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Activation of spinal neuropeptide FF and the neuropeptide FF receptor 2 during inflammatory hyperalgesia in rats
Neuroscience 118 (2003) 179 –187
ACTIVATION OF SPINAL NEUROPEPTIDE FF AND THE NEUROPEPTIDE FF RECEPTOR 2 DURING INFLAMMATORY HYPERALGESIA IN RATS H.-Y. T. YANG* AND M. J. IADAROLA
gested a variety of biological roles including nociception, cardiovascular regulation, opiate tolerance and neuroendocrine modulation (for review see Panula et al., 1996). The most widely explored functional role of NPFF has been on nociception because, in the rat CNS, the highest level of NPFF immunoreactivity is in the dorsal spinal cord (Majane et al., 1989) where it is highly localized in the superficial layers of the dorsal horn (Allard et al., 1991; Panula et al., 1987). Furthermore, NPFF was found to have anti- and pro-opioid activities; in general, NPFF was found to be anti-analgesic after i.c.v. injection but analgesic when injected intrathecally (for review see Roumy et al., 1998). The reason for this discrepancy has not been readily obvious, however, it is likely that NPFF represents only one of many RF-NH2 containing neuropeptides in the mammalian nervous system and, depending on the pharmacological doses used, NPFF may exhibit the biological activities of other structurally related RF-NH2 peptides. The gene coding for the NPFF precursor protein has been cloned and it codes for both NPFF and NPAF (neuropeptide AF) (AGEGLSSPFWSLAAPQRF-NH2) (Vilim et al.,1999; Perry et al., 1997). In addition to the NPFF gene, another Arg-Phe-NH2 peptide gene has recently been identified and peptides similar to NPFF were deduced from the precursor protein (Liu et al., 2001; Hinuma et al., 2000). The peptides deduced from the human precursor are NPVF (VPNLPQRF-NH2) and a longer, 37-amino acid peptide (SLNFEELKDWGPKNVIKMSTPAVNKMPHSFANLPLRF-NH2). Very recently, the rat equivalent of NPVF was identified as an octadecapeptide (ANMEAGTMSHFPSLPQRF-NH2) by immunoaffinity purification coupled with mass spectrometry (Ukena et al., 2002). Depending on the doses used, NPVF-related peptides and NPFFrelated peptides may exhibit similar non-specific, or their unique specific, biological activities thus complicating biological activity studies of the RF-NH2-containing peptides. Recently, two G-protein-coupled receptors were identified and characterized to be responsive to NPFF (Bonini et al., 2000; Elshourbagy et al., 2000; Hinuma et al., 2000), and one of these receptors was suggested to be specific for NPVF in the studies of Hinuma et al. (2000) and Liu et al. (2001). Characterization of the two precursor proteins coding for two structurally related peptides, NPFF and NPVF peptides, and their receptors allows the functional roles of PQRF-NH2-containing peptides to be explored more accurately. To further understand the possible role of PQRFNH2-containing neuronal systems in pain transmission, we have studied (1) the distributions of genes expressing NPFF, NPVF and NPFF receptors and (2) regulation of
Neuronal Gene Expression Unit, Pain and Neurosensory Mechanisms Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Building 49, Room 1A07, 49 Convent Drive, MSC 4410, Bethesda, MD 20892-4410, USA
Abstract—Several lines of evidence suggest that neuropeptide FF (NPFF) is involved in nociception and in the modulation of opioid-mediated analgesia. Following the identification of the precursor protein for NPFF, two NPFF receptors and a second PQRF-NH2 containing peptide, termed NPVF, were identified. To further explore the functional role of PQRF-NH2 peptides, we have studied their distribution and also the regulation of NPFF and NPVF systems in the spinal cord of rats with peripheral inflammation. The distribution of NPFF gene expression is very similar to that of NPFF immunoreactive peptide but is distinct from NPVF gene expression. In the rat spinal cord, gene expression of NPFF but not that of NPVF was up-regulated by persistent pain induced by carrageenan inflammation. The distribution of NPFF receptor 2 gene expression is very similar to that of the NPFF peptide with a striking localization in the superficial layer of spinal cord. In rats with carrageenan inflammation of the hind paw, expression of both NPFF and NPFF receptor 2 genes was up-regulated in the spinal cord, while expression of NPVF and NPFF receptor 1 genes was not affected. The results of this study demonstrate a coordinated involvement of the spinal NPFF system in the persistent nociceptive pain states. Several studies have found a potentiation and prolongation of morphine analgesia by NPFF, therefore, it is highly possible that the endogenous spinal NPFF system contributes to the enhanced analgesic potency of morphine in animals with peripheral inflammation. Published by Elsevier Science Ltd on behalf of IBRO. Key words: nociception, pain, inflammation, dorsal spinal cord, NPVF.
Neuropeptide FF (NPFF; FLFQPQRF-NH2) was originally detected by an antiserum raised against FMRF-NH2 and subsequently isolated from bovine brain and sequenced (Yang et al., 1985). Biological studies of this peptide and the distribution of NPFF-immunoreactive peptides sug*Corresponding author: Neuronal Gene Expression Unit, Pain and Neurosensory Mechanisms Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Building 49, Room 1A07, 49 Convent Drive, MSC 4410, Bethesda, MD 208924410, USA. Tel: ⫹1-301-402-4981; fax: ⫹1-301-402-0667. E-mail address: [email protected] (H.-Y. T. Yang). Abbreviations: CCK, cholecystokinin; GPDH, glyceraldehyde 3-phosphate dehydrogenase; NPAF, neuropeptide AF; NPFF, neuropeptide FF; NPFF-R1, neuropeptide FF receptor 1; NPFF-R2, neuropeptide FF receptor 2; NPVF, VPNLPQRF-NH2; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction. 0306-4522/03$30.00⫹0.00 Published by Elsevier Science Ltd on behalf of IBRO. doi:10.1016/S0306-4522(02)00931-4
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NPFF and NPVF transcripts as well as NPFF receptor gene expression at the spinal cord level in an animal model of persistent peripheral inflammation induced by carrageenan. The data from the rat show a strong enrichment of both NPFF and NPFF receptor 2 in superficial layer of dorsal spinal cord as well as a local and concurrent upregulation of both peptide and receptor by persistent nociceptive activity.
EXPERIMENTAL PROCEDURES Animal, carrageenan treatment and tissue dissection Male Sprague–Dawley Rats (250 –350 g) were housed with a 12-h light/dark cycle and given water and food ad libitum. Procedures for all animals used were in accordance with the National Institute of Health (NIH) Guidelines for the Care and Use of Laboratory Animals, and were approved by the National Institute of Dental and Craniofacial Research (NIDCR) Animal Care and Use Committee. All efforts were made to minimize both animal numbers and suffering in the experiments. Carrageenan Lambda type IV (Sigma, St Louis, MO, USA), 6 mg in 150 l phosphate-buffered saline per paw, was injected into the planter surface of the hind paw bilaterally or unilaterally to induce a long-lasting inflammation as previously described (Iadarola et al., 1988). Rats were killed by carbon dioxide exposure at the specified time after the carrageenan treatments as indicated in legends to figures. Spinal cords were removed according to the method of DeSousa and Horrocks (1979). Spinal cord (whole cord or dorsal cord as indicated in figure legends) segments of 6 – 8 mm containing L4 –L5 were rapidly dissected, divided into dorsal and ventral halves (for bilateral carrageenan) or left and right halves (for unilateral carrageenan), frozen immediately in dry ice and stored at ⫺80 °C until processed for RNA extraction. For the bilateral carrageenan injection, untreated rats were use as control and for unilateral carrageenan injection, control sides were not injected. Controls were not injected because the aim of this study was to compare between absolutely normal animals and animals with long-lasting persistent inflammation. For the regional distributions of NPFF, NPVF and NPFF receptor 2 mRNAs, rats were killed with CO2 and the brain, spinal cord, pituitary gland and trigeminal ganglia were rapidly removed. Brains were dissected into various regions (cerebellum, striatum, medulla, olfactory bulb, substantia nigra, hypothalamus, hippocampus, thalamus, septum, frontal cortex, entorhinal cortex, inferior colliculus, superior colliculus, periaqueductal gray, nucleus accumbens) as described in Naranjo et al., 1986, and pituitary was divided into anterior and neurointermediate lobes. Spinal cords were removed as described above and dorsal and ventral halves, superficial layer and central canal were dissected out immediately on a cold plate (see Fig. 3). Tissues were frozen immediately in dry ice after dissection and stored at ⫺80 °C until processed for RNA extraction.
