. The findings generate interest in the type of primary sensory neurons expressing PPN mRNA after peripheral inflammation.
Vanilloid receptor subtype 1 (VR1) was cloned as a capsaicin receptor (Caterina et al., 1997). VR1 receptor is activated by capsaicin, noxious heat, extracellular acidification, anandamide and lipoxygenase products and induces the influx of cations, particularly Na+ and Ca2+ ions (Caterina et al., 1997, Tominaga et al., 1998, Zygmunt et al., 1999, Hwang et al., 2000). A high level of VR1 mRNA is expressed in the DRG and trigeminal ganglia (Caterina et al., 1997). Little or no level of VR1 mRNA is expressed in the brain and spinal cord (Caterina et al., 1997, Tominaga et al., 1998), although RT-PCR reveals the expression in some brain regions (Sasamura et al., 1998, Sasamura and Kuraishi, 1999). VR1-expressing primary sensory neurons are associated with nociceptive transmission. Therefore, in the present study, we investigated the localization of PPN mRNA in primary sensory neurons of rats after peripheral inflammation and co-expression of PPN mRNA and VR1 receptors in the sensory neurons.
Section snippets
Animals
Male Sprague–Dawley rats (Japan SLC Ltd., Shizuoka) were used at 5 weeks old. The animals were kept under controlled temperature (22±1°C), humidity (55±10%), and light (lights on 07:00–19:00 h). Food and water were freely available.
Materials
The materials were obtained from the following sources; alkaline phosphate-conjugated anti-digoxigenin (DIG) antibody, 5-bromo-4-chloro-4-indonyl-phosphate 4-toluidine salt, 4-nitroblue tetrazolium chloride, and proteinase K were from Boehringer Mannheim
Carrageenan-induced expression of PPN mRNA
There were no cells positive for PPN mRNA in the DRG of untreated rat (Fig. 1A). Half an hour after carrageenan injection, there were many neurons strongly positive for PPN mRNA in the DRG on the treated side (Fig. 1B). Positive neurons were also observed at 1 and 3 h after carrageenan injection, but signal intensity and the number of positive cells were less than those at 0.5 h (Fig. 1C and D). The positive neurons were small or medium in diameter (<30 μm) (Fig. 1B–D). The percentage of PPN
Discussion
Although there were no cells positive for PPN mRNA in the DRGs of normal rats, an injection of carrageenan into the hind paw produced the induction of PPN mRNA in DRG neurons. The induction was rapid in onset and short-lasting, and the time course of the induction was similar to the detection using RT-PCR (Andoh et al., 1997), in which the induction level peaked 30 min after carrageenan, then decreased rapidly, and returned to normal by 6 h.
The sensory neurons that expressed PPN mRNA after
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