Nitric oxide (NO) regulates a wide range of physiological processes. Recent studies show that NO can regulate the release of catecholamines (CA) from the adrenal medulla. In the current study, the PC12 cell line was used to examine the effect of NO on the regulation of the CA biosynthetic enzymes: tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH) and phenylethanolamine Nmethyltransferase (PNMT). Treatment of PC12 cells with the NO donor, sodium nitroprusside (SNP) for 6 hours significantly increased TH, DBH and PNMT mRNA levels. In addition, NO potentiates the regulation of gene expression of all three CA biosynthetic enzymes by glucocorticoids and cholinergic agonists. The signaling pathways involved in NO regulation of CA biosynthetic enzymes were investigated with the use of specific kinase activators and inhibitors, with results supporting a contributing role of PKA, PKC and PKG in SNP-mediated induction for all three CA genes (p < 0.01). In addition, inhibitors of transcription and translation inhibited SNP-mediated induction of all three genes (p < 0.001) suggesting that both transcriptional and translational mechanisms may be involved in CA gene regulation by NO. Results from this study show that in addition to regulating CA biosynthetic enzymes, NO can also potentiate cholinergic and glucocorticoid activation of CA genes.
Nitric oxide (NO) is a volatile gas that has been associated with a wide range of physiological processes. First identified as an endothelial derived factor (EDRF) involved in blood vessel relaxation, NO has recently been recognized as an important intercellular messenger in the central and peripheral nervous systems, involved in events such as neurotransmitter release, long-term potentiation and gene transcription [
The complete L-arginine/NO/cGMP pathway exists in the adrenal medulla, a tissue that is critically important in the regulation, synthesis and release of catecholamines (CA) during stress response [
In-vivo, CA synthesis is mediated by hormonal regulation by the hypothalamic-pituitary-adrenal (HPA) axis via secretion of glucocorticoids and by neural regulation via the sympatho-adrenal (SA) system where the splanchnic nerve synthesizes and secretes a number of neurotransmitters, including acetylcholine [
The current study provides evidence that NO is capable of regulating genes encoding for the CA biosynthetic enzymes in the PC12 cell culture model. Furthermore, results demonstrate that NO is also capable of potentiating both neural and hormonal regulation of CA biosynthetic enzymes.
PC12 cells (Dr. O’Connor, UCSD, San Diego, CA, USA) were cultured in Dulbeco’s modified Eagle’s medium (DMEM) (Hyclone, Thermo Fisher Scientific, Nepean, ON, Canada) supplemented with 5% equine serum, 5% bovine calf serum (Hyclone Inc., Logan, UT, USA) and gentamycin sulphate (50 μg/mL) (Fisher Scientific, Ottawa, ON, Canada). Cells were maintained in a humidified incubator at 37˚C in an atmosphere of 5% CO2 - 95% air and grown to 80% - 90% confluency before being passed or used in an experiment. Prior to experimentation, cells were washed twice with PBS, trypsinized, and transferred to DMEM containing charcoal-treated serum. For RNA extraction, cells were seeded in 60 mm culture plates at a density of 1.6 × 106 cells/mL. Following seeding, cells were allowed to adhere to plates for 16 - 24 h prior to beginning experiments.
PC12 cells were treated with the NO donor sodium nitroprusside (SNP) to assess the effects of NO on the regulation of the CA biosynthetic enzymes. To determine the impact of SNP on cholinergic regulation of CA enzymes, cells were treated with a combination of SNP with either 100 μM nicotine (Nic), muscarine (Mus) or carbamylcholine (CCh). To determine whether SNP potentiates the hormonal activation of CA enzymes, cells were treated with SNP in combination with 1 μM dexamethasone (DEX). The effects of NO on intracellular kinase activators of CA enzymes was investigated by treating cells with 10 μM forskolin (Fn) (PKA activator), 80 nM PMA (PKC activator) and 100 μM 8-Br-cGMP (cGMP-PKG activator) in combination with SNP. In subsequent experiments, cells pre-treated with the inhibitors for PKA (30 μM H-89), PKC (100 nM GF109203X), cGMP (200 nM A6563) and PKG (100 nM DT-2) for 30 min prior to treatments with SNP. To assess whether the mRNA induction caused by SNP could be attenuated by inhibitors of transcription and translation, cells were pre-treated with 50 nM actinomycin-D (actino-D) and 1 μM cycloheximide (cyclohex) respectively and a combination of each inhibitor with SNP. All reagents were purchased from Sigma Aldrich (Oakville, ON, Canada).