RNA extraction and RT-PCR To assess mRNAs coding for NPFF, NPVF and NPFF receptors, reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out with RNA prepared from tissues dissected as described above. RNA was extracted using RNeasy Mini or Midi Kit (Qiagen Inc., Valencia, CA, USA) with an additional step of RNase-free DNase treatment. RNA was quantified by the RiboGreen reagent (Molecular Probes, Eugene, OR, USA) using a 96-well fluorometric plate reader (Molecular Devices, Sunnyvale, CA, USA). RT-PCR was carried out using the Access RT-PCR system (Promega, Madison, WI, USA) with specific primers custom synthesized by Gene Probe Technologies Inc (Gaithersburg, MD, USA). The primer pairs are (1) ACTGCTGCTGCTGAG-
GAACT and CACTTTATTTGGGGGCACAC (384-bp product) for rat NPFF, (2) GGAATCCCAAAAGGGGTAAA and GGGTCATGGCATAGAGCAAT (331-bp product) for rat NPVF, (3) GCCAAACCAGGAAATGAGGA and TCATCAGAGTCCACAGGGGA (254bp product) and second set GAGCAGTGGCATGTATCCAA and TACATCCCAAGTTTTCCCCA (411-bp product) for rat NPFF receptor 2, (4) AACCAGCCTCACCTTCTCCT and GTTACGAGCATCCAGCATGAA (505-bp product) for rat NPFF receptor 1, (5) GCCAAATCAGGAAATGAGGA and TCATTAGAGTCCACAGGGGC (254-bp product) for human NPFF receptor 2, (6) ACCACAGTCCATGCCATCAC and TCCACCACCCTGTTGCTGTA (452-bp product) for rat glyceraldehydes-3-phosphate dehydrogenase (GPDH) and, (7) GACCCCTTCATTGACCTCAACT and CATCGCCCCACTTGATTTTG (166-bp product) for human GPDH. The RT-PCR reaction was carried out according to the manufacture’s instruction (Promega, Madison, WI, USA) in 25 l reaction mixture containing 2– 8 ng total RNA in the RoboCycler Gradient 96 Temperature Cycler (Stratagene, La Jolla, CA, USA). Reverse transcription and PCR cycling profile were one cycle of 45 min at 48 °C for reverse transcription, one cycle of 2 min at 94 °C for transcriptase inactivation, 28 –36 cycles of 94 °C denaturation for 30 s, 55 °C annealing for 1 min, 68 °C extension for 2 min and one cycle of 68 °C final extension for 7 min. To determine the amount of RNA and cycle amplification number that would yield reliable relative values for each target transcript, preliminary experiments were carried out with each pair of primers and RNA preparations. The optimal conditions for each target transcript and primer pair were applied in the assessment of differential levels of expression. The RT-PCR products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. For the estimation of relative quantities of RT-PCR products, image acquisition was performed on the gel with an AlphaImager (Alpha Innotech, San Leandre, CA, USA). The relative intensities of the bands corresponding to the specific RT-PCR products were analyzed quantitatively using ImageQuant 5 software (Molecular Dynamics, Piscataway, NJ, USA). The results, normalized to GPDH, are presented in the figures. For the verification of the RT-PCR products, two representative NPFF receptor 2 RT-PCR products from rat and human spinal cords were sequenced. Human NPFF receptor 2 primer and rat NPFF receptor 2 primer yield 254-base pair RT-PCR products. These cDNAs were cloned using the TA cloning kit (Clontech, Palo Alto, CA, USA, Advantage PCR Cloning kit) and sequenced by the NIDCR Sequencing Core Facility; the sequences were then compared with the sequences of NPFF receptors in Gene bank to verify the RT-PCR products.
RESULTS Distribution of mRNA encoding NPFF, NPVF and NPFF receptors The regional distributions of NPFF and NPVF gene expression in the rat CNS and several peripheral tissues were examined by semi-quantitative RT-PCR and results are shown in Fig. 1. The experiment was carried out twice and very similar patterns of distribution were observed. The results of one experiment are presented in Fig. 1. The distribution of NPFF mRNA is strikingly distinct from that of NPVF mRNA. The highest level of NPFF transcript is in the spinal cord and medulla oblongata with much lower levels in the cerebral cortex. In contrast, NPVF transcript is most highly localized in the hypothalamus with a moderate level in the spinal cord. However, further analysis of spinal cord revealed that the NPVF transcript is nearly exclusively localized in the dorsal horn (Fig. 6, bottom).
H.-Y. T. Yang and M. J. Iadarola / Neuroscience 118 (2003) 179 –187
Fig. 1. Distribution of NPFF and NPVF mRNAs in brain regions, spinal cord and some peripheral tissues. Total RNA preparations from tissues were subjected to semi quantitative RT-PCR analysis using specific primers for NPFF, NPVF and GPDH. The relative levels of NPFF and NPVF mRNAs were presented as normalized to GPDH. Top panel, NPFF mRNA; bottom panel, NPVF mRNA.
Regional distribution of NPFF receptor 2 mRNA was examined by RT-PCR with NPFF receptor 2 primers. The results shown in Fig. 2 revealed a markedly uneven distribution of NPFF receptor 2 transcript in the CNS. With a moderate number of PCR cycles (33 cycles), the regions which gave clear RT-PCR products for NPFF receptor 2 were limited to spinal cord and hypothalamus (Fig. 2) indicating a striking localization of NPFF receptor 2 transcript in the spinal cord in the rat CNS. The experiment was repeated with a different set of PCR primers (set 2)
Fig. 2. Distribution of NPFF receptor 2 mRNAs in brain regions, spinal cord and some peripheral tissues. Total RNA preparations from tissues were subjected to RT-PCR analysis using specific primers for NPFF receptor 2 and GPDH. The number of PCR cycle used for NPFF receptor 2 in this experiment was 35. The RT-PCR products were examined by a 2.0% agarose/ethidium bromide gel. The gel images, not sharpened or filtered, were cropped to show bands corresponding to NPFF receptor 2 and GPDH (bottom panel). The lanes on the gel correspond to the bars on the graph. The relative levels of NPFF receptor 2 mRNA are expressed as normalized to GPDH (top panel). The very high level of expression is in spinal cord.
and again the two regions found to show RT-PCR products were spinal cord and hypothalamus (results not shown). Further analysis of the spinal cord shows that NPFF receptor 2 expression is highest in the superficial layer of dorsal horn, moderate in the central canal (Fig. 3, left) and nearly undetectable in the ventral horn. In the human spinal cord, expression of NPFF receptor 1 was much more readily detected than that of NPFF receptor 2 (result not shown); however, the existence of NPFF receptor 2
Abbreviations used in the figures Accum Cerebell Ent cort Front cort Hippo Hypoth Inf Colli
accumbens cerebellum entorhinal cortex frontal cortex hippocampus hypothalamus inferior colliculus
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Olf bulb PAG Sp cord Sub nigra Sup colli Trig ganglia
olfactory bulb periaqueductal gray spinal cord substantia nigra superior colliculus trigeminal ganglia
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Fig. 3. Enrichment of NPFF receptor 2 mRNA in the dorsal spinal cord. Total RNA preparations from the rat and human spinal cords were subjected to RT-PCR analysis using specific primers for NPFF receptor 2 and GPDH. The RT-PCR products were analyzed by a 2.0% agarose/ethidium bromide gel. The gel images were photographed and prints were scanned by U-Max Power Look 2000. The scanned images, not sharpened or filtered, were cropped to show bands corresponding to NPFF receptor 2 and GPDH (left panel). The superficial layer (SL) represents the highest expressing sub region of the cord, and a moderate level of expression is in the central canal (CC). Various regions, dorsal cord, ventral cord, superficial layer and central canal were dissected as summarized in the summary figure (right panel).
