Scholarly article on topic 'Effects of norepinephrine on galanin expression in dorsal root ganglion neurons in vitro'

Effects of norepinephrine on galanin expression in dorsal root ganglion neurons in vitro Academic research paper on "Biological sciences"

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{norepinephrine / adrenoreceptor / galanin / "dorsal root ganglion"}

Abstract of research paper on Biological sciences, author of scientific article — Xiangdong Yang, Zhen Liu, Zhenzhong Li

Abstract Background: Norepinephrine (NE) is a key neurotransmitter that functionally activates adrenoreceptors expressed in sympathetic neurons. Functional α1-adrenoreceptors are also expressed in dorsal root ganglion (DRG) primary sensory neurons and regulate neurogenic inflammation and nociceptive responses. Galanin is involved in inflammation and nociception. It has been suggested that galanin receptor (GalR) 1 and GalR3 activation induces analgesia at the level of the spinal cord, while activation of GalR2 has a pronociceptive role in the periphery. Whether activation or inhibition of α-adrenoreceptors influences galanin expression remains unknown. Objective: The aim of the present study was to investigate whether the α-adrenoreceptor agonist NE, the α1-adrenoreceptor antagonist prazosin, and the α2-adrenoreceptor antagonist yohimbine affect galanin expression in primary cultured DRG neurons. Methods: DRG was dissected from 240 embryonic 15-day-old Wistar rats, cultured as dissociated cells for 2 days, and then exposed to NE (10-4 mol/L) for another 4 days. In the NE + prazosin group and the NE + yohimbine group, DRG neurons were pretreated with prazosin (10-6 mol/L) and yohimbine (10-5 mol/L), respectively, 10 minutes prior to the NE challenge. The neurons cultured continuously in media served as the controls. All of the cultured samples were processed to detect galanin mRNA and galanin peptide expression by reverse transcriptase-polymerase chain reaction and Western blot, respectively. Five samples were tested for each procedure. Results: Forty samples were prepared for this study and included in the analysis. After 4 days of incubation, mean (SD) galanin mRNA/β-actin mRNA concentration ratio was significantly increased with NE compared with controls (0.3349 [0.0413] vs 0.2411 [0.0519]; P < 0.05). Pretreatment with prazosin seemed to block the effects of NE (0.2522 [0.0496]; P < 0.05 vs NE), while yohimbine did not appear to significantly alter the effects of NE on elevation of galanin mRNA/β-actin mRNA concentration (0.3154 [0.0239]; P < 0.05 vs controls). After 4 days of incubation, galanin/β-actin concentration ratio was significantly higher with NE compared with controls (0.4406 [0.0655] vs 0.2295 [0.0794]; P < 0.01). Pretreatment with prazosin appeared to inhibit NE-induced galanin peptide expression (0.3156 [0.0942]; P < 0.05 vs NE), while yohimbine did not appear to alter the effects of NE on elevation of galanin peptide concentration (0.3700 [0.0533]; P < 0.05 vs controls). Coclusions: In this small in vitro study, NE, likely due to action on α1-adrenoreceptors but not α2-adrenoreceptors, was associated with an increase in galanin mRNA concentration and galanin peptide expression in these DRG neurons. These findings might be relevant to noradrenergic pain modulation.

Academic research paper on topic "Effects of norepinephrine on galanin expression in dorsal root ganglion neurons in vitro"

Current Therapeutic Research

Volume 70, Number i, February 2009

Effects of Norepinephrine on Galanin Expression in Dorsal Root Ganglion Neurons in Vitro

Xiangdong Yang, MD1; Zhen Lin, MD, PhD2; and Zhenzhong Li, MD, PhD2

1 Department of Nephrology, Shandong University Qilu Hospital, Jinan, People's Republic of China; and 2 Department of Anatomy, Shandong University School of Medicine, Jinan, People's Republic of China

ABSTRACT

Background: Norepinephrine (NE) is a key neurotransmitter that functionally activates adrenoreceptors expressed in sympathetic neurons. Functional o^-adrenoreceptors are also expressed in dorsal root ganglion (DRG) primary sensory neurons and regulate neurogenic inflammation and nociceptive responses. Galanin is involved in inflammation and nociception. It has been suggested that galanin receptor (GalR) 1 and GalR3 activation induces analgesia at the level of the spinal cord, while activation of GalR2 has a pronociceptive role in the periphery. Whether activation or inhibition of a-adrenoreceptors influences galanin expression remains unknown.

