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Distribution of galanin-1, -2 and -3 receptor messenger RNAs in central and peripheral rat tissues
Neuroscience Vol. 95, No. 1, pp. 265–271, 2000 265 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
Quantitative rat galanin receptor mRNA distribution
Pergamon PII: S0306-4522(99)00407-8 www.elsevier.com/locate/neuroscience
DISTRIBUTION OF GALANIN-1, -2 AND -3 RECEPTOR MESSENGER RNAS IN CENTRAL AND PERIPHERAL RAT TISSUES S. M. WATERS* and J. E. KRAUSE Department of Biochemistry and Molecular Biology, Neurogen Corporation, Branford, CT 06405, U.S.A.
Abstract—Galanin is a neuropeptide widely expressed in the central nervous system and periphery. In rat, three galanin-binding receptors have been cloned and characterized. We report the qualitative and quantitative distribution of galanin-1, galanin-2, and galanin-3 messenger RNAs in central and peripheral rat tissues by reverse transcription–polymerase chain reaction and solution hybridization/RNase protection assays, respectively. Galanin-1 messenger RNA was detected exclusively in the central and peripheral nervous system with highest expression in hypothalamus, amygdala, spinal cord and dorsal root ganglia. Galanin-2 messenger RNA was highly expressed in hypothalamus, dorsal root ganglia, and kidney with moderate expression in several other tissues. Galanin-3 messenger RNA was widely distributed at low to moderate levels in many central and peripheral tissues. The observed expression of multiple galanin receptors in several tissues including hypothalamus, anterior pituitary and spinal cord supports earlier pharmacological studies suggesting the presence of more than one receptor subtype in these regions. The presence of multiple galanin receptors in these tissues in conjunction with the detection of a single subtype, galanin-2, in tissues such as heart and intestine, illustrates the potential complexity of galanin-associated actions in rat central nervous system and periphery. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: galanin, receptor, distribution, central nervous system, rat, solution hybridization/RNase protection assay.
Galanin, a 29–30 amino acid neuropeptide first described in pig intestine, 39 is a widely distributed transmitter in the CNS and periphery. 2,27 As a neurotransmitter/neuromodulator, galanin is associated with many biological activities. In hypothalamus, i.c.v. administration of galanin increases food intake with a preference for fat, 11 whereas galanin receptor peptidic antagonists reduce feeding. 9,24 In hippocampus, galanin decreases acetylcholine release presynaptically 14 and transmission postsynaptically 29 suggesting a role in memory. In spinal cord, galanin depresses spinal nociceptive reflexes 45,46 and galanin receptor peptidic antagonists inhibit spinal antinociception induced by morphine. 33 Additionally, galanin decreases glucose-dependent insulin release in species other than man, 1 stimulates growth hormone 3,7 and prolactin 22,47 release from the anterior pituitary, and induces smooth muscle contraction in the intestine. 12 The actions of galanin are mediated by at least three G-protein-coupled receptors termed galanin-1 (GalR1), GalR2 and GalR3. GalR1, originally isolated from human Bowes melanoma cells, 16 was subsequently cloned from rat 6,31 and mouse. 44 Activation of GalR1 results in pertussis toxin-sensitive inhibition of adenylyl cyclase through interaction with Gai/Gao G-proteins, 6,16,31 and can increase intracellular Ca 21 via bg subunit activation of phospholipase C b2 isoforms. Northern blot analysis and in situ hybridization show that GalR1 is predominantly located in the brain and spinal cord, 6,31 although a broader distribution has been suggested by the northern blot analyses of Sullivan et al. 38 GalR2 has been cloned from rat, 13,19,36,42 human, 4,21 and mouse. 30 In contrast to GalR1, activation of GalR2 increases
intracellular calcium levels through coupling to Gaq/11 G-proteins 21,36 and has a wider tissue distribution than GalR1. 13,21,36,42 More recently, the cloning of a galanin receptor homologue, GalR3, was reported in rat 37,43 and human. 21,37 Like GalR1, GalR3 is coupled to Gai/Gao G-proteins as expression of GalR3 in Xenopus oocytes results in pertussis toxin-sensitive, inward K 1 currents through GIRK channels. 37 However, the reported broad tissue distribution of GalR3 is comparable to GalR2. 21,37,43 Because GalR1, GalR2, and GalR3 likely mediate many of galanin’s actions, it is of interest to determine relative levels of the three receptors in galanin-responsive tissues. Differential distribution of galanin receptors in target organs may provide insight into receptor selectivity of galanin-mediated effects. To address this question, the objective of the present study was to quantitatively determine and compare the tissue distribution of rat GalR1, GalR2 and GalR3 mRNAs. EXPERIMENTAL PROCEDURES
Experimental animals Twenty male Sprague–Dawley rats (200–300 g) were used for central and peripheral tissue collection in this study. Animals were killed by CO2 inhalation and decapitated before tissue removal. All animal procedures followed USDA guidelines and protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Neurogen Corporation. Galanin receptor cloning Full-length coding regions of GalR1, GalR2 and GalR3 receptors were cloned by polymerase chain reaction (PCR) and subcloned into vectors containing DNA-dependent, RNA polymerase promoter sites. Specifically, rat GalR1 (Genebank no. U33193) 6 was cloned from a rat hypothalamus cDNA library using primers spanning nucleotides 1–18 (coding sequence, forward) and 1065–1087 (3 0 -UTR, reverse) and subcloned into the pGEMT cloning vector (Promega). The rat hypothalamus cDNA library was constructed in Zap Express (Stratagene) using poly A 1 RNA isolated from 200–250 g Sprague–Dawley rats. The library consisted of approximately 2.5 × 10 6 independent
*To whom correspondence should be addressed. Abbreviations: DRG, dorsal root ganglia; EDTA, ethylenediaminetetraacetate; GalR1, galanin-1 receptor; GalR2, galanin-2 receptor; GalR3, galanin-3 receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; PNS, peripheral nervous system; RT, reverse transcription. 265
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Solution hybridization/RNase protection assay
Fig. 1. Targeted region of galanin receptors for RT–PCR and solution hybridization/RNase protection analysis. Amplified RT–PCR fragments are the hatched bars and solution hybridization/RNase protection assay fragments are the solid bars.
recombinants with an average insert size of 1.5 kb. Rat GalR2 (Genebank no. AF010318) 36 was cloned from a Life Technologies/ Gibco/BRL rat brain cDNA library using primers spanning nucleotides 1–22 (coding sequence, forward) and 1100–1119 (coding sequence, reverse) and subcloned into pcDNA 3.0 (Invitrogen). Rat GalR3 (Genebank no. AF079844) was cloned from the Zap Express rat hypothalamus cDNA library using primers spanning nucleotides 1–21 (coding sequence, forward) and 1091–1113 (coding sequence, reverse) and subcloned into pBluescript II SK- (Stratagene). Of note, the independent clones (n 3) we obtained for rat GalR3 differ from previously published sequences by five (Genebank no. AF031522) 43 and three (Genebank no. AF073798) 37 nucleotides, respectively. Specifically, the differences noted from our clones and Wang et al., 43 respectively, were nucleotides 380 (C for A), 546–550 (CGCGC for GCGCT), 705 (C for G) and 931 (C for T). The differences between our clones and Smith et al., 37 respectively, were nucleotides 162 (G for C), 480 (C for T) and 783 (T for C). We did not observe these previously reported nucleotides in any of our three clones after multiple sequencing reactions of both strands.
Reverse transcription–polymerase chain reaction analysis Tissue distribution of GalR mRNA expression was first determined by reverse transcription (RT)–PCR. Total RNA was isolated from rat tissues as previously described. 17 Total RNA (5 mg) was treated with RQ1 DNase (Promega) for 15 min at 378C to remove potential genomic DNA contamination. RT product was generated by annealing random primers (1 mg) by slow cooling and incubating samples for 60 min at 428C with Superscript II RT enzyme (Life Technologies/ Gibco/BRL). PCRs consisted of rat gene-specific primers for GalR1 (coding sequence nucleotides 1–18 forward and 402–419 reverse), GalR2 (coding sequence nucleotides 435–452 forward and 1090– 1107 reverse), GalR3 (coding sequence 78–97 forward and 289–310 reverse) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control (coding sequence nucleotides 79–100 forward and 889–910 reverse), 250 ng RT product (or 7.5 pg standard) and rTth DNA polymerase, XL (Perkin Elmer). The GalR region amplified by RT–PCR is shown in Fig. 1. PCR cycles were as follows: 948C × 15 s, 658C × 3 min decreasing by 0.58C each cycle for 20 cycles, then 948C × 15 s, 558C × 30 s, 698C × 3 min for either 12 (GalRs) or 5 (GAPDH) cycles. Samples were run on 1.5% agarose gels containing ethidium bromide and photographed on a ultraviolet light box for detection.