transcript in the dorsal horn was definitely demonstrated by RT-PCR analysis (Fig. 3, left). While the highest NPFF receptor 2 gene expression was detected in the spinal cord, lower levels of NPFF receptor 2 gene expression were clearly demonstrated in regions other than spinal cord and hypothalamus when the PCR cycle number was increased (38 cycles) (Fig. 4, top). The experiment was repeated, a similar distribution was observed and results of one experiment were shown in Fig. 4, top. Based on the distribution of NPFF expression and NPFF localization (Majane et al., 1989), the pituitary was further dissected. A much higher level of NPFF receptor 2 expression was detected in neurointermediate lobe in comparison with the anterior lobe (Fig. 4, bottom). Effect of carrageenan inflammation on NPFF and NPVF system in the spinal cord To explore the role of spinal NPFF in chronic pain, rats were treated with carrageenan bilaterally for 4, 8, 16 and 24 h, total RNA was extracted from dorsal spinal cords and analyzed by RT-PCR with NPFF primers. For the control, spinal cords from untreated rats were used. NPFF gene expression was found to be significantly elevated 16 h after carrageenan injection and the effect lasted at least for 24 h (Fig. 5). Another group of rats consisting of untreated and carrageenan treated for 48 h were also analyzed for NPFF gene expression by RT-PCR and the carrageenan effect, though statistically significant, was clearly diminished (Fig. 5). In the spinal cord, NPVF transcript is localized at the dorsal horn and furthermore, NPFF and NPVF are identical
in their C-terminal PQRF-NH2 sequence. Because of this, the effect of peripheral carrageenan inflammation on spinal cord NPVF gene expression was also examined by RTPCR. Rats were treated with carrageenan unilaterally and killed after 7, 24, and 48 h. Spinal cord segments (L4 –L5) were dissected into two halves (ipsilateral and contralateral sides) and total RNAs were extracted for RT-PCR analysis. In contrast to NPFF, carrageenan inflammation failed to alter NPVF gene expression throughout the course of carrageenan effect (Fig. 6, top). The rats used for the NPVF mRNA study were also tested for paw-withdrawal latency and measured for their paw thickness to assess their inflammation-induced sensitivities to thermal pain. The paw-withdrawal latencies (second) measured at 7, 24 and 48 h after carrageenan were 7.22⫾0.6, 9.46⫾0.84, and 8.88⫾0.63 respectively for the control sides and 2.72⫾0.15, 4.72⫾0.92 and 3.86⫾0.18 respectively for the carrageenan-treated sides. The paw thickness was 6.0⫾0.16 mm for the untreated side and 11.36⫾0.63 for the carrageenan-treated sides measured at 48 h. The result are consistent with previous behavior work (Iadarola et al., 1988). For the effect of carrageenan inflammation on NPFF receptor, in the experiment shown in Fig. 7 top, dorsal or ventral spinal cords were pooled from 20 rats, total RNA extracted and used for RT-PCR analysis of NPFF receptor mRNAs. The reason for using pooled samples was that, in a preliminary experiments, variability was observed for the NPFF receptor mRNA levels normalized to GPDH mRNA among dorsal samples from individual animals. The large variability is likely due to a combination of the striking
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Fig. 4. Detection of low levels of NPFF receptor 2 mRNAs in regions other than spinal cord and hypothalamus. Total RNA preparations from tissues were subjected to RT-PCR analysis using specific primers for NPFF receptor 2 and GPDH and the RT-PCR products were examined by a 2.0% agarose/ethidium bromide gel. The gel images, not sharpened or filtered, were cropped to show bands corresponding to NPFF receptor 2 and GPDH (bottom two panels). The number of PCR cycles used in this experiment was 38. The quantified relative levels of NPFF receptor 2 mRNA in brain regions are expressed as normalized to GPDH (top panel)
localization of the NPFF receptor 2 mRNA in the superficial layer of the dorsal spinal cord (Fig. 3, left) and the difficulty of dissecting out homogenous individual samples. Thus, in the subsequent study (Fig. 7, bottom), the spinal cord segments from L-4 to L-5 regions of separate dorsal spinal cord samples were used for the RT-PCR analysis. A significant up-regulation of NPFF receptor 2 was observed in the inflamed group in comparison with the controls 48 h after carrageenan inflammation.
DISCUSSION NPFF is highly unevenly distributed in the rat CNS with higher concentrations being found in the spinal cord, hypothalamus and pons-medulla (Majane et al., 1989; Panula et al., 1996). Though a high level of NPFF-immunoreactive peptide was found in the rat hypothalamus, it should be noted that, in the earlier study, the molecular form of NPFF-immunoreactive peptide in the rat hypothalamus was found to be different from that in the spinal cord
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Fig. 5. Effect of carrageenan inflammation on NPFF mRNA levels in rat dorsal spinal cord. Rats were injected with carrageenan (6 mg in 150 l phosphate-buffered saline) bilaterally into the plantar surface of the hind paw and killed 4, 8, 16 and 24 h after treatments. Another group of rats were treated as described above and killed 48 h after carrageenan injection. Two independent groups of untreated rats were use as controls for the two experiments shown in left and right panels. Segments of spinal cords were dissected out from the L4 and L5 regions, and separated into dorsal and ventral parts. Total RNAs were extracted from the dorsal cords and analyzed for NPFF and GPDH mRNA levels by RT-PCR. The relative levels of NPFF mRNA are presented as normalized to GPDH. Values are means⫾S.E.M. (n⫽5). Differences between control and treated groups were analyzed by two-tailed Student’s t-test. * denotes P⬍0.05; ** denotes P⬍0.01.
(Majane et al., 1989). In the rat spinal cord, NPFF-immunoreactive peptides are concentrated in the superficial layer of the dorsal horn though there is also NPFF immunoreactivity in the lateral funiculi and around the central canal (Allard et al., 1991; Panula et al., 1987). In the dorsal spinal cord, NPFF was localized mainly in interneurons by immunohistochemical (Kivipelto and Panula, 1991) and in situ hybridization studies (Vilim et al., 1999). This striking localization of NPFF in the superficial layer of the dorsal horn distinguishes this peptide from other peptides such as NPY, enkephalins, somatostatin, substance P, cholecystokinin (CCK) and dynorphin which are localized in dorsal spinal cord but are also widely distributed at high levels in various brain regions (e.g. basal ganglia and cortex). In rats, intrathecal injection of NPFF or its analog can produce a long-lasting analgesia, which is apparently mediated by the opioid system. Though, this analgesic effect was reduced by both naloxone (Gouarde¨res et al., 1993) and naltrindole (Gouarde¨res et al., 1996), it should be noted that NPFF does not bind to - or ␦-opioid receptor (Allard et al., 1989; Payza and Yang, 1993). However, this
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Fig. 6. (Top panel) Effect of carrageenan inflammation on NPVF mRNA levels in rat dorsal spinal cords. (Bottom panel) Differential distribution of NPVF mRNA in rat dorsal spinal cord. (Top panel) Rats were injected with carrageenan (6 mg in 150 l phosphate-buffered saline) unilaterally into the plantar surface of the hind paw and killed 7, 24 and 48 h after treatments. Segments of spinal cords were dissected out from the L4 and L5 regions, and separated into left and right halves. Total RNAs were extracted from the tissues and analyzed for NPVF and GPDH mRNA levels by RT-PCR. The relative levels of NPVF mRNA are presented as normalized to GPDH. Values are means⫾S.E.M. (n⫽5). Differences between ipsilateral and contralateral sides were analyzed by two-tailed Student’s t-test. (Bottom panel) Spinal cords from two rats were dissected into dorsal and ventral horns and central canal (combined from two spinal cords) from segments L4 and L5 regions. Total RNAs were extracted from the tissues and analyzed for NPVF and GPDH mRNA levels by RT-PCR using NPVF primer. The RT-PCR products as visualized by 2% agarose/ethidium bromide gel electrophoresis were photographed and the print was then scanned by U-Max Power Look 2000. The image, not sharpened or filtered, was cropped to show NPVF band.
analgesic effect of NPFF was not observed by other investigators (Kontinen and Kalso, 1995). In order to further understand the functional role of the endogenous spinal cord NPFF system, we focused this study on the regulation of the NPFF system including NPFF and its recently identified receptors in dorsal spinal cord of rats with peripheral inflammation. While this study was in progress, a novel Arg-Phe-NH2 peptide gene was cloned and another NPFF-like peptide termed NPVF (VPNLPQRF-NH2) was deduced from the precursor (Liu et al., 2000). Interestingly, i.c.v.-injected NPVF was found to potently reduce morphine analgesia, similarly to the i.c.v.
Fig. 7. Effect of carrageenan inflammation on NPFF receptor 2 mRNA levels in rat spinal cords. (Top panel) Dorsal and ventral spinal cord (L4 –L5 regions) were dissected from rats treated with carrageenan for 24 h (24) and 48 h (48) and control rats (C). Each group consisted of 20 rats and tissues from the 20 rats were pooled; total RNAs were extracted from the tissues and subjected to RT-PCR analysis for NPFF receptor 2 and NPFF receptor 1 and GPDH mRNAs. RT-PCR products analyzed as described in Fig. 6 were cropped to show bands corresponding to NPFF receptor 1, NPFF receptor 2 and GPDH. (Bottom panel) Dorsal spinal cords (L4 –L5 regions) were dissected from control and carrageenan-treated rats. Total RNAs from these tissues were subjected to RT-PCR analysis for NPFF receptor 2 and GPDH mRNAs. The relative levels of NPFF receptor 2 mRNA are presented as normalized to GPDH. Values are means⫾S.E.M. (n⫽7). Difference between control and carrageenan treated was analyzed by two-tailed Student’s t-test. * denotes P⬍0.01.