Objective: The aim of the present study was to investigate whether the a-adrenoreceptor agonist NE, the o^-adrenoreceptor antagonist prazosin, and the a2-adrenoreceptor antagonist yohimbine affect galanin expression in primary cultured DRG neurons.

Methods: DRG was dissected from 240 embryonic 15-day-old Wistar rats, cultured as dissociated cells for 2 days, and then exposed to NE (10~4 mol/L) for another 4 days. In the NE + prazosin group and the NE + yohimbine group, DRG neurons were pretreated with prazosin (10~6 mol/L) and yohimbine (10~5 mol/L), respectively, 10 minutes prior to the NE challenge. The neurons cultured continuously in media served as the controls. All of the cultured samples were processed to detect galanin mRNA and galanin peptide expression by reverse transcriptase-polymerase chain reaction and Western blot, respectively. Five samples were tested for each procedure.

results: Forty samples were prepared for this study and included in the analysis. After 4 days of incubation, mean (SD) galanin mRNA/P-actin mRNA concentration ratio was significantly increased with NE compared with controls (0.3349 [0.0413] vs 0.2411 [0.0519]; P < 0.05). Pretreatment with prazosin seemed to block the effects of NE (0.2522 [0.0496]; P < 0.05 vs NE), while yohimbine did not appear to significantly alter the effects of NE on elevation of galanin mRNA/P-actin mRNA concentration (0.3154 [0.0239]; P < 0.05 vs controls). After 4 days of incubation, galanin/P-actin concentration ratio was significantly higher with NE compared with controls (0.4406 [0.0655] vs 0.2295 [0.0794]; P < 0.01). Pretreatment with prazosin appeared to in-

Acceptedforpublication December 22, 2008. doi: 10.1016/j.curtheres.2009.02.002

© 2009 Excerpta Medica Inc. All rights reserved. 0011-393X/$ - see front matter

hibir NE-induced galanin peptide expression (0.3156 [0.0942]; P < 0.05 vs NE), while yohimbine did nor appear ro alrer rhe effects of NE on elevation of galanin peptide concentration (0.3700 [0.0533]; P < 0.05 vs controls).

Conclusions: In this small in vitro study, NE, likely due to action on o^-adrenoreceptors but not a2-adrenoreceptors, was associated with an increase in galanin mRNA concentration and galanin peptide expression in these DRG neurons. These findings might be relevant to noradrenergic pain modulation. (Curr Ther Res Clin Exp. 2009;70:19-28) © 2009 Excerpta Medica Inc.

key words: norepinephrine, adrenoreceptor, galanin, dorsal root ganglion.