The level of GalR mRNA expression was quantified by solution hybridization/RNase protection assays. Full-length, sense RNA transcripts for GalR1, GalR2 and GalR3 were prepared from the aforementioned plasmids to generate GalR mRNA quantitation standards. Antisense probes were generated for each galanin receptor and b-actin using a[ 32P]-UTP and 200 ng linearized template. GalR1 template was linearized with ApaLI to generate a probe of 487 bases (441 base protected fragment), GalR2 template was linearized with MluI to generate a probe of 380 bases (320 base protected fragment), and GalR3 template was linearized with XbaI to generate a probe of 393 bases (373 base protected fragment) (Fig. 1). Assayed samples consisted of 25 mg tissue total RNA (10 mg for DRG, spinal cord comparison experiment), sense standard (10 pg to 1 pg) or yeast transfer RNA negative control (25 mg) co-precipitated with 2 × 10 5 c.p.m. of GalR probe (GalR-1 and GalR-2 specific activity 3.67 × 10 8 c.p.m./mg, GalR-3 6.67 × 10 8 c.p.m./mg) and 2.5 × 104 c.p.m. b-actin probe (specific activity 1.36 × 10 8 c.p.m./mg). Samples were resuspended in hybridization buffer, denatured at 958C for 5 min and allowed to hybridize to completion for 14–16 h at 458C. GalR1 and GalR2 samples were digested with RNase A and RNase T1 (25 mg and 85 units, respectively, Boehringer–Mannheim) for 45 min at 378C, proteinase K (200 mg, Boehringer–Mannheim) and sodium dodecyl sulfate (1%) for 30 min at 378C, then phenol/chloroform extracted and precipitated with an equal volume of isopropanol. GalR3 samples were digested with S1 nuclease (260 units, Boehringer–Mannheim) for 45 min at 378C, and precipitated with ammonium acetate, EDTA and isopropanol. All samples were run on 6% polyacrylamide, 8 M urea gels at 450 V for 2.5 h. Gels were dried and exposed to Kodak Biomax MS X-ray film for 16 h at 2808C. Receptor and b-actin-specific bands, as determined by autoradiographs, were isolated from the gels, placed in scintillation cocktail, analysed in a beta scintillation counter and compared to sense standard curves run in parallel for quantitation of mRNA expression. RESULTS
Reverse transcription–polymerase chain reaction analysis The central and peripheral tissue distribution of mRNA for the three characterized rat galanin receptors GalR1, GalR2 and GalR3 was determined by RT–PCR. GalR1 mRNA was present exclusively in CNS and peripheral nervous system (PNS) (Fig. 2). GalR1 mRNA was detected in all CNS regions examined including hippocampus, hypothalamus, cortex, amygdala, spinal cord and dorsal root ganglia (DRG). Strongest signals were seen in the hypothalamus, amygdala, and DRG. No signal was noted in peripheral tissues including anterior pituitary. GalR2 mRNA was widely distributed in central and peripheral tissues (Fig. 2). Strong signals were noted in hippocampus, hypothalamus, cortex, amygdala, spinal cord, DRG, anterior pituitary, lung and kidney. Lesser levels of GalR2 mRNA were seen in large intestine and spleen. Detectable levels of GalR2 mRNA were found in heart and liver. As observed for GalR2, GalR3 mRNA was noted in both central and peripheral tissues (Fig. 2). Hippocampus, hypothalamus, lung, kidney, and liver expressed the strongest mRNA signal for GalR3. Cortex, amygdala, spinal cord, DRG, anterior pituitary, large intestine and spleen expressed lower mRNA levels. Solution hybridization/RNase protection analysis Using solution hybridization/RNase protection analysis, GalR mRNA levels in rat tissues were quantitated. Standard curves of sense RNAs were used to determine mRNA concentrations and all samples were normalized to b-actin signals. As noted by RT–PCR, GalR1 mRNA was only detected in
Quantitative rat galanin receptor mRNA distribution
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Fig. 2. RT–PCR analysis of GalR1, GalR2, and GalR3 mRNA distribution. RT products (250 ng) from isolated rat tissues were used in PCR reactions with galanin receptor or GAPDH primers. Plasmid DNA (7.5 pg) was used as a size and signal standard.
CNS and PNS tissues (Figs 3A, 4). Quantitation of excised bands showed that hypothalamus had the greatest expression of GalR1 mRNA (14.4 pg/25 mg total RNA, Table 1). Amygdala, spinal cord and DRG also had high levels of GalR1 mRNA (9.0–10.0 pg/25 mg total RNA), whereas hippocampus and cortex had lower expression (3.4–4.1 pg/25 mg total RNA, Table 1). GalR2 mRNA was found to be distributed across many central and peripheral tissues (Figs 3B, 4). Highest levels of mRNA expression were found in hypothalamus, DRG and kidney (8.4–10.6 pg/25 mg total RNA, Table 1). Hippocampus, cortex, amygdala, spinal cord, pituitary and spleen had lower levels (3.3–5.4 pg/25 mg total RNA) whereas heart, lung and large intestine had detectable levels (0.9–2.0 pg/ 25 mg total RNA, Table 1) of GalR2 mRNA. GalR3 mRNA was broadly distributed with moderate levels in CNS, PNS and periphery, relative to GalR1 and GalR2 (Figs 3C, 4). Highest expression was observed in DRG, lung, kidney and liver (4.3–5.0 pg/25 mg total RNA, Table 1). Slightly lower levels of GalR3 mRNA were measured in brain regions, anterior pituitary and spleen (3.0–3.9 pg/25 mg total RNA, Table 1). No GalR3 mRNA was detected in heart or large intestine by solution hybridization/RNase protection analysis (Fig. 3C). DISCUSSION
Pharmacological studies have suggested the presence of
multiple galanin receptor subtypes in responsive tissues. To date, three such receptors have been cloned and characterized. The goal of the present study was to determine the relative level of galanin receptor mRNAs in tissues associated with galanin peptide activity. Although previous studies have determined the qualitative distribution of rat GalR1, 6,15,31,38 GalR2, 13,19 and GalR3, 37,43 the current use of solution hybridization/RNase protection assays allowed for quantitative assessment of receptor mRNA. In our present studies, RT– PCR, a qualitative to semi-quantitative assay, correlated well with quantitative solution hybridization/RNase protection assays for GalR tissue distribution. Rat GalR1 mRNA was expressed at high levels in hypothalamus, spinal cord, DRG and amygdala. GalR1 mRNA was detected solely in CNS and PNS with no peripheral tissue expression observed, as noted in previous studies. 6,15,31 This contrasts with results reported by Sullivan and co-workers who describe a broad central and peripheral tissue distribution for GalR1 mRNA by northern blot analysis. 38 However, the use of more selective and sensitive assays in the present study would suggest an exclusive CNS and PNS distribution for GalR1 mRNA exists in rat tissues. GalR2 and GalR3 mRNAs were present in both central and peripheral tissues as noted by earlier studies. 13,19,37,42,43 GalR2 was highly expressed in hypothalamus, DRG and kidney. In the present study, GalR3 mRNA was more widely distributed at moderate levels than previously noted by Wang et al. 43 who detected significant expression of this receptor only in spleen and testis
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Table 1. Quantitation of solution hybridization/RNase protection assays for rat galanin receptors Tissue
Galanin-1 R
Galanin-2 R
Galanin-3 R
Hippocampus Hypothalamus Cortex Amygdala Spinal cord Dorsal root ganglia Anterior pituitary Heart Lung Kidney Liver Large intestine Spleen
3.35 ^ 0.28 14.39 ^ 3.34 4.05 ^ 1.61 9.97 ^ 0.41 8.96 ^ 0.23 9.57 ^ 0.56 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
4.21 ^ 0.45 10.60 ^ 2.30 5.30 ^ 0.86 5.37 ^ 0.79 4.39 ^ 0.47 8.88 ^ 2.30 4.56 ^ 0.30 0.90 ^ 0.16 2.00 ^ 0.17 8.43 ^ 0.86 0.20 ^ 0.56 2.04 ^ 0.25 3.29 ^ 0.80
3.24 ^ 0.40 3.37 ^ 0.85 3.03 ^ 0.22 3.53 ^ 0.57 3.26 ^ 1.14 4.99 ^ 1.37 3.89 ^ 0.35 n.d. 4.87 ^ 0.68 4.65 ^ 0.56 4.30 ^ 0.72 n.d. 3.10 ^ 0.24
Values are expressed as mean pg/25 mg total RNA ^S.E.M. (n 3–4 samples). Receptor and b-actin-specific bands were isolated from assay gels (Figs 3, 4), analysed in a beta scintillation counter and compared to sense standard curves run in parallel assays for quantitation of mRNA expression. Average b-actin signals from all tissues within an experiment (except heart where different isoforms of actin are mainly expressed) were used for normalization of galanin receptor signals. n.d., not detected.
and Smith et al. 37 who observed strong signals only in hypothalamus and anterior pituitary. In addition to these differences in mRNA distribution noted between studies, the role of GalR3 in galanin-mediated effects remains uncertain. In oocyte functional assays, high nanomolar to micromolar concentrations of galanin are required to obtain EC50s for GIRK channel activation. 37 Further, Ki values for galanin peptides are 10–100 nM depending on the cell line used for binding analysis. 37,43 From these studies documenting the apparent low affinity and efficacy of galanin at the so-called GalR3, it may be that this receptor sequence is not functionally relevant for galanin and perhaps another ligand exists for GalR3 in vivo. The disparity of the current GalR mRNA distribution results and those previously reported may be largely attributed to the assays used for mRNA detection. Northern blot analyses in several previous reports show inconsistent distribution and transcript size of GalR mRNAs. 31,38,42,43 This may be a result of probe selection and quality of RNA blots, including commercial sources of the blots. These potential complicating factors were avoided by use of solution hybridization/RNase protection assays which provide a specific analysis of individual GalR mRNAs. Galanin is associated with many central and peripheral receptor-mediated effects including feeding, cognition, pain and anterior pituitary hormone regulation. The role of galanin in feeding is attributed to receptor activation in both hypothalamus and amygdala. 23,35 Interestingly, recent pharmacological studies using i.c.v. administration of the reported GalR2 and GalR3 selective peptide galanin(2–29) suggest that these two receptors play little role in feeding compared to GalR1. 41 In the present study, GalR1 mRNA was highly expressed in both hypothalamus and amygdala whereas GalR2 was markedly expressed only in hypothalamus and GalR3 was present at low levels in both regions. Therefore, the relative distribution of these receptor mRNAs supports a greater role for GalR1 in the feeding response.