Total RNA was isolated from PC12 cells using TRI Reagent (Sigma Aldrich Canada) according to the manufacturer’s instructions. Total RNA pellets were resuspended in diethylpyrocarbonate-treated water and stored at −80˚C. Prior to cDNA synthesis, 2 μg of total RNA was subjected to deoxyribonuclease I treatment (Sigma Aldrich Canada) to eliminate possible genomic DNA contamination. cDNA synthesis was performed using 100 U Revert Aid Moloney Murine Leukemia Virus reverse transcriptase (Fermentas, Burlington, ON), 0.5 μg/μL of random hexamers, 200 μmol/μL dNTPs in a total reaction volume of 35 μL. In selected tubes the reverse transcriptase was omitted as a control of amplification from contaminating cDNA or genomic DNA. The reaction was carried out at 42˚C for 60 min and was terminated at 70˚C for 10 min in a MG Mini Personal Thermocycler from Biorad (Hercules, CA, USA).
PCR was performed in 25 μL reaction volumes containing 78 ng of cDNA, using 50 U GoTaq Flexi DNA polymerase (Promega) containing 200 μM of dNTPs, 1.5 mM MgCl2 and 25 ng of forward and reverse primer sequences specific for the following genes: tyrosine hydroxylase (TH) (5’-GCGACAGAGTCTCATCGAGGAT-3’ and 5’-AAGAGCAGGTTGAGAACAGCATT-3’ for 20 cycles at 52˚C), dopamine-β-hydroxylase (DBH) (5’-GACAGGACCTACTTTGCGAC-3’ and 5’-AGCTGTGTAGTGTAGACGGATGC-3’ for 27 cycles at 58˚C), phenylethanolamine-N-methyltransferase (PNMT) (5’-CAGACTTCTTGGAGGTCAACCG-3’ and 5’- AGCAGCGTCGTGATATGATAC-3’ for 35 cycles at 58˚C) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (5’-ATGGTGGTGCTGAGTATGTCG-3’ and 5’-CATGTCAGATCCACAACGGATAC-3’ for 21 cycles at 58˚C). PCR products (10 μL) were electrophoresed on a 1.5% agarose gel in 40 mM Tris-acetate and 2 mM EDTA buffer, pH 8.0; stained with ethidium bromide (EtBr); and documented using Chemidoc XRS (Biorad) imaging system and densitometric analysis performed with Quantity One software (Biorad).
Total protein was isolated and extracted from PC12 cells by the addition of RIPA lysis buffer (25 mM Tris HCl, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 0.5 mM PMSF, protease inhibitors, pH 7.6). Cell lysates were incubated on ice for 10 min prior to centrifugation at 12000 × g for 20 min at 4˚C, supernatants collected and stored at −80˚C until use. Protein concentrations of the extracts were determined by the Bradford method as previously described [
Protein samples (50 μg) were prepared using RIPA lysis buffer with the addition of 6x loading dye (300 mM Tris HCl, 12% SDS, 12 mM EDTA, 6% β-mercaptoethanol, 60% glycerol, 6% bromophenol blue, pH 6.8) and denatured for 2 min at 95˚C. Samples were resolved on 7% and 12% sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) and resolved for 1.5 h at 100 V. Proteins were transferred to polyvinylidene fluoride membranes (Whatman) (1.5 h at 100 V). Membranes were stained using Ponceau S (Sigma) to confirm equal loading of each sample. Membranes were blocked with 10% skim milk dissolved in 1X TBS-T (10 mM Tris HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) for one hour, washed (3 × 10 min) with TBS-T, then incubated for one hour with the TH, DBH (Novus) or PNMT (Immunostar) primary antibodies. (1:8000 dilution in 10% skim milk). The GAPDH primary antibody was diluted 1:5000 and incubated overnight. Membranes were washed (3 × 10 min) with TBS-T, followed by incubation with secondary antibody conjugated with horseradish peroxidase (1:5000 for 45 min). Membranes were then washed (3 × 10 min) with TBS-T and enhanced chemiluminescence detection was subsequently performed, followed by autoradiography.
All data are presented as the mean ± SEM. Experiments were repeated at least three times. Statistical significance between experimental and control groups was determined by one-way ANOVA followed by post hoc comparisons using the Student-Newman-Keuls multiple comparison test. Results were considered statistically significant with values of p < 0.05.