INTRODUCTION

The noradrenergic system is subject to plastic changes that influence its antinociceptive efficacy after injury or inflammation. Norepinephrine (NE) is a key neurotransmitter released by sympathetic postganglionic nerve fibers and is crucial in noradrenergic pain modulation.1 NE functionally activates adrenoceptors expressed in sympathetic neurons in a variety of physiologic and pathophysiologic conditions. Functional a-adrenoreceptors are expressed in primary sensory neurons and regulate neurogenic inflammation and nociceptive responses.2-4 The a-adrenoreceptors have been found to mediate depolarization in cultured rat dorsal root ganglion (DRG) neurons5; a2-adrenoreceptors are also expressed in DRG neurons.6-8 In a study of transgenic mice lacking functional a2A-adrenoreceptors, the a2-adrenoreceptor antagonist was associated with attenuated nerve injury—induced heat hyperalgesia, suggesting that it might facilitate the mediation of NE-induced pain.9 In other studies, a-adrenoreceptor expression in DRG neurons changed after peripheral nerve injury or inflammation.7,8,10 In 1 study,7 a2A-adrenoceptor mRNA increased to 45% in all DRG neuron profiles after unilateral peripheral nerve injury. In another study,8 excitatory a2-adrenoreceptors on peripheral nociceptors and their DRG cell bodies were observed after nerve injury. In the last study,10 an increase in the mRNAs for a1B-, a2A-, and a2D-adrenoreceptors was found with the ipsilateral DRG after sciatic nerve injury. The a-adrenoreceptors have a key role in mediating the regulatory effects of NE on pain.1 The expression of different subtypes of a-adrenoreceptors increased after nerve injury.6,11 These adrenoceptors that are functionally active may vary with the presence of nerve injury, inflammation, or other physiologic or pathophysiologic conditions.6,8,11

Galanin expression was reported to be regulated by NE in the magnocellular neurons of supraoptic nuclei in an ex vivo acute model of rat hypothalamic slices.12 Some freshly dissociated and cultured DRG neurons from adjuvant inflamed rats were found to have a small depolarization in response to NE.13 Galanin, a 29—amino acid neuropeptide in most species (a 30—amino acid neuropeptide in humans), is widely distributed throughout the nervous system, including the DRG neurons, and is involved in the regulation of various functions, including nociception and developmental and trophic effects.14-19 It has been suggested that galanin receptor (GalR) 1 and GalR3 activation induces analgesia at the spinal cord level, while activation of

GalR2 has a pronociceptive role in the periphery.16,20,21 Whether activation or inhibition of a-adrenoreceptors influences galanin expression remains unknown.

In the present study, we investigated whether the a-adrenoreceptor agonist NE, the a-adrenoreceptor antagonist prazosin, and the a2-adrenoreceptor antagonist yohimbine affect galanin expression in primary cultured DRG neurons.

MATERIALS AND METHODS

study design

Dorsal Root Ganglion Cell Culture Preparations

DRG was dissected from 240 embryonic 15-day-old Wistar rats. The animals were obtained from the Experimental Animal Center of Shandong University (Jinan, People's Republic of China). Forty samples were prepared for all experiments. Before establishment in the culture, DRG was digested with 0.25% trypsin (Sigma-Aldrich, St. Louis, Missouri) in D-Hanks solution at 37°C for 10 minutes and centrifuged for 5 minutes at 1 x 103 rpm. The supernatants were removed and the pellets were resuspended in Dulbecco's Modified Eagle Medium with F-12 supplement (DMEM/F-12) media (Grand Island Biological Company [GIBCO], Invitrogen, Carlsbad, California) and triturated using a sterile modified Pasteur's glass pipette. Cells were filtered using a 130-pm filter and counted. Dissociated DRG cells were cultured in flasks (Costar, Corning Incorporated, Corning, New York) to detect the expression of galanin mRNA by reverse transcriptase—polymerase chain reaction (RT-PCR) and galanin peptide by Western blot. The flasks were precoated with poly-L-lysine prior to plating the DRG cells. DRG cells were plated at a density of 5 x 105 cells/mL in flasks. DRG cells were then incubated in culture media at 37°C with 5% carbon dioxide for 24 hours and then maintained in culture media containing cytarabine (5 pg/mL) for another 24 hours to inhibit growth of nonneuronal cells. They were then cultured in culture media for another 4 days, with the media being changed every 2 days. The composition of the culture media was DMEM/F-12 (1:1) supplemented with 5% fetal bovine serum, 2% B-27 supplement (GIBCO), insulin (0.25 jig/mL, Sigma-Aldrich), L-glutamine (0.1 mg/mL, Sigma-Aldrich), penicillin (100 U/mL), and streptomycin (100 pg/mL).