Fig. 3. Autoradiographs of representative solution hybridization/RNase protection assays for rat (A) GalR1, (B) GalR2, and (C) GalR3 mRNAs in central and peripheral tissues. Twenty-five micrograms of total RNA was hybridized with [ 32P]-labeled antisense RNA probes for galanin receptors and b-actin, digested with RNase A and T1 (GalR1 and GalR2), or S1 nuclease (GalR3) and run on 6% polyacrylamide, 8 M urea gels. Receptor and b-actin-specific bands were isolated from the gels, analysed in a beta scintillation counter and compared to sense standard curves run in parallel for quantitation of mRNA expression (Table 1).
Cognitive deficits induced by central galanin administration are attributed to inhibition of both acetylcholine release from basal forebrain structures projecting to cortex and hippocampus 10,14 and postsynaptic cholinergic signal
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Quantitative rat galanin receptor mRNA distribution
Fig. 4. Autoradiograph of a representative solution hybridization/RNase protection assay for rat GalR1 (1), GalR2 (2), and GalR3 (3) mRNAs in spinal cord and DRG. Ten micrograms of total RNA was hybridized with [ 32P]-labeled antisense RNA probes for galanin receptors and b-actin (b), digested with RNase A and T1 (GalR1 and GalR2), or S1 nuclease (GalR3) and run on 6% polyacrylamide, 8 M urea gels. Receptor and b-actin-specific bands were isolated from the gels, analysed in a beta scintillation counter and compared to sense standard curves run in parallel for quantitation of mRNA expression (Table 1).
transduction. 29 Further, administration of the galanin receptor antagonist, M35, enhances performance in the Morris swim test supporting galanin’s role in tonic cholinergic inhibition. 28 However, a complicating factor of using peptidic galanin receptor antagonists such as M35 is the noted partial agonist and non-selective activity of these compounds in the CNS and periphery. 20 In the present study, all three rat galanin receptor mRNAs were present in low to moderate levels in hippocampus and cortex. Therefore, more discrete localization studies in hippocampus, cortex and basal forebrain are required to determine the galanin receptor subtype or subtypes involved in cognitive deficits induced by galanin. Although galanin-like immunoreactivity and mRNA are expressed at low levels in spinal cord and DRG of naive rats, several lines of evidence suggest a role for galanin in pain transmission and nerve regeneration. Despite low endogenous peptide levels, i.t. galanin administration inhibits spinal nociception to noxious stimuli. 45 Additionally, following nerve injury, galanin peptide and mRNA are dramatically up-regulated in small DRG neurons 18 suggesting a role for galanin as an endogenous analgesic or nerve regeneration factor. 26 The change in galanin levels after nerve injury is likely mediated by the release of the cytokine, leukemia inhibitory factor, and removal of nerve growth factor which increase galanin peptide mRNA as a result of injury. 8,49 Targeted removal of GalR1 by nucleic acid antisense constructs decreases the ability of galanin to inhibit spinal reflexes providing evidence for GalR1 involvement in spinal excitability. 32 After axotomy, GalR1 and GalR2 mRNAs are
decreased in DRG neurons, 34 suggesting down-regulation of receptor induced by increased local peptide concentrations. In contrast, both galanin peptide and GalR2 mRNAs are increased in the facial nucleus after facial nerve crush. 5 This suggests a differential regulation of GalRs dependent on the site of nerve injury. We observed GalR1 as the dominant galanin receptor mRNA present in spinal cord whereas GalR2 and GalR1 were highly expressed in DRG. GalR3 was present in lower levels in both spinal cord and DRG. Based on this distribution, GalR2 may play a role in the modulation of sensory input from the periphery and GalR1 in the spinal transmission of nociceptive stimuli. The role of GalR3 in pain perception is not clear due to the relative paucity of receptor mRNA in spinal cord and DRG and thus requires further investigation. Pharmacological manipulation of specific receptor subtypes and subsequent changes in receptor mRNA and protein levels should provide further insight into the relevant GalRs involved in nociception. In the periphery, galanin is present at high levels in anterior pituitary and is associated with increasing growth hormone, leutinizing hormone and prolactin release. The effects of galanin on growth hormone and leutinizing hormone are presumably mediated by inhibition of somatostatin 7 and stimulation of gonadotropin-releasing hormone 25 release from hypothalamus, respectively. Galanin likely has a direct effect on prolactin release as application of galanin to dispersed pituitary cells results in prolactin secretion. 48 The galanin receptor or receptors which mediate the pituitary actions of galanin is unclear. GalR2 and GalR3 mRNAs were expressed in anterior pituitary whereas GalR1 mRNA was not detectable. Further, a novel galanin receptor has been postulated in anterior pituitary based on the ability of galanin(3–29) to induce prolactin 48 and decrease gonadotrophin 40 release in dispersed pituitary cells. This peptide does not show high-affinity binding to either GalR1, GalR2 or GalR3. Therefore, the complex pharmacology of galanin in anterior pituitary may be a result of activating the multiple known and potentially novel GalR isoforms expressed in this tissue. CONCLUSIONS
The diverse actions of galanin make this peptidergic system an attractive target for modulating obesity, cognitive deficits, analgesia and pituitary dysfunction. The presence of at least three G-protein-coupled receptors responsive to galanin underlines the complexity of galanin-mediated biology. The detection of GalR1 mRNA exclusively in CNS and PNS tissues implicates this receptor in the central and peripheral nervous system effects of galanin, especially in the hypothalamus, amygdala, spinal cord and DRG where high level expression was observed. The relatively widespread distribution of GalR2 and GalR3 mRNAs in rat tissues suggests involvement in both central and peripheral galanin effects. The development of selective receptor agonists and antagonists will be required for direct association of galanin action and specific receptor subtypes. This study documents the distribution of galanin receptor mRNAs that are localized to cell bodies that may project to other brain regions or tissues. Information from quantitative solution hybridization/RNase protection assays, coupled with the use of recently generated galanin receptor subtype selective antibodies, will define the precise location of receptor synthesis and galanin peptide action throughout the neuraxis.
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REFERENCES
1. Ahren B. and Lindskog S. (1992) Galanin and the regulation of islet hormone secretion. Int. J. Pancreat. 11, 147–160. 2. Bartfai T., Hokfelt T. and Langel U. (1993) Galanin—A neuroendocrine peptide. Crit. Rev. Neurobiol. 7, 229–274. 3. Bauer F. E., Ginsberg L., Venetikou M., MacKay D. J., Burrin J. M. and Bloom S. R. (1986) Growth hormone release in man induced by galanin, a new hypothalamic peptide. Lancet 2, 192–195. 4. Bloomquist B. T., Beauchamp M. R., Zhelnin L., Brown S.-E., Gore-Willse A. R., Gregor P. and Cornfield L. J. (1998) Cloning and expression of the human galanin receptor GalR2. Biochem. biophys. Res. Commun. 243, 474–479. 5. Burazin T. C. D. and Gundlach A. L. (1998) Inducible galanin and GalR2 receptor system in motor neuron injury and regeneration. J. Neurochem. 71, 879–882. 6. Burgevin M.-C., Loquet I., Quarteronet D. and Habert-Ortoli E. (1995) Cloning, pharmacological characterization, and anatomical distribution of a rat cDNA encoding for a galanin receptor. J. molec. Neurosci. 6, 33–41. 7. Chan Y. Y., Grafstein-Dunn E., Delemarre-Van De Waal H. A., Burton K. A., Clifton D. K. and Steiner R. A. (1996) The role of galanin and its receptor in the feedback regulation of growth hormone secretion. Endocrinology 137, 5303–5310. 8. Corness J., Stevens B., Douglas Fields R. and Hokfelt T. (1998) NGF and LIF both regulate galanin gene expression in primary DRG cultures. NeuroReport 9, 1533–1536. 9. Corwin R. L., Robinson J. K. and Crawley J. N. (1993) Galanin antagonists block galanin-induced feeding in the hypothalamus and amygdala of the rat. Eur. J. Neurosci. 5, 1528–1533. 10. Crawley J. N. (1996) Galanin–acetylcholine interactions: relevance to memory and Alzheimer’s disease. Life Sci. 58, 2185–2199. 11. Crawley J. N., Austin M. C., Fiske S. M., Martin B., Consolo S., Berthold M., Langel U., Fisone G. and Bartfai T. (1990) Activity of centrally administered galanin fragments on stimulation of feeding behavior and on galanin receptor binding in the rat hypothalamus. J. Neurosci 10, 3695–3700. 12. Ekblad E., Hakanson R., Sundler F. and Wahlestedt C. (1985) Galanin: neuromodulatory and direct contractile effects on smooth muscle preparations. Br. J. Pharmac. 86, 241–246. 