injection of NPFF. In view of the identical C-terminal tetrapeptide sequence, PQRF-NH2, for both NPFF and NPVF and their apparently similar morphine modulating activities, we have also studied the distribution and regulation of NPVF gene expression. NPFF gene expression is highly localized to spinal cord and medulla (Fig. 1, top) which is consistent with in situ hybridization results showing the highest levels of gene expression in the nucleus of solitary tract and the superficial layer of dorsal spinal cord and its medullary equivalent (Vilim et al., 1999). In contrast, NPVF was found to be highly localized in the hypothalamus (Fig. 1, bottom) in good agreement with other investigators showing an exclusive expression of NPVF gene in the hypothalamus between the ventromedial nucleus and dorsalmedial nucleus (Hinuma et al., 2000; Liu et al., 2001). However, using RT-PCR analysis, NPVF mRNA was readily detected in dorsal horn of the rat spinal cord (Fig. 6, bottom). The existence of NPVF-like immunoreactive peptide in superficial layer of mouse dorsal spinal cord has
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also been noted using antiserum confirmed to cross-react with FPSLPQRF-NH2 (the mouse sequence of NPVF) but not with NPFF (Ukena and Tsutsui, 2001). This dorsal spinal cord localization, together with the potent anti-morphine activity of NPVF, prompted us to explore the gene regulation of these two neuropeptides by inflammatory pain. We detected an up-regulation of NPFF gene expression 16 h after carrageenan inflammation (Fig. 5), while NPVF gene expression was unaffected throughout the course of the study (Fig. 6, top). This up-regulation of spinal NPFF gene expression seems to be in harmony with the study of Vilim et al. (1999) reporting an increase in number of NPFF mRNA-expressing cells in dorsal spinal cord by acute inflammation, i.e. 3 h after carrageenan injection into hind paws. Though, in this study, we failed to observe an increase in mRNA level 3 h after carrageenan treatment as analyzed by RT-PCR, the discrepancy may be due to in situ hybridization being more sensitive in detecting gene expressing alterations in highly localized anatomical regions (i.e. single cells). Interestingly, the immunohistochemical study of Kontinen et al. (1997) has found an increase in the number of NPFF immunoreactive neuronal cell bodies in the spinal cord 2 h after carrageenan inflammation of the rat paw, while no change was observed in the NPFF-immunoreactive nerve fibers and terminal-like thickenings. The increase in the number of NPFF-immunoreactive neuronal perikarya is in agreement with the up-regulation of NPFF gene expression by carrageenan inflammation. Our data suggest that these authors observed the initiation of a more sustained physiological up-regulation. NPFF specific binding sites were demonstrated in rat brain and spinal cord membranes (Allard et al., 1989) and this binding was demonstrated to be inhibited by guanine nucleotide suggesting a G-protein-coupled receptor(s) for NPFF (Payza and Yang, 1993). Recently two G-proteincoupled receptors were isolated and characterized to be responsive to NPFF; NPFF receptor 2 was found to have somewhat higher affinity for NPFF than NPFF receptor 1 (Bonini et al., 2000). The distribution of NPFF receptor 2 mRNA (Fig. 2) is very similar to that of NPFF immunoreactivity (Majane et al., 1989; Panula et al., 1996); both NPFF receptor 2 mRNA (Fig. 3, left) and NPFF are highly enriched in the superficial layer of the dorsal spinal cord compared with much lower levels in other regions of the CNS. In parallel with NPFF gene expression, NPFF receptor 2 mRNA was also up-regulated by carrageenan inflammation (Fig. 7). This result is consistent with the study of Lombard et al. (1999) reporting an increased 125I-1-dimethyl-FLFQPQRF-NH2 binding in the spinal cords of rats with joint inflammation induced by killed Mycobacterium butyricum suspended in Freund adjuvant injected into tibio-tarsal joint (Lombard et al., 1999). NPFF injected into L5 and L6 attenuated the allodynic response to mechanical stimulation applied to an inflamed paw but was without effect on acute pain in normal animals (Altier et al., 2000). The distribution as well as the regulation of NPFF seems to parallel that of NPFF receptor 2. The distribution of NPFF receptor 2 mRNA, as shown in Fig. 2 and Fig. 3
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is consistent with the studies of other investigators showing high levels of NPFF receptor 2 mRNA in the spinal cord and hypothalamus (Bonini et al., 2000) by RT-PCR analysis and strong expression in the superficial layer of dorsal horn by in situ hybridization study (Liu et al., 2001). This parallel distribution of NPFF receptor 2 and NPFF may indicate that NPFF is a likely endogenous ligand for NPFF receptor 2 in the dorsal spinal cord of the rat. After the identification of the NPVF coding precursor, the in vitro study using cells expressing NPFF receptor 1 or NPFF receptor 2 has revealed that NPFF receptor 2 is responsive to and also more selectively binds NPFF-related peptides, while NPFF receptor 1 is activated by and binds better NPVF-related peptides (Liu et al., 2001); consequently, it was suggested that the NPFF-related peptides and NPVF-related peptides are for NPFF receptor 2 and NPFF receptor 1 respectively. In a separate study, an NPVF-related peptide was also identified by data base search followed by isolation of cDNAs encoding this peptides from various species; furthermore, a seven-transmembrane-domain receptor 0T7T022 (identical to NPFF receptor 1) with which NPVF-related peptides interact was identified (Hinuma et al., 2000). Functional studies have indicated that NPVF-related peptide can regulate prolactin secretion from anterior pituitary via activation of NPFF receptor 1 within the hypothalamus (Hinuma et al., 2000) further supporting the suggestion that NPVF-related peptides are physiological ligands for NPFF receptor 1. NPFF receptor 2 mRNA is in much lower abundance in the human spinal cord in comparison with the rat, but it is detected in human spinal cord by RT-PCR (Fig. 3, left). Interestingly, using [125]YLFQPQRF-NH2 as a ligand, high-affinity binding sites for NPFF were demonstrated in postmortem specimens of human spinal cord with the highest density in the superficial layer of the dorsal horn and the spinal trigeminal nucleus (Allard et al., 1994), though whether this high density of [125]YLFQPQRF-NH2 binding sites is due to NPFF receptor 1, NPFF receptor 2 or other as yet unidentified NPFF receptors remains unclear. While NPFF receptor 2 gene is highly expressed in the spinal cord, low levels of NPFF receptor 2 mRNA exist in other regions (Fig. 4, top). For example, in the pituitary gland, NPFF receptor 2 gene expression was found to be markedly higher in the posterior lobe (Fig. 4, bottom) which contains a high concentration of NPFF and numerous NPFF-positive nerve terminals (Boersma et al., 1993; Majane et al., 1989). Several lines of evidence suggest an interaction between NPFF and vasopressin within the neural lobe. For example, localization of NPFF in some vasopressin-containing cells in the hypothalamo-neurohypophyseal system (Boersma et al., 1993; Majane et al., 1993), absence of NPFF in neural lobe of homozygous Brattleboro rats (unable to synthesize normal vasopressin) (Majane and Yang, 1990), inhibition of arg-vasopressin release by NPFF from conscious rats (Arima et al., 1996; Yokoi et al., 1998) were reported. Electron microscopic studies have revealed that, in the neural lobe, NPFFcontaining nerve terminals form synaptoid contact with pituicytes; consequently, it was proposed that NPFF may
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have a modulator role on vasopressin and oxytocin secretion locally at the neural lobe with pituicytes as intermediate cells (Boersma et al., 1993). This proposal is in harmony with the finding of NPFF receptor 2 in the neural lobe, though whether NPFF receptor 2 is localized in pituicytes remains to be determined. Nevertheless, the results are consistent with the idea that NPFF may interact with vasopressin in modulating water balance through the receptor in the neural lobe. Further evidence for a modulator role of NPFF in vasopressin secretion is that NPFF can cause a large increase in water intake (Sunter et al., 2001). The up-regulation of both NPFF and NPFF receptor 2 gene expression by peripheral carrageenan inflammation as shown in this study strongly supports a role of spinal cord NPFF in modulating inflammatory pain. It is widely known that peripheral inflammation increases the spinal potency of morphine (Przewłocki and Przewłocka., 2001; Hylden et al., 1991). Several mechanisms have been suggested for this change in opioid activity including CCK and the descending noradrenergic system. It has been proposed that, in spinal cord of normal animals, morphine induces release of CCK which modulates morphine effects; however, in animals with peripheral inflammation, this morphine-mediated CCK release may be reduced to enhance the morphine potency (Stanfa et al., 1993). Other investigators found that the enhanced antinociceptive activity of morphine during carrageenan inflammation was blocked by intrathecal application of an ␣2-adrenoceptor antagonist idazoxan, suggesting a role of spinal noradrenergic pathways in morphine antinociception during inflammation (Hyden et al., 1991). In this study we have found an increased activity of the spinal NPFF system as shown by the up-regulation of NPFF and NPFF receptor 2 gene expressions in an animal model of persistent pain. Although physiological consequences of up-regulation of the spinal NPFF system during the peripheral inflammation still remain to be examined, several studies have found that, intrathecally injected NPFF at a non-analgesic dose can potentiate and prolong morphine analgesia in normal rats (Gouarde¨res et al., 1993, 1996; Kontinen and Kalso, 1995). Taken together, these studies and our findings, it is highly possible that NPFF system also contributes to the increased spinal analgesic potency of morphine in animals with peripheral inflammation. Acknowledgements—This research was supported by the Division of Intramural Research, NIDCR, NIH.