Exposure of Dorsal Root Ganglion neurons to Norepinephrine

DRG cell cultures were prepared as described earlier and allowed to grow for 2 days, after which NE (10~4 mol/L) was added. Cultures were incubated for an additional 4 days, with the media being changed every 2 days. The culture media contained NE (10~4 mol/L) during the 4 days of incubation. In the NE + prazosin group and the NE + yohimbine group, DRG neurons were pretreated with prazosin (10-6 mol/L) and yohimbine (10~5 mol/L), respectively, 10 minutes before the NE challenge. The neurons cultured continuously in media served as the controls. Five samples were tested for each procedure.

MRNA Extraction and RT-PCR

The galanin mRNA concentration was analyzed using RT-PCR after the 4-day incubation period in the different agents. The expression of P-actin was also deter-

mined as an internal control. Total DRG cell RNA in each flask was isolated using TRIzol® (Invitrogen) reagent. cDNA synthesis was performed using Moloney murine leukemia virus RT. The gene-specific primers were synthesized using the published cDNA sequences for galanin and P-actin.22 The synthetic oligonucleotide primer sequences for galanin were 5'-ATG CCA ACA AAG GAG AAG AG-3' (upper primer) and 5'-AGG TGC AAG AAA CTG AGA AA-3' (lower primer), and for P-actin they were 5 '-ATC ATG TTT GAG ACC TTC AAC-3' (upper primer) and 5'-CAT CTC TTG CTC GAA GTC CA-3' (lower primer).

The predicted size of the amplified galanin and P-actin DNA products were 224 and 317 bp, respectively.

PCR amplification was performed for 35 cycles. The cycle profile included dena-turation for 45 seconds at 94°C, annealing for 60 seconds at 53°C, and extension for 45 seconds at 72°C. PCR was performed within the range that demonstrated a linear correlation between the amount of cDNA and the yield of PCR products.

The amplified products were analyzed using 1.5% standard agarose gel electrophoresis and stained with ethidium bromide; they were then visualized using an ultraviolet transilluminator and photographed. DNA marker DL2000 was used as a molecular standard. The photographs were scanned and the electrophoresis gel images were analyzed quantitatively using ImageJ 1.39u image analysis software (National Institutes of Health, Bethesda, Maryland). Galanin mRNA concentration was expressed as the ratio of the gene to P-actin.

western blot analysis for galanin peptide expression

Galanin peptide expression was analyzed using Western blot. After 4 days of treatment with the different agents, fresh cultured DRG neurons were homogenized in 10 mmol/L Tris homogenization buffer (pH 7.4) with protease inhibitors (Sigma-Aldrich). The samples were centrifuged at 12,000 rpm (4°C) for 20 minutes and the supernatant was then collected for analysis. After determining the protein concentrations of the supernatants (bicinchoninic acid method and bovine serum albumin [standard]), 50 jig of protein from each sample was loaded onto the 8% sodium dodecyl sulfate gel, separated by electrophoresis, and transferred to polyvinylidene fluoride membrane. The membranes were blocked in blocking buffer (5% nonfat milk) for 2 hours at room temperature (20°C) and were incubated with goat antirat galanin polyclonal immunoglobulin G (IgG) (1:1000, Santa Cruz Biotechnology [SCB], Inc., Santa Cruz, California) or mouse antirat P-actin monoclonal IgG (1:1000, SCB, Inc.) overnight at 4°C. After being washed 3 times for 10 minutes with washing solution, the membranes were incubated with donkey antigoat IgG-horseradish peroxidase (HRP) (1:2000, SCB, Inc.) or goat antimouse IgG-HRP (1:2000, SCB, Inc.) for 2 hours. After being washed 3 times for 10 minutes with washing solution, the immunoreactive bands were visualized using an enhanced chemiluminescent Western blotting detection kit (Pierce Biotechnology, a division of Thermo Fisher Scientific, Rockford, Illinois) on light-sensitive film. The film was scanned and the images were analyzed quantitatively using ImageJ 1.39u image analysis software. Galanin peptide concentration was expressed as the ratio of the protein to P-actin.