13. Fathi Z., Cunningham A. M., Iben L. G., Battaglino P. B., Ward S. A., Nichol K. A., Pine K. A., Wang J., Goldstein M. E., Iismaa T. P. and Zimanyi I. A. (1997) Cloning, pharmacological characterization and distribution of a novel galanin receptor. Molec. Brain Res. 51, 49–59. 14. Fisone G., Wu C. F., Consolo S., Nordstrom O., Brynne N., Bartfai T., Melander T. and Hokfelt T. (1987) Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo and in vitro studies. Proc. natn. Acad. Sci. U.S.A. 84, 7339–7343. 15. Gustafson E. L., Smith K. E., Durkin M. M., Gerald C. and Branchek T. A. (1996) Distribution of a rat galanin receptor mRNA in rat brain. NeuroReport 7, 953–957. 16. Habert-Ortoli E., Amiranoff B., Loquet I., Laburthe M. and Mayaux J.-F. (1994) Molecular cloning of a functional human galanin receptor. Proc. natn. Acad. Sci. U.S.A. 91, 9780–9783. 17. Hershey A. D., Dykema P. E. and Krause J. E. (1991) Organization, structure, and expression of the gene encoding the rat substance P receptor. J. biol. Chem. 266, 4366–4374. 18. Hokfelt T., Wiesenfeld-Hallin Z., Villar M. J. and Melander T. (1987) Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy. Neurosci. Lett. 83, 217–220. 19. Howard A. D., Tan C., Shiao L.-L., Palya O. C., McKee K. K., Weinberg D. H., Feighner S. D., Cascieri M. A., Smith R. G., Van Der Ploeg L. H. T. and Sullivan K. A. (1997) Molecular cloning and characterization of a new receptor for galanin. Fedn Eur. biochem. Socs Lett. 405, 285–290. 20. Kinney G. A., Emmerson P. J. and Miller R. J. (1998) Galanin receptor-mediated inhibition of glutamate release in the arcuate nucleus of the hypothalamus. J. Neurosci. 18, 3489–3500. 21. Kolakowski L. F. Jr., O’Neill G. P., Howard A. D., Broussard S. R., Sullivan K. A., Feighner S. D., Sawzdargo M., Nguyen T., Kargman S., Shiao L.-L., Hreniuk D. L., Tan C. P., Evans J., Abramovitz M., Chateauneuf A., Coulombe N., Ng G., Johnson M. P., Tharian A., Khoshbouei H., George S. R., Smith R. G. and O’Dowd B. F. (1998) Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3. J. Neurochem. 71, 2239–2251. 22. Koshiyama H., Shimatsu A., Kato Y., Assadian H., Hattori N., Ishikawa Y., Tanoh T., Yanaihara N. and Imura H. (1990) Galanin-induced prolactin release in rats: pharmacological evidence for the involvement of alpha-adrenergic and opioidergic mechanisms. Brain Res. 507, 321–324. 23. Kyrkouli S. E., Stanley B. G., Seirafi R. D. and Leibowitz S. F. (1990) Stimulation of feeding by galanin: anatomical localization and behavioral specificity of this peptide’s effects in brain. Peptides 11, 995–1001. 24. Leibowitz S. F. and Kim T. (1992) Impact of galanin antagonist on exogenous galanin and natural patterns of fat ingestion. Brain Res. 599, 148–152. 25. Lopez F. J., Merchenthaler I., Ching M., Wisniewski M. G. and Negro-Vilar A. (1991) Galanin: a hypothalamic-hypophysiotrophic hormone modulating reproductive functions. Proc. natn. Acad. Sci. U.S.A. 88, 4508–4512. 26. Mahoney R. P., Siegel R. and Zigmond R. E. (1994) Galanin and vasoactive intestinal peptide messenger RNA expression increase following axotomy of the adult rat SCG. J. Neurobiol. 25, 108–118. 27. Merchenthaler I., Lopez F. J. and Negro-Vilar A. (1993) Anatomy and physiology of central galanin-containing pathways. Prog. Neurobiol. 40, 711–769. 28. Ogren S. O., Hokfelt T., Kask K., Langel U. and Bartfai T. (1992) Evidence for a role of the neuropeptide galanin in spatial learning. Neuroscience 51, 1–5. 29. Palazzi E., Felinska S., Zambelli M., Fisone G., Bartfai T. and Consolo S. (1991) Galanin reduces carbachol stimulation of phosphoinositide turnover in rat ventral hippocampus by lowering Ca 21 influx through voltage sensitive Ca 21 channels. J. Neurochem. 56, 739–747. 30. Pang L., Hashemi T., Lee H.-J. J., Maguire M., Graziano M. P., Bayne M., Hawes B., Wong G. and Wang S. (1998) The mouse GalR2 galanin receptor: genomic organization, cDNA cloning, and functional characterization. J. Neurochem. 71, 2252–2259. 31. Parker E. M., Izzarelli D. G., Nowak H. P., Mahle C. D., Iben L. G., Wang J. and Goldstein M. E. (1995) Cloning and characterization of the rat GALR1 galanin receptor from Rin14B insulinoma cells. Molec. Brain Res. 34, 179–189. 32. Pooga M., Soomets U., Hallbrink M., Valkna A., Saar K., Rezaei K., Kahl U., Hao J.-X., Xu X.-J., Wiesenfeld-Hallin Z., Hokfelt T., Bartfai T. and Langel U. (1998) Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo. Nature Biotech. 16, 857–861. 33. Reimann W., Engelberger W., Friderichs E., Selve N. and Wilffert B. (1994) Spinal antinociception by morphine in rats is antagonised by galanin receptor antagonists. Naunyn-Schmiedeberg’s Arch. Pharmac. 350, 380–386. 34. Shi T.-J. S., Zhang X., Holmberg K., Xu Z.-Q. D. and Hokfelt T. (1997) Expression and regulation of galanin-R2 receptors in rat primary sensory neurons: effect of axotomy and inflammation. Neurosci. Lett. 237, 57–60. 35. Smith B. K., York D. A. and Bray G. A. (1996) Effects of dietary preference and galanin administration in the paraventricular or amygdaloid nucleus on diet self-selection. Brain Res. Bull. 39, 149–154. 36. Smith K. E., Forray C., Walker M. W., Jones K. A., Tamm J. A., Bard J., Branchek T. A., Linemeyer D. L. and Gerald C. (1997) Expression cloning of a rat hypothalamic galanin receptor coupled to phosphoinositide turnover. J. biol. Chem. 272, 24,612–24,616. 37. Smith K. E., Walker M. W., Artmyshyn R., Bard J., Borowsky B., Tamm J. A., Yao W.-J., Vaysse P. J.-J., Branchek T. A., Gerald C. and Jones K. A. (1998) Cloned human and rat galanin GALR3 receptors: pharmacology and activation of G-protein inwardly rectifying K 1 channels. J. biol. Chem. 273, 23,321–23,326. 38. Sullivan K. A., Shiao L.-L. and Cascieri M. A. (1997) Pharmacological characterization and tissue distribution of the human and rat GALR1 receptors. Biochem. biophys. Res. Commun. 233, 823–828.
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39. Tatemoto K., Rokaeus A., Jornwall H., McDonald T. J. and Mutt V. (1983) Galanin—A novel biologically active peptide from porcine intestine. Fedn Eur. biochem. Socs Lett. 164, 124–128. 40. Todd J. F., Small C. J., Akinsanya K. O., Stanley S. A., Smith D. M. and Bloom S. R. (1998) Galanin is a paracrine inhibitor of gonadotroph function in the female rat. Endocrinology 139, 4222–4229. 41. Wang S., Ghibaudi L., Hashemi T., He C., Strader C., Bayne M., Davis H. and Hwa J. J. (1998) The GalR2 galanin receptor mediates galanin-induced jejunal contraction, but not feeding behavior, in the rat: differentiation of central and peripheral effects of receptor subtype activation. Fedn Eur. biochem. Socs Lett. 434, 277–282. 42. Wang S., Hashemi T., He C., Strader C. and Bayne M. (1997) Molecular cloning and pharmacological characterization of a new galanin receptor subtype. Molec. Pharmac. 52, 337–343. 43. Wang S., He C., Hashemi T. and Bayne M. (1997) Cloning and expressional characterization of a novel galanin receptor: identification of different pharmacophores within galanin for the three galanin receptor subtypes. J. biol. Chem. 272, 31,949–31,952. 44. Wang S., He C., Maguire M. T., Clemmons A. L., Burrier R. E., Guzzi M. F., Strader C. D., Parker E. M. and Bayne M. L. (1997) Genomic organization and functional characterization of the mouse GalR1 galanin receptor. Fedn Eur. biochem. Socs Lett. 411, 225–230. 45. Wiesenfeld-Hallin Z., Xu X.-J., Hao J.-X. and Hokfelt T. (1993) The behavioural effects of intrathecal galanin on tests of thermal and mechanical nociception in the rat. Acta physiol. scand. 147, 457–458. 46. Wiesenfeld-Hallin Z., Xu X. J., Hakanson R., Feng D. M., Folkers K., Kristensson K., Villar M. J., Fahrenkrug J. and Hokfelt T. (1991) On the role of substance P, galanin, vasoactive intestinal peptide, and calcitonin gene-related peptide in mediation of spinal reflex excitability in rats with intact and sectioned peripheral nerves. Ann. N.Y. Acad. Sci. 632, 198–211. 47. Wynick D., Small C. J., Bacon A., Holmes F. E., Norman M., Ormandy C. J., Kilic E., Kerr N. C. H., Ghatei M., Talamantes F., Bloom S. R. and Pachnis V. (1998) Galanin regulates prolactin release and lactotroph proliferation. Proc. natn. Acad. Sci. U.S.A. 95, 12,671–12,676. 48. Wynick D., Smith D. M., Ghatei M., Akinsanya K., Bhogal R., Purkiss P., Byfield P., Yanaihara N. and Bloom S. R. (1993) Characterization of a highaffinity galanin receptor in the rat anterior pituitary: absence of biological effect and reduced membrane binding of the antagonist M15 differentiate it from the brain/gut receptor. Proc. natn. Acad. Sci. U.S.A. 90, 4231–4235. 49. Zigmond R. E. (1997) LIF, NGF, and the cell body response to axotomy. Neuroscientist 3, 176–185. (Accepted 2 August 1999)
Quantitative rat galanin receptor mRNA distribution
Pergamon PII: S0306-4522(99)00407-8 www.elsevier.com/locate/neuroscience
DISTRIBUTION OF GALANIN-1, -2 AND -3 RECEPTOR MESSENGER RNAS IN CENTRAL AND PERIPHERAL RAT TISSUES S. M. WATERS* and J. E. KRAUSE Department of Biochemistry and Molecular Biology, Neurogen Corporation, Branford, CT 06405, U.S.A.