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Wilson S, Burke JF (1997) A human gene encoding morphine modulating peptides related to NPFF and FMRFamide. FEBS Lett 409:426 –430. Przewlocki R, Przewlocka B (2001) Opioids in chronic pain. Eur J Pharmacol 429:79 –91. Roumy M, Zajac J-M (1998) Neuropeptide FF, pain and analgesia. Eur J Pharmacol 345:1–11. Stanfa LC, Dickenson AH (1993) Cholecystokinin as a factor in the enhanced potency of spinal morphine following carrageenin inflammation. Br J Pharmacol 108:967–973. Sunter D, Hewson AK, Lynam S, Dickson SL (2001) Intracerebroventricular injection of neuropeptide FF, an opioid modulating neuropeptide, acutely reduces food intake and stimulates water intake in the rat. Neurosci Lett 313:145–148. Ukena K, Iwakoshi E, Minakata H, Tsutsui K (2002) A novel rat hypothalamic RFamide-related peptide identified by immunoaffinity chromatography and mass spectrometry. FEBS Lett 512:255–258. Ukena K, Tsutsui K (2001) Distribution of novel RFamide-related peptide-like immunoreactivity in the mouse central nervous system. Neurosci Lett 300:153–156. Vilim FS, Aarnisalo AA, Nieminenn M-L, Lintunen M, Karlsstedt K, Kontinen VK, Kalso E, States B, Panula P, Ziff E (1999) Gene for pain modulatory neuropeptide NPFF: induction in spinal cord by noxious stimuli. Mol Pharmacol 55:804 –811. Yang H-YT, Fratta W, Majane EA, Costa E (1985) Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci USA 93:60025–60030. Yokoi H, Arima H, Kondo K, Murase T, Iwasaki Y, Yang H-YT, Oiso Y (1998) Antiserum against neuropeptide FF augments vasopressin release in conscious rats. Peptides 19:393–395.
(Accepted 8 November 2002)
ACTIVATION OF SPINAL NEUROPEPTIDE FF AND THE NEUROPEPTIDE FF RECEPTOR 2 DURING INFLAMMATORY HYPERALGESIA IN RATS H.-Y. T. YANG* AND M. J. IADAROLA
gested a variety of biological roles including nociception, cardiovascular regulation, opiate tolerance and neuroendocrine modulation (for review see Panula et al., 1996). The most widely explored functional role of NPFF has been on nociception because, in the rat CNS, the highest level of NPFF immunoreactivity is in the dorsal spinal cord (Majane et al., 1989) where it is highly localized in the superficial layers of the dorsal horn (Allard et al., 1991; Panula et al., 1987). Furthermore, NPFF was found to have anti- and pro-opioid activities; in general, NPFF was found to be anti-analgesic after i.c.v. injection but analgesic when injected intrathecally (for review see Roumy et al., 1998). The reason for this discrepancy has not been readily obvious, however, it is likely that NPFF represents only one of many RF-NH2 containing neuropeptides in the mammalian nervous system and, depending on the pharmacological doses used, NPFF may exhibit the biological activities of other structurally related RF-NH2 peptides. The gene coding for the NPFF precursor protein has been cloned and it codes for both NPFF and NPAF (neuropeptide AF) (AGEGLSSPFWSLAAPQRF-NH2) (Vilim et al.,1999; Perry et al., 1997). In addition to the NPFF gene, another Arg-Phe-NH2 peptide gene has recently been identified and peptides similar to NPFF were deduced from the precursor protein (Liu et al., 2001; Hinuma et al., 2000). The peptides deduced from the human precursor are NPVF (VPNLPQRF-NH2) and a longer, 37-amino acid peptide (SLNFEELKDWGPKNVIKMSTPAVNKMPHSFANLPLRF-NH2). Very recently, the rat equivalent of NPVF was identified as an octadecapeptide (ANMEAGTMSHFPSLPQRF-NH2) by immunoaffinity purification coupled with mass spectrometry (Ukena et al., 2002). Depending on the doses used, NPVF-related peptides and NPFFrelated peptides may exhibit similar non-specific, or their unique specific, biological activities thus complicating biological activity studies of the RF-NH2-containing peptides. Recently, two G-protein-coupled receptors were identified and characterized to be responsive to NPFF (Bonini et al., 2000; Elshourbagy et al., 2000; Hinuma et al., 2000), and one of these receptors was suggested to be specific for NPVF in the studies of Hinuma et al. (2000) and Liu et al. (2001). Characterization of the two precursor proteins coding for two structurally related peptides, NPFF and NPVF peptides, and their receptors allows the functional roles of PQRF-NH2-containing peptides to be explored more accurately. To further understand the possible role of PQRFNH2-containing neuronal systems in pain transmission, we have studied (1) the distributions of genes expressing NPFF, NPVF and NPFF receptors and (2) regulation of
Neuronal Gene Expression Unit, Pain and Neurosensory Mechanisms Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Building 49, Room 1A07, 49 Convent Drive, MSC 4410, Bethesda, MD 20892-4410, USA
Abstract—Several lines of evidence suggest that neuropeptide FF (NPFF) is involved in nociception and in the modulation of opioid-mediated analgesia. Following the identification of the precursor protein for NPFF, two NPFF receptors and a second PQRF-NH2 containing peptide, termed NPVF, were identified. To further explore the functional role of PQRF-NH2 peptides, we have studied their distribution and also the regulation of NPFF and NPVF systems in the spinal cord of rats with peripheral inflammation. The distribution of NPFF gene expression is very similar to that of NPFF immunoreactive peptide but is distinct from NPVF gene expression. In the rat spinal cord, gene expression of NPFF but not that of NPVF was up-regulated by persistent pain induced by carrageenan inflammation. The distribution of NPFF receptor 2 gene expression is very similar to that of the NPFF peptide with a striking localization in the superficial layer of spinal cord. In rats with carrageenan inflammation of the hind paw, expression of both NPFF and NPFF receptor 2 genes was up-regulated in the spinal cord, while expression of NPVF and NPFF receptor 1 genes was not affected. The results of this study demonstrate a coordinated involvement of the spinal NPFF system in the persistent nociceptive pain states. Several studies have found a potentiation and prolongation of morphine analgesia by NPFF, therefore, it is highly possible that the endogenous spinal NPFF system contributes to the enhanced analgesic potency of morphine in animals with peripheral inflammation. Published by Elsevier Science Ltd on behalf of IBRO. Key words: nociception, pain, inflammation, dorsal spinal cord, NPVF.
Neuropeptide FF (NPFF; FLFQPQRF-NH2) was originally detected by an antiserum raised against FMRF-NH2 and subsequently isolated from bovine brain and sequenced (Yang et al., 1985). Biological studies of this peptide and the distribution of NPFF-immunoreactive peptides sug*Corresponding author: Neuronal Gene Expression Unit, Pain and Neurosensory Mechanisms Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Building 49, Room 1A07, 49 Convent Drive, MSC 4410, Bethesda, MD 208924410, USA. Tel: ⫹1-301-402-4981; fax: ⫹1-301-402-0667. E-mail address: [email protected] (H.-Y. T. Yang). Abbreviations: CCK, cholecystokinin; GPDH, glyceraldehyde 3-phosphate dehydrogenase; NPAF, neuropeptide AF; NPFF, neuropeptide FF; NPFF-R1, neuropeptide FF receptor 1; NPFF-R2, neuropeptide FF receptor 2; NPVF, VPNLPQRF-NH2; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction. 0306-4522/03$30.00⫹0.00 Published by Elsevier Science Ltd on behalf of IBRO. doi:10.1016/S0306-4522(02)00931-4
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NPFF and NPVF transcripts as well as NPFF receptor gene expression at the spinal cord level in an animal model of persistent peripheral inflammation induced by carrageenan. The data from the rat show a strong enrichment of both NPFF and NPFF receptor 2 in superficial layer of dorsal spinal cord as well as a local and concurrent upregulation of both peptide and receptor by persistent nociceptive activity.
EXPERIMENTAL PROCEDURES Animal, carrageenan treatment and tissue dissection Male Sprague–Dawley Rats (250 –350 g) were housed with a 12-h light/dark cycle and given water and food ad libitum. Procedures for all animals used were in accordance with the National Institute of Health (NIH) Guidelines for the Care and Use of Laboratory Animals, and were approved by the National Institute of Dental and Craniofacial Research (NIDCR) Animal Care and Use Committee. All efforts were made to minimize both animal numbers and suffering in the experiments. Carrageenan Lambda type IV (Sigma, St Louis, MO, USA), 6 mg in 150 l phosphate-buffered saline per paw, was injected into the planter surface of the hind paw bilaterally or unilaterally to induce a long-lasting inflammation as previously described (Iadarola et al., 1988). Rats were killed by carbon dioxide exposure at the specified time after the carrageenan treatments as indicated in legends to figures. Spinal cords were removed according to the method of DeSousa and Horrocks (1979). Spinal cord (whole cord or dorsal cord as indicated in figure legends) segments of 6 – 8 mm containing L4 –L5 were rapidly dissected, divided into dorsal and ventral halves (for bilateral carrageenan) or left and right halves (for unilateral carrageenan), frozen immediately in dry ice and stored at ⫺80 °C until processed for RNA extraction. For the bilateral carrageenan injection, untreated rats were use as control and for unilateral carrageenan injection, control sides were not injected. Controls were not injected because the aim of this study was to compare between absolutely normal animals and animals with long-lasting persistent inflammation. For the regional distributions of NPFF, NPVF and NPFF receptor 2 mRNAs, rats were killed with CO2 and the brain, spinal cord, pituitary gland and trigeminal ganglia were rapidly removed. Brains were dissected into various regions (cerebellum, striatum, medulla, olfactory bulb, substantia nigra, hypothalamus, hippocampus, thalamus, septum, frontal cortex, entorhinal cortex, inferior colliculus, superior colliculus, periaqueductal gray, nucleus accumbens) as described in Naranjo et al., 1986, and pituitary was divided into anterior and neurointermediate lobes. Spinal cords were removed as described above and dorsal and ventral halves, superficial layer and central canal were dissected out immediately on a cold plate (see Fig. 3). Tissues were frozen immediately in dry ice after dissection and stored at ⫺80 °C until processed for RNA extraction.