Statistical Analysis

The ratio of galanin mRNA to P-actin mRNA and the ratio of galanin to P-actin protein in each group were compared. One-way analysis of variance (ANOVA) was used according to the 1 influence factor and test of normality. Data are expressed as mean (SD). Statistical analyses were performed using SPSS software version 13.0 (SPSS Inc., Chicago, Illinois) by 1-way ANOVA followed by the Student-Newman-Keuls test for significance to compare the between-group differences. P < 0.05 was considered statistically significant.

RESULTS

sample size

Forty samples were prepared for the experiments and were all included in the analysis.

galanin mrna expression

After 4 days of incubation, the galanin mRNA/P-actin mRNA concentration ratio was found to be significantly increased with NE compared with controls (mean [SD], 0.3349 to.0413] vs 0.2411 [0.0519]; P < 0.05). Pretreatment with prazosin appeared to block the effects of NE (0.2522 [0.0496]; P < 0.05 vs NE), while yohimbine was not associated with significantly altered effects of NE on the increase in galanin mRNA/P-actin mRNA ratio (0.3154 [0.0239]; P < 0.05 vs controls) (Figure 1).

galanin peptide expression

After 4 days of incubation, the galanin/P-actin concentration ratio was significantly increased with NE compared with controls (mean [SD], 0.4406 [0.0655] vs 0.2295 [0.0794]; P < 0.01). Pretreatment with prazosin appeared to inhibit NE-induced galanin peptide expression (0.3156 [0.0942]; P < 0.05 vs NE), while yohimbine did not appear to alter the effects of NE on elevation of galanin peptide expression (0.3700 [0.0533]; P < 0.05 vs controls) (Figure 2).

DISCUSSION

NE, a key neurotransmitter released by sympathetic postganglionic nerve fibers, is crucial in noradrenergic pain modulation.1 The physiologic and pathophysiologic aspects of the effects of NE have been extensively studied.23-25 Based on a literature search, reports addressing the changes caused by NE in galanin mRNA and galanin peptide concentration in cultured DRG neurons have not been identified. In the present study, the findings suggest that exposure to NE promoted galanin mRNA and galanin peptide expression in cultured embryonic DRG neurons and that pretreatment with the o^-adrenoreceptor antagonist prazosin inhibited NE-induced galanin peptide expression, whereas the a2-adrenoreceptor antagonist yohimbine did not affect galanin expression.

Galanin might play a role in the adaptive response of the peripheral nervous system to injury and may modulate pain transmission.26 Galanin is normally expressed at low concentrations in sensory neurons in adult DRG27 and is markedly

''S 0.35-

Ç 0.259 0.20-

I 0.15 H

224 bp

NE + Prazosin

NE + Yohimbine

Normal Control

Galanin

317 bp

ß-Actin

Figure 1. Effects of norepinephrine (NE) on galanin mRNA expression in primary cultured dorsal root ganglion neurons: (A) graph and (B) reverse transcriptase-polymerase chain reaction findings (Lane 1: normal control [mean (SD), galanin mRNA/p-actin mRNA ratio = 0.2411 (0.0519)]; Lane 2: NE [0.3349 (0.0413)]; Lane 3: NE + prazosin [0.2522 (0.0496)]; and Lane 4: NE + yohimbine [0.3154 (0.0239)]). *P < 0.05 versus controls; tP < 0.05 versus NE.

upregulated within DRG neurons after peripheral nerve injury or inflammation in adults.!5.26,28-33 Furthermore, galanin is recognized as one of the markers of DRG injury.34 DRG neurons in vitro have a phenotype similar to that of DRG neurons after axotomy (ie, a phenotype distinctly different from normal DRG neurons).35 Both galanin mRNA and galanin peptide were expressed to some extent in primary cultured DRG neurons in the present study. NE appeared to induce an increase in the expression of galanin mRNA and the neuropeptide in primary cultured DRG neurons.