Abstract—Galanin is a neuropeptide widely expressed in the central nervous system and periphery. In rat, three galanin-binding receptors have been cloned and characterized. We report the qualitative and quantitative distribution of galanin-1, galanin-2, and galanin-3 messenger RNAs in central and peripheral rat tissues by reverse transcription–polymerase chain reaction and solution hybridization/RNase protection assays, respectively. Galanin-1 messenger RNA was detected exclusively in the central and peripheral nervous system with highest expression in hypothalamus, amygdala, spinal cord and dorsal root ganglia. Galanin-2 messenger RNA was highly expressed in hypothalamus, dorsal root ganglia, and kidney with moderate expression in several other tissues. Galanin-3 messenger RNA was widely distributed at low to moderate levels in many central and peripheral tissues. The observed expression of multiple galanin receptors in several tissues including hypothalamus, anterior pituitary and spinal cord supports earlier pharmacological studies suggesting the presence of more than one receptor subtype in these regions. The presence of multiple galanin receptors in these tissues in conjunction with the detection of a single subtype, galanin-2, in tissues such as heart and intestine, illustrates the potential complexity of galanin-associated actions in rat central nervous system and periphery. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: galanin, receptor, distribution, central nervous system, rat, solution hybridization/RNase protection assay.
Galanin, a 29–30 amino acid neuropeptide first described in pig intestine, 39 is a widely distributed transmitter in the CNS and periphery. 2,27 As a neurotransmitter/neuromodulator, galanin is associated with many biological activities. In hypothalamus, i.c.v. administration of galanin increases food intake with a preference for fat, 11 whereas galanin receptor peptidic antagonists reduce feeding. 9,24 In hippocampus, galanin decreases acetylcholine release presynaptically 14 and transmission postsynaptically 29 suggesting a role in memory. In spinal cord, galanin depresses spinal nociceptive reflexes 45,46 and galanin receptor peptidic antagonists inhibit spinal antinociception induced by morphine. 33 Additionally, galanin decreases glucose-dependent insulin release in species other than man, 1 stimulates growth hormone 3,7 and prolactin 22,47 release from the anterior pituitary, and induces smooth muscle contraction in the intestine. 12 The actions of galanin are mediated by at least three G-protein-coupled receptors termed galanin-1 (GalR1), GalR2 and GalR3. GalR1, originally isolated from human Bowes melanoma cells, 16 was subsequently cloned from rat 6,31 and mouse. 44 Activation of GalR1 results in pertussis toxin-sensitive inhibition of adenylyl cyclase through interaction with Gai/Gao G-proteins, 6,16,31 and can increase intracellular Ca 21 via bg subunit activation of phospholipase C b2 isoforms. Northern blot analysis and in situ hybridization show that GalR1 is predominantly located in the brain and spinal cord, 6,31 although a broader distribution has been suggested by the northern blot analyses of Sullivan et al. 38 GalR2 has been cloned from rat, 13,19,36,42 human, 4,21 and mouse. 30 In contrast to GalR1, activation of GalR2 increases
intracellular calcium levels through coupling to Gaq/11 G-proteins 21,36 and has a wider tissue distribution than GalR1. 13,21,36,42 More recently, the cloning of a galanin receptor homologue, GalR3, was reported in rat 37,43 and human. 21,37 Like GalR1, GalR3 is coupled to Gai/Gao G-proteins as expression of GalR3 in Xenopus oocytes results in pertussis toxin-sensitive, inward K 1 currents through GIRK channels. 37 However, the reported broad tissue distribution of GalR3 is comparable to GalR2. 21,37,43 Because GalR1, GalR2, and GalR3 likely mediate many of galanin’s actions, it is of interest to determine relative levels of the three receptors in galanin-responsive tissues. Differential distribution of galanin receptors in target organs may provide insight into receptor selectivity of galanin-mediated effects. To address this question, the objective of the present study was to quantitatively determine and compare the tissue distribution of rat GalR1, GalR2 and GalR3 mRNAs. EXPERIMENTAL PROCEDURES
Experimental animals Twenty male Sprague–Dawley rats (200–300 g) were used for central and peripheral tissue collection in this study. Animals were killed by CO2 inhalation and decapitated before tissue removal. All animal procedures followed USDA guidelines and protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Neurogen Corporation. Galanin receptor cloning Full-length coding regions of GalR1, GalR2 and GalR3 receptors were cloned by polymerase chain reaction (PCR) and subcloned into vectors containing DNA-dependent, RNA polymerase promoter sites. Specifically, rat GalR1 (Genebank no. U33193) 6 was cloned from a rat hypothalamus cDNA library using primers spanning nucleotides 1–18 (coding sequence, forward) and 1065–1087 (3 0 -UTR, reverse) and subcloned into the pGEMT cloning vector (Promega). The rat hypothalamus cDNA library was constructed in Zap Express (Stratagene) using poly A 1 RNA isolated from 200–250 g Sprague–Dawley rats. The library consisted of approximately 2.5 × 10 6 independent
*To whom correspondence should be addressed. Abbreviations: DRG, dorsal root ganglia; EDTA, ethylenediaminetetraacetate; GalR1, galanin-1 receptor; GalR2, galanin-2 receptor; GalR3, galanin-3 receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; PNS, peripheral nervous system; RT, reverse transcription. 265
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Solution hybridization/RNase protection assay
Fig. 1. Targeted region of galanin receptors for RT–PCR and solution hybridization/RNase protection analysis. Amplified RT–PCR fragments are the hatched bars and solution hybridization/RNase protection assay fragments are the solid bars.
recombinants with an average insert size of 1.5 kb. Rat GalR2 (Genebank no. AF010318) 36 was cloned from a Life Technologies/ Gibco/BRL rat brain cDNA library using primers spanning nucleotides 1–22 (coding sequence, forward) and 1100–1119 (coding sequence, reverse) and subcloned into pcDNA 3.0 (Invitrogen). Rat GalR3 (Genebank no. AF079844) was cloned from the Zap Express rat hypothalamus cDNA library using primers spanning nucleotides 1–21 (coding sequence, forward) and 1091–1113 (coding sequence, reverse) and subcloned into pBluescript II SK- (Stratagene). Of note, the independent clones (n 3) we obtained for rat GalR3 differ from previously published sequences by five (Genebank no. AF031522) 43 and three (Genebank no. AF073798) 37 nucleotides, respectively. Specifically, the differences noted from our clones and Wang et al., 43 respectively, were nucleotides 380 (C for A), 546–550 (CGCGC for GCGCT), 705 (C for G) and 931 (C for T). The differences between our clones and Smith et al., 37 respectively, were nucleotides 162 (G for C), 480 (C for T) and 783 (T for C). We did not observe these previously reported nucleotides in any of our three clones after multiple sequencing reactions of both strands.
Reverse transcription–polymerase chain reaction analysis Tissue distribution of GalR mRNA expression was first determined by reverse transcription (RT)–PCR. Total RNA was isolated from rat tissues as previously described. 17 Total RNA (5 mg) was treated with RQ1 DNase (Promega) for 15 min at 378C to remove potential genomic DNA contamination. RT product was generated by annealing random primers (1 mg) by slow cooling and incubating samples for 60 min at 428C with Superscript II RT enzyme (Life Technologies/ Gibco/BRL). PCRs consisted of rat gene-specific primers for GalR1 (coding sequence nucleotides 1–18 forward and 402–419 reverse), GalR2 (coding sequence nucleotides 435–452 forward and 1090– 1107 reverse), GalR3 (coding sequence 78–97 forward and 289–310 reverse) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control (coding sequence nucleotides 79–100 forward and 889–910 reverse), 250 ng RT product (or 7.5 pg standard) and rTth DNA polymerase, XL (Perkin Elmer). The GalR region amplified by RT–PCR is shown in Fig. 1. PCR cycles were as follows: 948C × 15 s, 658C × 3 min decreasing by 0.58C each cycle for 20 cycles, then 948C × 15 s, 558C × 30 s, 698C × 3 min for either 12 (GalRs) or 5 (GAPDH) cycles. Samples were run on 1.5% agarose gels containing ethidium bromide and photographed on a ultraviolet light box for detection.