RNA extraction and RT-PCR To assess mRNAs coding for NPFF, NPVF and NPFF receptors, reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out with RNA prepared from tissues dissected as described above. RNA was extracted using RNeasy Mini or Midi Kit (Qiagen Inc., Valencia, CA, USA) with an additional step of RNase-free DNase treatment. RNA was quantified by the RiboGreen reagent (Molecular Probes, Eugene, OR, USA) using a 96-well fluorometric plate reader (Molecular Devices, Sunnyvale, CA, USA). RT-PCR was carried out using the Access RT-PCR system (Promega, Madison, WI, USA) with specific primers custom synthesized by Gene Probe Technologies Inc (Gaithersburg, MD, USA). The primer pairs are (1) ACTGCTGCTGCTGAG-
GAACT and CACTTTATTTGGGGGCACAC (384-bp product) for rat NPFF, (2) GGAATCCCAAAAGGGGTAAA and GGGTCATGGCATAGAGCAAT (331-bp product) for rat NPVF, (3) GCCAAACCAGGAAATGAGGA and TCATCAGAGTCCACAGGGGA (254bp product) and second set GAGCAGTGGCATGTATCCAA and TACATCCCAAGTTTTCCCCA (411-bp product) for rat NPFF receptor 2, (4) AACCAGCCTCACCTTCTCCT and GTTACGAGCATCCAGCATGAA (505-bp product) for rat NPFF receptor 1, (5) GCCAAATCAGGAAATGAGGA and TCATTAGAGTCCACAGGGGC (254-bp product) for human NPFF receptor 2, (6) ACCACAGTCCATGCCATCAC and TCCACCACCCTGTTGCTGTA (452-bp product) for rat glyceraldehydes-3-phosphate dehydrogenase (GPDH) and, (7) GACCCCTTCATTGACCTCAACT and CATCGCCCCACTTGATTTTG (166-bp product) for human GPDH. The RT-PCR reaction was carried out according to the manufacture’s instruction (Promega, Madison, WI, USA) in 25 l reaction mixture containing 2– 8 ng total RNA in the RoboCycler Gradient 96 Temperature Cycler (Stratagene, La Jolla, CA, USA). Reverse transcription and PCR cycling profile were one cycle of 45 min at 48 °C for reverse transcription, one cycle of 2 min at 94 °C for transcriptase inactivation, 28 –36 cycles of 94 °C denaturation for 30 s, 55 °C annealing for 1 min, 68 °C extension for 2 min and one cycle of 68 °C final extension for 7 min. To determine the amount of RNA and cycle amplification number that would yield reliable relative values for each target transcript, preliminary experiments were carried out with each pair of primers and RNA preparations. The optimal conditions for each target transcript and primer pair were applied in the assessment of differential levels of expression. The RT-PCR products were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. For the estimation of relative quantities of RT-PCR products, image acquisition was performed on the gel with an AlphaImager (Alpha Innotech, San Leandre, CA, USA). The relative intensities of the bands corresponding to the specific RT-PCR products were analyzed quantitatively using ImageQuant 5 software (Molecular Dynamics, Piscataway, NJ, USA). The results, normalized to GPDH, are presented in the figures. For the verification of the RT-PCR products, two representative NPFF receptor 2 RT-PCR products from rat and human spinal cords were sequenced. Human NPFF receptor 2 primer and rat NPFF receptor 2 primer yield 254-base pair RT-PCR products. These cDNAs were cloned using the TA cloning kit (Clontech, Palo Alto, CA, USA, Advantage PCR Cloning kit) and sequenced by the NIDCR Sequencing Core Facility; the sequences were then compared with the sequences of NPFF receptors in Gene bank to verify the RT-PCR products.
RESULTS Distribution of mRNA encoding NPFF, NPVF and NPFF receptors The regional distributions of NPFF and NPVF gene expression in the rat CNS and several peripheral tissues were examined by semi-quantitative RT-PCR and results are shown in Fig. 1. The experiment was carried out twice and very similar patterns of distribution were observed. The results of one experiment are presented in Fig. 1. The distribution of NPFF mRNA is strikingly distinct from that of NPVF mRNA. The highest level of NPFF transcript is in the spinal cord and medulla oblongata with much lower levels in the cerebral cortex. In contrast, NPVF transcript is most highly localized in the hypothalamus with a moderate level in the spinal cord. However, further analysis of spinal cord revealed that the NPVF transcript is nearly exclusively localized in the dorsal horn (Fig. 6, bottom).
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Fig. 1. Distribution of NPFF and NPVF mRNAs in brain regions, spinal cord and some peripheral tissues. Total RNA preparations from tissues were subjected to semi quantitative RT-PCR analysis using specific primers for NPFF, NPVF and GPDH. The relative levels of NPFF and NPVF mRNAs were presented as normalized to GPDH. Top panel, NPFF mRNA; bottom panel, NPVF mRNA.
Regional distribution of NPFF receptor 2 mRNA was examined by RT-PCR with NPFF receptor 2 primers. The results shown in Fig. 2 revealed a markedly uneven distribution of NPFF receptor 2 transcript in the CNS. With a moderate number of PCR cycles (33 cycles), the regions which gave clear RT-PCR products for NPFF receptor 2 were limited to spinal cord and hypothalamus (Fig. 2) indicating a striking localization of NPFF receptor 2 transcript in the spinal cord in the rat CNS. The experiment was repeated with a different set of PCR primers (set 2)
Fig. 2. Distribution of NPFF receptor 2 mRNAs in brain regions, spinal cord and some peripheral tissues. Total RNA preparations from tissues were subjected to RT-PCR analysis using specific primers for NPFF receptor 2 and GPDH. The number of PCR cycle used for NPFF receptor 2 in this experiment was 35. The RT-PCR products were examined by a 2.0% agarose/ethidium bromide gel. The gel images, not sharpened or filtered, were cropped to show bands corresponding to NPFF receptor 2 and GPDH (bottom panel). The lanes on the gel correspond to the bars on the graph. The relative levels of NPFF receptor 2 mRNA are expressed as normalized to GPDH (top panel). The very high level of expression is in spinal cord.
and again the two regions found to show RT-PCR products were spinal cord and hypothalamus (results not shown). Further analysis of the spinal cord shows that NPFF receptor 2 expression is highest in the superficial layer of dorsal horn, moderate in the central canal (Fig. 3, left) and nearly undetectable in the ventral horn. In the human spinal cord, expression of NPFF receptor 1 was much more readily detected than that of NPFF receptor 2 (result not shown); however, the existence of NPFF receptor 2
Abbreviations used in the figures Accum Cerebell Ent cort Front cort Hippo Hypoth Inf Colli
accumbens cerebellum entorhinal cortex frontal cortex hippocampus hypothalamus inferior colliculus
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Olf bulb PAG Sp cord Sub nigra Sup colli Trig ganglia
olfactory bulb periaqueductal gray spinal cord substantia nigra superior colliculus trigeminal ganglia
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Fig. 3. Enrichment of NPFF receptor 2 mRNA in the dorsal spinal cord. Total RNA preparations from the rat and human spinal cords were subjected to RT-PCR analysis using specific primers for NPFF receptor 2 and GPDH. The RT-PCR products were analyzed by a 2.0% agarose/ethidium bromide gel. The gel images were photographed and prints were scanned by U-Max Power Look 2000. The scanned images, not sharpened or filtered, were cropped to show bands corresponding to NPFF receptor 2 and GPDH (left panel). The superficial layer (SL) represents the highest expressing sub region of the cord, and a moderate level of expression is in the central canal (CC). Various regions, dorsal cord, ventral cord, superficial layer and central canal were dissected as summarized in the summary figure (right panel).