NE activates adrenoceptors to modulate noradrenergic pain. Both and a9-adrenoreceptors have been identified in DRG neurons. A facilitatory role of peripheral pain modulation mediated by (Xj-adrenoreceptors has been found in other

_CÜ CD

0.60.50.40.30.2-

ro 0.1-

NE + Prazosin

NE + Yohimbine

Normal Control

13 kDa

43 kDa

Galanin

ß-Actin

Figure 2. Effects of norephinephrine (NE) on galanin peptide expression in primary cultured dorsal root ganglion neurons: (A) graph and (B) Western blot findings (Lane 1: normal control [galanin/p-actin = 0.2295 (0.0794)]; Lane 2: NE [0.4406 (0.0655)]; Lane 3: NE + prazosin [0.3156 (0.0942)]; and Lane 4: NE + yohimbine [0.3700 (0.0533)]). *P < 0.01 versus control; tp < 0.05 versus NE; +P < 0.05 versus control.

studies. For example, sympathetic efferents acting on peripheral a ^adrenoceptors facilitate triggering of a dorsal root reflex in nociceptive nerve fibers, suggesting that NE acting on peripheral o^-adrenoreceptors predominantly facilitates nociceptors.36 In addition, a whole cell patch clamp study found that the a ^adrenoceptor agonist phenylephrine increased excitability of cultured DRG neurons.5 However, evidence has accumulated that a9-adrenoreceptors have both a facilitatory and an inhibitory role in peripheral pain modulation. In a study of chronic inflammation-induced NE sensitivity, NE-induced hypersensitivity was attenuated by an a9-adrenoreceptor antagonist, suggesting that a9-adrenoreceptors may induce pain in hypersensitivity.37

Perineural administration of an a2-adrenoreceptor agonist attenuated axotomy-induced hyperexcitability of DRG neurons, suggesting a pain-inhibitory role of peripheral a2-adrenoreceptors.38 Whether a2-adrenoreceptors in the periphery contribute to the aggravation of pain or to the suppression of pain is still a matter of debate. In the present study, the o^-adrenoreceptor antagonist prazosin appeared to significantly inhibit NE-induced galanin expression, whereas the a2-adrenoreceptor antagonist yohimbine did not appear to have this effect. These results suggested that o^-adrenoreceptors, but not a2-adrenoreceptors, are involved in NE-induced galanin expression. Galanin expression might participate in the pain facilitatory role of o^-adrenoreceptors, whereas the pain facilitatory or inhibitory role of a2-adrenoreceptors might not be mediated by alterations in galanin expression.

NE has little influence on pain in healthy tissue, whereas the noradrenergic system is subject to various plastic changes that influence its antinociceptive efficacy after injury or inflammation.1 The findings in the present study in cultured embryonic DRG neuronal cells cannot be extrapolated to the regulation of galanin via a ^adrenoceptors after NE treatment in clinical practice. We speculate that NE might have little influence on galanin expression in normal adult DRG neurons in vivo, but the influence of NE on galanin expression in injury or inflammatory conditions needs to be further clarified.

limitations

This study had some limitations, including the small sample size and the lack of blinding of the study investigators.

CONCLUSIONS

In this small in vitro study, NE, likely due to activity on a ^adrenoceptors but not a2-adrenoreceptors, was associated with an increase in galanin mRNA concentration and galanin peptide expression in these DRG neurons. These findings might be relevant to noradrenergic pain modulation.

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Address correspondence to: Zhenzhong Li, MD, PhD, Department of Anatomy, Shandong University School of Medicine, Jinan 250012, People's Republic of China. E-mail: zli@sdu.edu.cn