The level of GalR mRNA expression was quantified by solution hybridization/RNase protection assays. Full-length, sense RNA transcripts for GalR1, GalR2 and GalR3 were prepared from the aforementioned plasmids to generate GalR mRNA quantitation standards. Antisense probes were generated for each galanin receptor and b-actin using a[ 32P]-UTP and 200 ng linearized template. GalR1 template was linearized with ApaLI to generate a probe of 487 bases (441 base protected fragment), GalR2 template was linearized with MluI to generate a probe of 380 bases (320 base protected fragment), and GalR3 template was linearized with XbaI to generate a probe of 393 bases (373 base protected fragment) (Fig. 1). Assayed samples consisted of 25 mg tissue total RNA (10 mg for DRG, spinal cord comparison experiment), sense standard (10 pg to 1 pg) or yeast transfer RNA negative control (25 mg) co-precipitated with 2 × 10 5 c.p.m. of GalR probe (GalR-1 and GalR-2 specific activity 3.67 × 10 8 c.p.m./mg, GalR-3 6.67 × 10 8 c.p.m./mg) and 2.5 × 104 c.p.m. b-actin probe (specific activity 1.36 × 10 8 c.p.m./mg). Samples were resuspended in hybridization buffer, denatured at 958C for 5 min and allowed to hybridize to completion for 14–16 h at 458C. GalR1 and GalR2 samples were digested with RNase A and RNase T1 (25 mg and 85 units, respectively, Boehringer–Mannheim) for 45 min at 378C, proteinase K (200 mg, Boehringer–Mannheim) and sodium dodecyl sulfate (1%) for 30 min at 378C, then phenol/chloroform extracted and precipitated with an equal volume of isopropanol. GalR3 samples were digested with S1 nuclease (260 units, Boehringer–Mannheim) for 45 min at 378C, and precipitated with ammonium acetate, EDTA and isopropanol. All samples were run on 6% polyacrylamide, 8 M urea gels at 450 V for 2.5 h. Gels were dried and exposed to Kodak Biomax MS X-ray film for 16 h at 2808C. Receptor and b-actin-specific bands, as determined by autoradiographs, were isolated from the gels, placed in scintillation cocktail, analysed in a beta scintillation counter and compared to sense standard curves run in parallel for quantitation of mRNA expression. RESULTS
Reverse transcription–polymerase chain reaction analysis The central and peripheral tissue distribution of mRNA for the three characterized rat galanin receptors GalR1, GalR2 and GalR3 was determined by RT–PCR. GalR1 mRNA was present exclusively in CNS and peripheral nervous system (PNS) (Fig. 2). GalR1 mRNA was detected in all CNS regions examined including hippocampus, hypothalamus, cortex, amygdala, spinal cord and dorsal root ganglia (DRG). Strongest signals were seen in the hypothalamus, amygdala, and DRG. No signal was noted in peripheral tissues including anterior pituitary. GalR2 mRNA was widely distributed in central and peripheral tissues (Fig. 2). Strong signals were noted in hippocampus, hypothalamus, cortex, amygdala, spinal cord, DRG, anterior pituitary, lung and kidney. Lesser levels of GalR2 mRNA were seen in large intestine and spleen. Detectable levels of GalR2 mRNA were found in heart and liver. As observed for GalR2, GalR3 mRNA was noted in both central and peripheral tissues (Fig. 2). Hippocampus, hypothalamus, lung, kidney, and liver expressed the strongest mRNA signal for GalR3. Cortex, amygdala, spinal cord, DRG, anterior pituitary, large intestine and spleen expressed lower mRNA levels. Solution hybridization/RNase protection analysis Using solution hybridization/RNase protection analysis, GalR mRNA levels in rat tissues were quantitated. Standard curves of sense RNAs were used to determine mRNA concentrations and all samples were normalized to b-actin signals. As noted by RT–PCR, GalR1 mRNA was only detected in
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Fig. 2. RT–PCR analysis of GalR1, GalR2, and GalR3 mRNA distribution. RT products (250 ng) from isolated rat tissues were used in PCR reactions with galanin receptor or GAPDH primers. Plasmid DNA (7.5 pg) was used as a size and signal standard.
CNS and PNS tissues (Figs 3A, 4). Quantitation of excised bands showed that hypothalamus had the greatest expression of GalR1 mRNA (14.4 pg/25 mg total RNA, Table 1). Amygdala, spinal cord and DRG also had high levels of GalR1 mRNA (9.0–10.0 pg/25 mg total RNA), whereas hippocampus and cortex had lower expression (3.4–4.1 pg/25 mg total RNA, Table 1). GalR2 mRNA was found to be distributed across many central and peripheral tissues (Figs 3B, 4). Highest levels of mRNA expression were found in hypothalamus, DRG and kidney (8.4–10.6 pg/25 mg total RNA, Table 1). Hippocampus, cortex, amygdala, spinal cord, pituitary and spleen had lower levels (3.3–5.4 pg/25 mg total RNA) whereas heart, lung and large intestine had detectable levels (0.9–2.0 pg/ 25 mg total RNA, Table 1) of GalR2 mRNA. GalR3 mRNA was broadly distributed with moderate levels in CNS, PNS and periphery, relative to GalR1 and GalR2 (Figs 3C, 4). Highest expression was observed in DRG, lung, kidney and liver (4.3–5.0 pg/25 mg total RNA, Table 1). Slightly lower levels of GalR3 mRNA were measured in brain regions, anterior pituitary and spleen (3.0–3.9 pg/25 mg total RNA, Table 1). No GalR3 mRNA was detected in heart or large intestine by solution hybridization/RNase protection analysis (Fig. 3C). DISCUSSION
Pharmacological studies have suggested the presence of
multiple galanin receptor subtypes in responsive tissues. To date, three such receptors have been cloned and characterized. The goal of the present study was to determine the relative level of galanin receptor mRNAs in tissues associated with galanin peptide activity. Although previous studies have determined the qualitative distribution of rat GalR1, 6,15,31,38 GalR2, 13,19 and GalR3, 37,43 the current use of solution hybridization/RNase protection assays allowed for quantitative assessment of receptor mRNA. In our present studies, RT– PCR, a qualitative to semi-quantitative assay, correlated well with quantitative solution hybridization/RNase protection assays for GalR tissue distribution. Rat GalR1 mRNA was expressed at high levels in hypothalamus, spinal cord, DRG and amygdala. GalR1 mRNA was detected solely in CNS and PNS with no peripheral tissue expression observed, as noted in previous studies. 6,15,31 This contrasts with results reported by Sullivan and co-workers who describe a broad central and peripheral tissue distribution for GalR1 mRNA by northern blot analysis. 38 However, the use of more selective and sensitive assays in the present study would suggest an exclusive CNS and PNS distribution for GalR1 mRNA exists in rat tissues. GalR2 and GalR3 mRNAs were present in both central and peripheral tissues as noted by earlier studies. 13,19,37,42,43 GalR2 was highly expressed in hypothalamus, DRG and kidney. In the present study, GalR3 mRNA was more widely distributed at moderate levels than previously noted by Wang et al. 43 who detected significant expression of this receptor only in spleen and testis
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Table 1. Quantitation of solution hybridization/RNase protection assays for rat galanin receptors Tissue
Galanin-1 R
Galanin-2 R
Galanin-3 R
Hippocampus Hypothalamus Cortex Amygdala Spinal cord Dorsal root ganglia Anterior pituitary Heart Lung Kidney Liver Large intestine Spleen
3.35 ^ 0.28 14.39 ^ 3.34 4.05 ^ 1.61 9.97 ^ 0.41 8.96 ^ 0.23 9.57 ^ 0.56 n.d. n.d. n.d. n.d. n.d. n.d. n.d.
4.21 ^ 0.45 10.60 ^ 2.30 5.30 ^ 0.86 5.37 ^ 0.79 4.39 ^ 0.47 8.88 ^ 2.30 4.56 ^ 0.30 0.90 ^ 0.16 2.00 ^ 0.17 8.43 ^ 0.86 0.20 ^ 0.56 2.04 ^ 0.25 3.29 ^ 0.80
3.24 ^ 0.40 3.37 ^ 0.85 3.03 ^ 0.22 3.53 ^ 0.57 3.26 ^ 1.14 4.99 ^ 1.37 3.89 ^ 0.35 n.d. 4.87 ^ 0.68 4.65 ^ 0.56 4.30 ^ 0.72 n.d. 3.10 ^ 0.24
Values are expressed as mean pg/25 mg total RNA ^S.E.M. (n 3–4 samples). Receptor and b-actin-specific bands were isolated from assay gels (Figs 3, 4), analysed in a beta scintillation counter and compared to sense standard curves run in parallel assays for quantitation of mRNA expression. Average b-actin signals from all tissues within an experiment (except heart where different isoforms of actin are mainly expressed) were used for normalization of galanin receptor signals. n.d., not detected.
and Smith et al. 37 who observed strong signals only in hypothalamus and anterior pituitary. In addition to these differences in mRNA distribution noted between studies, the role of GalR3 in galanin-mediated effects remains uncertain. In oocyte functional assays, high nanomolar to micromolar concentrations of galanin are required to obtain EC50s for GIRK channel activation. 37 Further, Ki values for galanin peptides are 10–100 nM depending on the cell line used for binding analysis. 37,43 From these studies documenting the apparent low affinity and efficacy of galanin at the so-called GalR3, it may be that this receptor sequence is not functionally relevant for galanin and perhaps another ligand exists for GalR3 in vivo. The disparity of the current GalR mRNA distribution results and those previously reported may be largely attributed to the assays used for mRNA detection. Northern blot analyses in several previous reports show inconsistent distribution and transcript size of GalR mRNAs. 31,38,42,43 This may be a result of probe selection and quality of RNA blots, including commercial sources of the blots. These potential complicating factors were avoided by use of solution hybridization/RNase protection assays which provide a specific analysis of individual GalR mRNAs. Galanin is associated with many central and peripheral receptor-mediated effects including feeding, cognition, pain and anterior pituitary hormone regulation. The role of galanin in feeding is attributed to receptor activation in both hypothalamus and amygdala. 23,35 Interestingly, recent pharmacological studies using i.c.v. administration of the reported GalR2 and GalR3 selective peptide galanin(2–29) suggest that these two receptors play little role in feeding compared to GalR1. 41 In the present study, GalR1 mRNA was highly expressed in both hypothalamus and amygdala whereas GalR2 was markedly expressed only in hypothalamus and GalR3 was present at low levels in both regions. Therefore, the relative distribution of these receptor mRNAs supports a greater role for GalR1 in the feeding response.