transcript in the dorsal horn was definitely demonstrated by RT-PCR analysis (Fig. 3, left). While the highest NPFF receptor 2 gene expression was detected in the spinal cord, lower levels of NPFF receptor 2 gene expression were clearly demonstrated in regions other than spinal cord and hypothalamus when the PCR cycle number was increased (38 cycles) (Fig. 4, top). The experiment was repeated, a similar distribution was observed and results of one experiment were shown in Fig. 4, top. Based on the distribution of NPFF expression and NPFF localization (Majane et al., 1989), the pituitary was further dissected. A much higher level of NPFF receptor 2 expression was detected in neurointermediate lobe in comparison with the anterior lobe (Fig. 4, bottom). Effect of carrageenan inflammation on NPFF and NPVF system in the spinal cord To explore the role of spinal NPFF in chronic pain, rats were treated with carrageenan bilaterally for 4, 8, 16 and 24 h, total RNA was extracted from dorsal spinal cords and analyzed by RT-PCR with NPFF primers. For the control, spinal cords from untreated rats were used. NPFF gene expression was found to be significantly elevated 16 h after carrageenan injection and the effect lasted at least for 24 h (Fig. 5). Another group of rats consisting of untreated and carrageenan treated for 48 h were also analyzed for NPFF gene expression by RT-PCR and the carrageenan effect, though statistically significant, was clearly diminished (Fig. 5). In the spinal cord, NPVF transcript is localized at the dorsal horn and furthermore, NPFF and NPVF are identical
in their C-terminal PQRF-NH2 sequence. Because of this, the effect of peripheral carrageenan inflammation on spinal cord NPVF gene expression was also examined by RTPCR. Rats were treated with carrageenan unilaterally and killed after 7, 24, and 48 h. Spinal cord segments (L4 –L5) were dissected into two halves (ipsilateral and contralateral sides) and total RNAs were extracted for RT-PCR analysis. In contrast to NPFF, carrageenan inflammation failed to alter NPVF gene expression throughout the course of carrageenan effect (Fig. 6, top). The rats used for the NPVF mRNA study were also tested for paw-withdrawal latency and measured for their paw thickness to assess their inflammation-induced sensitivities to thermal pain. The paw-withdrawal latencies (second) measured at 7, 24 and 48 h after carrageenan were 7.22⫾0.6, 9.46⫾0.84, and 8.88⫾0.63 respectively for the control sides and 2.72⫾0.15, 4.72⫾0.92 and 3.86⫾0.18 respectively for the carrageenan-treated sides. The paw thickness was 6.0⫾0.16 mm for the untreated side and 11.36⫾0.63 for the carrageenan-treated sides measured at 48 h. The result are consistent with previous behavior work (Iadarola et al., 1988). For the effect of carrageenan inflammation on NPFF receptor, in the experiment shown in Fig. 7 top, dorsal or ventral spinal cords were pooled from 20 rats, total RNA extracted and used for RT-PCR analysis of NPFF receptor mRNAs. The reason for using pooled samples was that, in a preliminary experiments, variability was observed for the NPFF receptor mRNA levels normalized to GPDH mRNA among dorsal samples from individual animals. The large variability is likely due to a combination of the striking
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Fig. 4. Detection of low levels of NPFF receptor 2 mRNAs in regions other than spinal cord and hypothalamus. Total RNA preparations from tissues were subjected to RT-PCR analysis using specific primers for NPFF receptor 2 and GPDH and the RT-PCR products were examined by a 2.0% agarose/ethidium bromide gel. The gel images, not sharpened or filtered, were cropped to show bands corresponding to NPFF receptor 2 and GPDH (bottom two panels). The number of PCR cycles used in this experiment was 38. The quantified relative levels of NPFF receptor 2 mRNA in brain regions are expressed as normalized to GPDH (top panel)
localization of the NPFF receptor 2 mRNA in the superficial layer of the dorsal spinal cord (Fig. 3, left) and the difficulty of dissecting out homogenous individual samples. Thus, in the subsequent study (Fig. 7, bottom), the spinal cord segments from L-4 to L-5 regions of separate dorsal spinal cord samples were used for the RT-PCR analysis. A significant up-regulation of NPFF receptor 2 was observed in the inflamed group in comparison with the controls 48 h after carrageenan inflammation.
DISCUSSION NPFF is highly unevenly distributed in the rat CNS with higher concentrations being found in the spinal cord, hypothalamus and pons-medulla (Majane et al., 1989; Panula et al., 1996). Though a high level of NPFF-immunoreactive peptide was found in the rat hypothalamus, it should be noted that, in the earlier study, the molecular form of NPFF-immunoreactive peptide in the rat hypothalamus was found to be different from that in the spinal cord
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Fig. 5. Effect of carrageenan inflammation on NPFF mRNA levels in rat dorsal spinal cord. Rats were injected with carrageenan (6 mg in 150 l phosphate-buffered saline) bilaterally into the plantar surface of the hind paw and killed 4, 8, 16 and 24 h after treatments. Another group of rats were treated as described above and killed 48 h after carrageenan injection. Two independent groups of untreated rats were use as controls for the two experiments shown in left and right panels. Segments of spinal cords were dissected out from the L4 and L5 regions, and separated into dorsal and ventral parts. Total RNAs were extracted from the dorsal cords and analyzed for NPFF and GPDH mRNA levels by RT-PCR. The relative levels of NPFF mRNA are presented as normalized to GPDH. Values are means⫾S.E.M. (n⫽5). Differences between control and treated groups were analyzed by two-tailed Student’s t-test. * denotes P⬍0.05; ** denotes P⬍0.01.
(Majane et al., 1989). In the rat spinal cord, NPFF-immunoreactive peptides are concentrated in the superficial layer of the dorsal horn though there is also NPFF immunoreactivity in the lateral funiculi and around the central canal (Allard et al., 1991; Panula et al., 1987). In the dorsal spinal cord, NPFF was localized mainly in interneurons by immunohistochemical (Kivipelto and Panula, 1991) and in situ hybridization studies (Vilim et al., 1999). This striking localization of NPFF in the superficial layer of the dorsal horn distinguishes this peptide from other peptides such as NPY, enkephalins, somatostatin, substance P, cholecystokinin (CCK) and dynorphin which are localized in dorsal spinal cord but are also widely distributed at high levels in various brain regions (e.g. basal ganglia and cortex). In rats, intrathecal injection of NPFF or its analog can produce a long-lasting analgesia, which is apparently mediated by the opioid system. Though, this analgesic effect was reduced by both naloxone (Gouarde¨res et al., 1993) and naltrindole (Gouarde¨res et al., 1996), it should be noted that NPFF does not bind to - or ␦-opioid receptor (Allard et al., 1989; Payza and Yang, 1993). However, this
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Fig. 6. (Top panel) Effect of carrageenan inflammation on NPVF mRNA levels in rat dorsal spinal cords. (Bottom panel) Differential distribution of NPVF mRNA in rat dorsal spinal cord. (Top panel) Rats were injected with carrageenan (6 mg in 150 l phosphate-buffered saline) unilaterally into the plantar surface of the hind paw and killed 7, 24 and 48 h after treatments. Segments of spinal cords were dissected out from the L4 and L5 regions, and separated into left and right halves. Total RNAs were extracted from the tissues and analyzed for NPVF and GPDH mRNA levels by RT-PCR. The relative levels of NPVF mRNA are presented as normalized to GPDH. Values are means⫾S.E.M. (n⫽5). Differences between ipsilateral and contralateral sides were analyzed by two-tailed Student’s t-test. (Bottom panel) Spinal cords from two rats were dissected into dorsal and ventral horns and central canal (combined from two spinal cords) from segments L4 and L5 regions. Total RNAs were extracted from the tissues and analyzed for NPVF and GPDH mRNA levels by RT-PCR using NPVF primer. The RT-PCR products as visualized by 2% agarose/ethidium bromide gel electrophoresis were photographed and the print was then scanned by U-Max Power Look 2000. The image, not sharpened or filtered, was cropped to show NPVF band.
analgesic effect of NPFF was not observed by other investigators (Kontinen and Kalso, 1995). In order to further understand the functional role of the endogenous spinal cord NPFF system, we focused this study on the regulation of the NPFF system including NPFF and its recently identified receptors in dorsal spinal cord of rats with peripheral inflammation. While this study was in progress, a novel Arg-Phe-NH2 peptide gene was cloned and another NPFF-like peptide termed NPVF (VPNLPQRF-NH2) was deduced from the precursor (Liu et al., 2000). Interestingly, i.c.v.-injected NPVF was found to potently reduce morphine analgesia, similarly to the i.c.v.
Fig. 7. Effect of carrageenan inflammation on NPFF receptor 2 mRNA levels in rat spinal cords. (Top panel) Dorsal and ventral spinal cord (L4 –L5 regions) were dissected from rats treated with carrageenan for 24 h (24) and 48 h (48) and control rats (C). Each group consisted of 20 rats and tissues from the 20 rats were pooled; total RNAs were extracted from the tissues and subjected to RT-PCR analysis for NPFF receptor 2 and NPFF receptor 1 and GPDH mRNAs. RT-PCR products analyzed as described in Fig. 6 were cropped to show bands corresponding to NPFF receptor 1, NPFF receptor 2 and GPDH. (Bottom panel) Dorsal spinal cords (L4 –L5 regions) were dissected from control and carrageenan-treated rats. Total RNAs from these tissues were subjected to RT-PCR analysis for NPFF receptor 2 and GPDH mRNAs. The relative levels of NPFF receptor 2 mRNA are presented as normalized to GPDH. Values are means⫾S.E.M. (n⫽7). Difference between control and carrageenan treated was analyzed by two-tailed Student’s t-test. * denotes P⬍0.01.
injection of NPFF. In view of the identical C-terminal tetrapeptide sequence, PQRF-NH2, for both NPFF and NPVF and their apparently similar morphine modulating activities, we have also studied the distribution and regulation of NPVF gene expression. NPFF gene expression is highly localized to spinal cord and medulla (Fig. 1, top) which is consistent with in situ hybridization results showing the highest levels of gene expression in the nucleus of solitary tract and the superficial layer of dorsal spinal cord and its medullary equivalent (Vilim et al., 1999). In contrast, NPVF was found to be highly localized in the hypothalamus (Fig. 1, bottom) in good agreement with other investigators showing an exclusive expression of NPVF gene in the hypothalamus between the ventromedial nucleus and dorsalmedial nucleus (Hinuma et al., 2000; Liu et al., 2001). However, using RT-PCR analysis, NPVF mRNA was readily detected in dorsal horn of the rat spinal cord (Fig. 6, bottom). The existence of NPVF-like immunoreactive peptide in superficial layer of mouse dorsal spinal cord has
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also been noted using antiserum confirmed to cross-react with FPSLPQRF-NH2 (the mouse sequence of NPVF) but not with NPFF (Ukena and Tsutsui, 2001). This dorsal spinal cord localization, together with the potent anti-morphine activity of NPVF, prompted us to explore the gene regulation of these two neuropeptides by inflammatory pain. We detected an up-regulation of NPFF gene expression 16 h after carrageenan inflammation (Fig. 5), while NPVF gene expression was unaffected throughout the course of the study (Fig. 6, top). This up-regulation of spinal NPFF gene expression seems to be in harmony with the study of Vilim et al. (1999) reporting an increase in number of NPFF mRNA-expressing cells in dorsal spinal cord by acute inflammation, i.e. 3 h after carrageenan injection into hind paws. Though, in this study, we failed to observe an increase in mRNA level 3 h after carrageenan treatment as analyzed by RT-PCR, the discrepancy may be due to in situ hybridization being more sensitive in detecting gene expressing alterations in highly localized anatomical regions (i.e. single cells). Interestingly, the immunohistochemical study of Kontinen et al. (1997) has found an increase in the number of NPFF immunoreactive neuronal cell bodies in the spinal cord 2 h after carrageenan inflammation of the rat paw, while no change was observed in the NPFF-immunoreactive nerve fibers and terminal-like thickenings. The increase in the number of NPFF-immunoreactive neuronal perikarya is in agreement with the up-regulation of NPFF gene expression by carrageenan inflammation. Our data suggest that these authors observed the initiation of a more sustained physiological up-regulation. NPFF specific binding sites were demonstrated in rat brain and spinal cord membranes (Allard et al., 1989) and this binding was demonstrated to be inhibited by guanine nucleotide suggesting a G-protein-coupled receptor(s) for NPFF (Payza and Yang, 1993). Recently two G-proteincoupled receptors were isolated and characterized to be responsive to NPFF; NPFF receptor 2 was found to have somewhat higher affinity for NPFF than NPFF receptor 1 (Bonini et al., 2000). The distribution of NPFF receptor 2 mRNA (Fig. 2) is very similar to that of NPFF immunoreactivity (Majane et al., 1989; Panula et al., 1996); both NPFF receptor 2 mRNA (Fig. 3, left) and NPFF are highly enriched in the superficial layer of the dorsal spinal cord compared with much lower levels in other regions of the CNS. In parallel with NPFF gene expression, NPFF receptor 2 mRNA was also up-regulated by carrageenan inflammation (Fig. 7). This result is consistent with the study of Lombard et al. (1999) reporting an increased 125I-1-dimethyl-FLFQPQRF-NH2 binding in the spinal cords of rats with joint inflammation induced by killed Mycobacterium butyricum suspended in Freund adjuvant injected into tibio-tarsal joint (Lombard et al., 1999). NPFF injected into L5 and L6 attenuated the allodynic response to mechanical stimulation applied to an inflamed paw but was without effect on acute pain in normal animals (Altier et al., 2000). The distribution as well as the regulation of NPFF seems to parallel that of NPFF receptor 2. The distribution of NPFF receptor 2 mRNA, as shown in Fig. 2 and Fig. 3
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is consistent with the studies of other investigators showing high levels of NPFF receptor 2 mRNA in the spinal cord and hypothalamus (Bonini et al., 2000) by RT-PCR analysis and strong expression in the superficial layer of dorsal horn by in situ hybridization study (Liu et al., 2001). This parallel distribution of NPFF receptor 2 and NPFF may indicate that NPFF is a likely endogenous ligand for NPFF receptor 2 in the dorsal spinal cord of the rat. After the identification of the NPVF coding precursor, the in vitro study using cells expressing NPFF receptor 1 or NPFF receptor 2 has revealed that NPFF receptor 2 is responsive to and also more selectively binds NPFF-related peptides, while NPFF receptor 1 is activated by and binds better NPVF-related peptides (Liu et al., 2001); consequently, it was suggested that the NPFF-related peptides and NPVF-related peptides are for NPFF receptor 2 and NPFF receptor 1 respectively. In a separate study, an NPVF-related peptide was also identified by data base search followed by isolation of cDNAs encoding this peptides from various species; furthermore, a seven-transmembrane-domain receptor 0T7T022 (identical to NPFF receptor 1) with which NPVF-related peptides interact was identified (Hinuma et al., 2000). Functional studies have indicated that NPVF-related peptide can regulate prolactin secretion from anterior pituitary via activation of NPFF receptor 1 within the hypothalamus (Hinuma et al., 2000) further supporting the suggestion that NPVF-related peptides are physiological ligands for NPFF receptor 1. NPFF receptor 2 mRNA is in much lower abundance in the human spinal cord in comparison with the rat, but it is detected in human spinal cord by RT-PCR (Fig. 3, left). Interestingly, using [125]YLFQPQRF-NH2 as a ligand, high-affinity binding sites for NPFF were demonstrated in postmortem specimens of human spinal cord with the highest density in the superficial layer of the dorsal horn and the spinal trigeminal nucleus (Allard et al., 1994), though whether this high density of [125]YLFQPQRF-NH2 binding sites is due to NPFF receptor 1, NPFF receptor 2 or other as yet unidentified NPFF receptors remains unclear. While NPFF receptor 2 gene is highly expressed in the spinal cord, low levels of NPFF receptor 2 mRNA exist in other regions (Fig. 4, top). For example, in the pituitary gland, NPFF receptor 2 gene expression was found to be markedly higher in the posterior lobe (Fig. 4, bottom) which contains a high concentration of NPFF and numerous NPFF-positive nerve terminals (Boersma et al., 1993; Majane et al., 1989). Several lines of evidence suggest an interaction between NPFF and vasopressin within the neural lobe. For example, localization of NPFF in some vasopressin-containing cells in the hypothalamo-neurohypophyseal system (Boersma et al., 1993; Majane et al., 1993), absence of NPFF in neural lobe of homozygous Brattleboro rats (unable to synthesize normal vasopressin) (Majane and Yang, 1990), inhibition of arg-vasopressin release by NPFF from conscious rats (Arima et al., 1996; Yokoi et al., 1998) were reported. Electron microscopic studies have revealed that, in the neural lobe, NPFFcontaining nerve terminals form synaptoid contact with pituicytes; consequently, it was proposed that NPFF may
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have a modulator role on vasopressin and oxytocin secretion locally at the neural lobe with pituicytes as intermediate cells (Boersma et al., 1993). This proposal is in harmony with the finding of NPFF receptor 2 in the neural lobe, though whether NPFF receptor 2 is localized in pituicytes remains to be determined. Nevertheless, the results are consistent with the idea that NPFF may interact with vasopressin in modulating water balance through the receptor in the neural lobe. Further evidence for a modulator role of NPFF in vasopressin secretion is that NPFF can cause a large increase in water intake (Sunter et al., 2001). The up-regulation of both NPFF and NPFF receptor 2 gene expression by peripheral carrageenan inflammation as shown in this study strongly supports a role of spinal cord NPFF in modulating inflammatory pain. It is widely known that peripheral inflammation increases the spinal potency of morphine (Przewłocki and Przewłocka., 2001; Hylden et al., 1991). Several mechanisms have been suggested for this change in opioid activity including CCK and the descending noradrenergic system. It has been proposed that, in spinal cord of normal animals, morphine induces release of CCK which modulates morphine effects; however, in animals with peripheral inflammation, this morphine-mediated CCK release may be reduced to enhance the morphine potency (Stanfa et al., 1993). Other investigators found that the enhanced antinociceptive activity of morphine during carrageenan inflammation was blocked by intrathecal application of an ␣2-adrenoceptor antagonist idazoxan, suggesting a role of spinal noradrenergic pathways in morphine antinociception during inflammation (Hyden et al., 1991). In this study we have found an increased activity of the spinal NPFF system as shown by the up-regulation of NPFF and NPFF receptor 2 gene expressions in an animal model of persistent pain. Although physiological consequences of up-regulation of the spinal NPFF system during the peripheral inflammation still remain to be examined, several studies have found that, intrathecally injected NPFF at a non-analgesic dose can potentiate and prolong morphine analgesia in normal rats (Gouarde¨res et al., 1993, 1996; Kontinen and Kalso, 1995). Taken together, these studies and our findings, it is highly possible that NPFF system also contributes to the increased spinal analgesic potency of morphine in animals with peripheral inflammation. Acknowledgements—This research was supported by the Division of Intramural Research, NIDCR, NIH.
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(Accepted 8 November 2002)