Fig. 3. Autoradiographs of representative solution hybridization/RNase protection assays for rat (A) GalR1, (B) GalR2, and (C) GalR3 mRNAs in central and peripheral tissues. Twenty-five micrograms of total RNA was hybridized with [ 32P]-labeled antisense RNA probes for galanin receptors and b-actin, digested with RNase A and T1 (GalR1 and GalR2), or S1 nuclease (GalR3) and run on 6% polyacrylamide, 8 M urea gels. Receptor and b-actin-specific bands were isolated from the gels, analysed in a beta scintillation counter and compared to sense standard curves run in parallel for quantitation of mRNA expression (Table 1).
Cognitive deficits induced by central galanin administration are attributed to inhibition of both acetylcholine release from basal forebrain structures projecting to cortex and hippocampus 10,14 and postsynaptic cholinergic signal
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Quantitative rat galanin receptor mRNA distribution
Fig. 4. Autoradiograph of a representative solution hybridization/RNase protection assay for rat GalR1 (1), GalR2 (2), and GalR3 (3) mRNAs in spinal cord and DRG. Ten micrograms of total RNA was hybridized with [ 32P]-labeled antisense RNA probes for galanin receptors and b-actin (b), digested with RNase A and T1 (GalR1 and GalR2), or S1 nuclease (GalR3) and run on 6% polyacrylamide, 8 M urea gels. Receptor and b-actin-specific bands were isolated from the gels, analysed in a beta scintillation counter and compared to sense standard curves run in parallel for quantitation of mRNA expression (Table 1).
transduction. 29 Further, administration of the galanin receptor antagonist, M35, enhances performance in the Morris swim test supporting galanin’s role in tonic cholinergic inhibition. 28 However, a complicating factor of using peptidic galanin receptor antagonists such as M35 is the noted partial agonist and non-selective activity of these compounds in the CNS and periphery. 20 In the present study, all three rat galanin receptor mRNAs were present in low to moderate levels in hippocampus and cortex. Therefore, more discrete localization studies in hippocampus, cortex and basal forebrain are required to determine the galanin receptor subtype or subtypes involved in cognitive deficits induced by galanin. Although galanin-like immunoreactivity and mRNA are expressed at low levels in spinal cord and DRG of naive rats, several lines of evidence suggest a role for galanin in pain transmission and nerve regeneration. Despite low endogenous peptide levels, i.t. galanin administration inhibits spinal nociception to noxious stimuli. 45 Additionally, following nerve injury, galanin peptide and mRNA are dramatically up-regulated in small DRG neurons 18 suggesting a role for galanin as an endogenous analgesic or nerve regeneration factor. 26 The change in galanin levels after nerve injury is likely mediated by the release of the cytokine, leukemia inhibitory factor, and removal of nerve growth factor which increase galanin peptide mRNA as a result of injury. 8,49 Targeted removal of GalR1 by nucleic acid antisense constructs decreases the ability of galanin to inhibit spinal reflexes providing evidence for GalR1 involvement in spinal excitability. 32 After axotomy, GalR1 and GalR2 mRNAs are
decreased in DRG neurons, 34 suggesting down-regulation of receptor induced by increased local peptide concentrations. In contrast, both galanin peptide and GalR2 mRNAs are increased in the facial nucleus after facial nerve crush. 5 This suggests a differential regulation of GalRs dependent on the site of nerve injury. We observed GalR1 as the dominant galanin receptor mRNA present in spinal cord whereas GalR2 and GalR1 were highly expressed in DRG. GalR3 was present in lower levels in both spinal cord and DRG. Based on this distribution, GalR2 may play a role in the modulation of sensory input from the periphery and GalR1 in the spinal transmission of nociceptive stimuli. The role of GalR3 in pain perception is not clear due to the relative paucity of receptor mRNA in spinal cord and DRG and thus requires further investigation. Pharmacological manipulation of specific receptor subtypes and subsequent changes in receptor mRNA and protein levels should provide further insight into the relevant GalRs involved in nociception. In the periphery, galanin is present at high levels in anterior pituitary and is associated with increasing growth hormone, leutinizing hormone and prolactin release. The effects of galanin on growth hormone and leutinizing hormone are presumably mediated by inhibition of somatostatin 7 and stimulation of gonadotropin-releasing hormone 25 release from hypothalamus, respectively. Galanin likely has a direct effect on prolactin release as application of galanin to dispersed pituitary cells results in prolactin secretion. 48 The galanin receptor or receptors which mediate the pituitary actions of galanin is unclear. GalR2 and GalR3 mRNAs were expressed in anterior pituitary whereas GalR1 mRNA was not detectable. Further, a novel galanin receptor has been postulated in anterior pituitary based on the ability of galanin(3–29) to induce prolactin 48 and decrease gonadotrophin 40 release in dispersed pituitary cells. This peptide does not show high-affinity binding to either GalR1, GalR2 or GalR3. Therefore, the complex pharmacology of galanin in anterior pituitary may be a result of activating the multiple known and potentially novel GalR isoforms expressed in this tissue. CONCLUSIONS
The diverse actions of galanin make this peptidergic system an attractive target for modulating obesity, cognitive deficits, analgesia and pituitary dysfunction. The presence of at least three G-protein-coupled receptors responsive to galanin underlines the complexity of galanin-mediated biology. The detection of GalR1 mRNA exclusively in CNS and PNS tissues implicates this receptor in the central and peripheral nervous system effects of galanin, especially in the hypothalamus, amygdala, spinal cord and DRG where high level expression was observed. The relatively widespread distribution of GalR2 and GalR3 mRNAs in rat tissues suggests involvement in both central and peripheral galanin effects. The development of selective receptor agonists and antagonists will be required for direct association of galanin action and specific receptor subtypes. This study documents the distribution of galanin receptor mRNAs that are localized to cell bodies that may project to other brain regions or tissues. Information from quantitative solution hybridization/RNase protection assays, coupled with the use of recently generated galanin receptor subtype selective antibodies, will define the precise location of receptor synthesis and galanin peptide action throughout the neuraxis.
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REFERENCES
1. Ahren B. and Lindskog S. (1992) Galanin and the regulation of islet hormone secretion. Int. J. Pancreat. 11, 147–160. 2. Bartfai T., Hokfelt T. and Langel U. (1993) Galanin—A neuroendocrine peptide. Crit. Rev. Neurobiol. 7, 229–274. 3. Bauer F. E., Ginsberg L., Venetikou M., MacKay D. J., Burrin J. M. and Bloom S. R. (1986) Growth hormone release in man induced by galanin, a new hypothalamic peptide. Lancet 2, 192–195. 4. Bloomquist B. T., Beauchamp M. R., Zhelnin L., Brown S.-E., Gore-Willse A. R., Gregor P. and Cornfield L. J. (1998) Cloning and expression of the human galanin receptor GalR2. Biochem. biophys. Res. Commun. 243, 474–479. 5. Burazin T. C. D. and Gundlach A. L. (1998) Inducible galanin and GalR2 receptor system in motor neuron injury and regeneration. J. Neurochem. 71, 879–882. 6. Burgevin M.-C., Loquet I., Quarteronet D. and Habert-Ortoli E. (1995) Cloning, pharmacological characterization, and anatomical distribution of a rat cDNA encoding for a galanin receptor. J. molec. Neurosci. 6, 33–41. 7. Chan Y. Y., Grafstein-Dunn E., Delemarre-Van De Waal H. A., Burton K. A., Clifton D. K. and Steiner R. A. (1996) The role of galanin and its receptor in the feedback regulation of growth hormone secretion. Endocrinology 137, 5303–5310. 8. Corness J., Stevens B., Douglas Fields R. and Hokfelt T. (1998) NGF and LIF both regulate galanin gene expression in primary DRG cultures. NeuroReport 9, 1533–1536. 9. Corwin R. L., Robinson J. K. and Crawley J. N. (1993) Galanin antagonists block galanin-induced feeding in the hypothalamus and amygdala of the rat. Eur. J. Neurosci. 5, 1528–1533. 10. Crawley J. N. (1996) Galanin–acetylcholine interactions: relevance to memory and Alzheimer’s disease. Life Sci. 58, 2185–2199. 11. Crawley J. N., Austin M. C., Fiske S. M., Martin B., Consolo S., Berthold M., Langel U., Fisone G. and Bartfai T. (1990) Activity of centrally administered galanin fragments on stimulation of feeding behavior and on galanin receptor binding in the rat hypothalamus. J. Neurosci 10, 3695–3700. 12. Ekblad E., Hakanson R., Sundler F. and Wahlestedt C. (1985) Galanin: neuromodulatory and direct contractile effects on smooth muscle preparations. Br. J. Pharmac. 86, 241–246. 13. Fathi Z., Cunningham A. M., Iben L. G., Battaglino P. B., Ward S. A., Nichol K. A., Pine K. A., Wang J., Goldstein M. E., Iismaa T. P. and Zimanyi I. A. (1997) Cloning, pharmacological characterization and distribution of a novel galanin receptor. Molec. Brain Res. 51, 49–59. 14. Fisone G., Wu C. F., Consolo S., Nordstrom O., Brynne N., Bartfai T., Melander T. and Hokfelt T. (1987) Galanin inhibits acetylcholine release in the ventral hippocampus of the rat: histochemical, autoradiographic, in vivo and in vitro studies. Proc. natn. Acad. Sci. U.S.A. 84, 7339–7343. 15. Gustafson E. L., Smith K. E., Durkin M. M., Gerald C. and Branchek T. A. (1996) Distribution of a rat galanin receptor mRNA in rat brain. NeuroReport 7, 953–957. 16. Habert-Ortoli E., Amiranoff B., Loquet I., Laburthe M. and Mayaux J.-F. (1994) Molecular cloning of a functional human galanin receptor. Proc. natn. Acad. Sci. U.S.A. 91, 9780–9783. 17. Hershey A. D., Dykema P. E. and Krause J. E. (1991) Organization, structure, and expression of the gene encoding the rat substance P receptor. J. biol. Chem. 266, 4366–4374. 18. Hokfelt T., Wiesenfeld-Hallin Z., Villar M. J. and Melander T. (1987) Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy. Neurosci. Lett. 83, 217–220. 19. Howard A. D., Tan C., Shiao L.-L., Palya O. C., McKee K. K., Weinberg D. H., Feighner S. D., Cascieri M. A., Smith R. G., Van Der Ploeg L. H. T. and Sullivan K. A. (1997) Molecular cloning and characterization of a new receptor for galanin. Fedn Eur. biochem. Socs Lett. 405, 285–290. 20. Kinney G. A., Emmerson P. J. and Miller R. J. (1998) Galanin receptor-mediated inhibition of glutamate release in the arcuate nucleus of the hypothalamus. J. Neurosci. 18, 3489–3500. 21. Kolakowski L. F. Jr., O’Neill G. P., Howard A. D., Broussard S. R., Sullivan K. A., Feighner S. D., Sawzdargo M., Nguyen T., Kargman S., Shiao L.-L., Hreniuk D. L., Tan C. P., Evans J., Abramovitz M., Chateauneuf A., Coulombe N., Ng G., Johnson M. P., Tharian A., Khoshbouei H., George S. R., Smith R. G. and O’Dowd B. F. (1998) Molecular characterization and expression of cloned human galanin receptors GALR2 and GALR3. J. Neurochem. 71, 2239–2251. 22. Koshiyama H., Shimatsu A., Kato Y., Assadian H., Hattori N., Ishikawa Y., Tanoh T., Yanaihara N. and Imura H. (1990) Galanin-induced prolactin release in rats: pharmacological evidence for the involvement of alpha-adrenergic and opioidergic mechanisms. Brain Res. 507, 321–324. 23. Kyrkouli S. E., Stanley B. G., Seirafi R. D. and Leibowitz S. F. (1990) Stimulation of feeding by galanin: anatomical localization and behavioral specificity of this peptide’s effects in brain. Peptides 11, 995–1001. 24. Leibowitz S. F. and Kim T. (1992) Impact of galanin antagonist on exogenous galanin and natural patterns of fat ingestion. Brain Res. 599, 148–152. 25. Lopez F. J., Merchenthaler I., Ching M., Wisniewski M. G. and Negro-Vilar A. (1991) Galanin: a hypothalamic-hypophysiotrophic hormone modulating reproductive functions. Proc. natn. Acad. Sci. U.S.A. 88, 4508–4512. 26. Mahoney R. P., Siegel R. and Zigmond R. E. (1994) Galanin and vasoactive intestinal peptide messenger RNA expression increase following axotomy of the adult rat SCG. J. Neurobiol. 25, 108–118. 27. Merchenthaler I., Lopez F. J. and Negro-Vilar A. (1993) Anatomy and physiology of central galanin-containing pathways. Prog. Neurobiol. 40, 711–769. 28. Ogren S. O., Hokfelt T., Kask K., Langel U. and Bartfai T. (1992) Evidence for a role of the neuropeptide galanin in spatial learning. Neuroscience 51, 1–5. 29. Palazzi E., Felinska S., Zambelli M., Fisone G., Bartfai T. and Consolo S. (1991) Galanin reduces carbachol stimulation of phosphoinositide turnover in rat ventral hippocampus by lowering Ca 21 influx through voltage sensitive Ca 21 channels. J. Neurochem. 56, 739–747. 30. Pang L., Hashemi T., Lee H.-J. J., Maguire M., Graziano M. P., Bayne M., Hawes B., Wong G. and Wang S. (1998) The mouse GalR2 galanin receptor: genomic organization, cDNA cloning, and functional characterization. J. Neurochem. 71, 2252–2259. 31. Parker E. M., Izzarelli D. G., Nowak H. P., Mahle C. D., Iben L. G., Wang J. and Goldstein M. E. (1995) Cloning and characterization of the rat GALR1 galanin receptor from Rin14B insulinoma cells. Molec. Brain Res. 34, 179–189. 32. Pooga M., Soomets U., Hallbrink M., Valkna A., Saar K., Rezaei K., Kahl U., Hao J.-X., Xu X.-J., Wiesenfeld-Hallin Z., Hokfelt T., Bartfai T. and Langel U. (1998) Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo. Nature Biotech. 16, 857–861. 33. Reimann W., Engelberger W., Friderichs E., Selve N. and Wilffert B. (1994) Spinal antinociception by morphine in rats is antagonised by galanin receptor antagonists. Naunyn-Schmiedeberg’s Arch. Pharmac. 350, 380–386. 34. Shi T.-J. S., Zhang X., Holmberg K., Xu Z.-Q. D. and Hokfelt T. (1997) Expression and regulation of galanin-R2 receptors in rat primary sensory neurons: effect of axotomy and inflammation. Neurosci. Lett. 237, 57–60. 35. Smith B. K., York D. A. and Bray G. A. (1996) Effects of dietary preference and galanin administration in the paraventricular or amygdaloid nucleus on diet self-selection. Brain Res. Bull. 39, 149–154. 36. Smith K. E., Forray C., Walker M. W., Jones K. A., Tamm J. A., Bard J., Branchek T. A., Linemeyer D. L. and Gerald C. (1997) Expression cloning of a rat hypothalamic galanin receptor coupled to phosphoinositide turnover. J. biol. Chem. 272, 24,612–24,616. 37. Smith K. E., Walker M. W., Artmyshyn R., Bard J., Borowsky B., Tamm J. A., Yao W.-J., Vaysse P. J.-J., Branchek T. A., Gerald C. and Jones K. A. (1998) Cloned human and rat galanin GALR3 receptors: pharmacology and activation of G-protein inwardly rectifying K 1 channels. J. biol. Chem. 273, 23,321–23,326. 38. Sullivan K. A., Shiao L.-L. and Cascieri M. A. (1997) Pharmacological characterization and tissue distribution of the human and rat GALR1 receptors. Biochem. biophys. Res. Commun. 233, 823–828.
Quantitative rat galanin receptor mRNA distribution
271
39. Tatemoto K., Rokaeus A., Jornwall H., McDonald T. J. and Mutt V. (1983) Galanin—A novel biologically active peptide from porcine intestine. Fedn Eur. biochem. Socs Lett. 164, 124–128. 40. Todd J. F., Small C. J., Akinsanya K. O., Stanley S. A., Smith D. M. and Bloom S. R. (1998) Galanin is a paracrine inhibitor of gonadotroph function in the female rat. Endocrinology 139, 4222–4229. 41. Wang S., Ghibaudi L., Hashemi T., He C., Strader C., Bayne M., Davis H. and Hwa J. J. (1998) The GalR2 galanin receptor mediates galanin-induced jejunal contraction, but not feeding behavior, in the rat: differentiation of central and peripheral effects of receptor subtype activation. Fedn Eur. biochem. Socs Lett. 434, 277–282. 42. Wang S., Hashemi T., He C., Strader C. and Bayne M. (1997) Molecular cloning and pharmacological characterization of a new galanin receptor subtype. Molec. Pharmac. 52, 337–343. 43. Wang S., He C., Hashemi T. and Bayne M. (1997) Cloning and expressional characterization of a novel galanin receptor: identification of different pharmacophores within galanin for the three galanin receptor subtypes. J. biol. Chem. 272, 31,949–31,952. 44. Wang S., He C., Maguire M. T., Clemmons A. L., Burrier R. E., Guzzi M. F., Strader C. D., Parker E. M. and Bayne M. L. (1997) Genomic organization and functional characterization of the mouse GalR1 galanin receptor. Fedn Eur. biochem. Socs Lett. 411, 225–230. 45. Wiesenfeld-Hallin Z., Xu X.-J., Hao J.-X. and Hokfelt T. (1993) The behavioural effects of intrathecal galanin on tests of thermal and mechanical nociception in the rat. Acta physiol. scand. 147, 457–458. 46. Wiesenfeld-Hallin Z., Xu X. J., Hakanson R., Feng D. M., Folkers K., Kristensson K., Villar M. J., Fahrenkrug J. and Hokfelt T. (1991) On the role of substance P, galanin, vasoactive intestinal peptide, and calcitonin gene-related peptide in mediation of spinal reflex excitability in rats with intact and sectioned peripheral nerves. Ann. N.Y. Acad. Sci. 632, 198–211. 47. Wynick D., Small C. J., Bacon A., Holmes F. E., Norman M., Ormandy C. J., Kilic E., Kerr N. C. H., Ghatei M., Talamantes F., Bloom S. R. and Pachnis V. (1998) Galanin regulates prolactin release and lactotroph proliferation. Proc. natn. Acad. Sci. U.S.A. 95, 12,671–12,676. 48. Wynick D., Smith D. M., Ghatei M., Akinsanya K., Bhogal R., Purkiss P., Byfield P., Yanaihara N. and Bloom S. R. (1993) Characterization of a highaffinity galanin receptor in the rat anterior pituitary: absence of biological effect and reduced membrane binding of the antagonist M15 differentiate it from the brain/gut receptor. Proc. natn. Acad. Sci. U.S.A. 90, 4231–4235. 49. Zigmond R. E. (1997) LIF, NGF, and the cell body response to axotomy. Neuroscientist 3, 176–185. (Accepted 2 August 1999)