RT-PCR analysis from PC12 cells treated for 6 h with the NO donor (100 μM SNP) significantly increased TH mRNA 2.0-fold (p < 0.01) and DBH mRNA levels 1.3-fold (p < 0.05) compared to untreated control (
To determine the impact of NO elevations on cholinergic regulation of CA biosynthetic enzymes, RT-PCR analysis from PC12 cells treated for 6 h with specific cholinergic receptor agonists (carbamylcholine, nicotine and muscarine) and SNP was conducted (
To determine whether NO can potentiate the glucocorticoid activation of CA biosynthetic enzymes, RT-PCR analysis was performed from PC12 cells treated with 1 μM DEX and 100 μM SNP for 6 h (
The intracellular signaling pathways involved in the NO regulation of CA biosynthetic enzymes were investigated with specific kinase activators (
8-Br-cGMP (cGMP-PKG activator) with 100 μM SNP for 6 h and RT-PCR analysis performed. Fn treatment significantly increased all four genes compared to untreated control (p < 0.01 for TH, DBH and PNMT splice variants). The addition of SNP in combination with Fn increased TH mRNA levels compared to SNP alone (1.3- fold; p < 0.001). PMA treatment significantly increased TH and PNMT intron-retaining mRNA levels compared to untreated control (p < 0.01). When SNP was added in combination with PMA, TH mRNA levels were further increased compared to SNP alone (1.4-fold; p < 0.001), whereas DBH and PNMT intron-retaining and intronless mRNA levels were unchanged. 8-Br-cGMP treatment significantly increased mRNA levels for all three genes compared to untreated control (p < 0.05). Treatments with 8-Br-cGMP and SNP significantly increased TH, DBH and PNMT intronless mRNA levels compared to SNP alone (1.2, 1.5, 1.3-fold; p < 0.05).
Subsequently, specific kinase inhibitors for PKA (30 μM H-89), PKC (100 nM GF109203X), cGMP (200 nM) and PKG (100 nM DT-2) were used prior to treatment of PC12 cells with SNP for 6 h. H-89 treatment alone only decreased PNMT intron-retaining basal mRNA levels (p < 0.01). H-89 significantly attenuated the increase in TH, DBH and PNMT intron-retaining and intronless mRNA levels induced by SNP (0.3, 0.5, 0.8, 0.5-fold; p < 0.05) compared to SNP alone. GF treatment alone altered PNMT intron-retaining mRNA levels (p < 0.001). GF significantly attenuated the increase in TH, DBH and PNMT mRNA levels induced by SNP (0.3, 0.8, 0.8, 0.6-fold; p < 0.05) compared to SNP alone. cGMP inhibitor alone altered PNMT intron-retaining mRNA levels (p < 0.05). cGMP inhibitor significantly attenuated the increase in TH, DBH and PNMT intronless mRNA levels (0.4, 0.6, 0.5-fold; p < 0.01) compared to SNP alone. Lastly, DT-2 did not alter basal mRNA levels for any of the four genes. DT-2 significantly attenuated the increase in TH, DBH and PNMT intron-retaining and intronless mRNA levels (0.4, 0.6, 0.5, 0.5-fold; p < 0.01) compared to SNP alone.
To assess whether the mRNA induction caused by NO was associated with de novo synthesis of mRNA and/or specific protein, 50 nM Actino-D, an inhibitor of RNA polymerase, and 1 μM cycloheximide, an inhibitor of protein biosynthesis was added to media prior to treatment of cells with SNP (
Nitric oxide has been associated with a wide range of physiological processes and also functions as a central and peripheral neuronal messenger or neurotransmitter [
The in vitro PC12 cell line was used in the current study to examine the effect of SNP-derived NO on the regulation of the CA biosynthetic enzymes: TH, DBH and PNMT (intron-retaining and intronless splice variants). Both splice variants of PNMT were investigated to assess the functional implications of alternative splicing by intron-retention. Proteins translated from intron-retained mRNA species may exert a dominant negative effect on normal protein activity [
CA enzyme genes can be neurally regulated by the release of neurotransmitters from the splanchnic nerve and cholinergic agonists have been used to induce CA gene expression via nicotinic and muscarinic receptors [
In addition to neural regulation, CA enzyme genes, specifically PNMT, can be regulated via hormonal stimulus such as glucocorticoids [
Stimulation of the splanchnic nerve activates a number of secondary messenger systems within the adrenal medullary cells, including that of cAMP-PKA, PKC and PKG that can modulate CA gene regulation [
In summary, the findings reported here provide evidence that NO plays an important role in CA biosynthetic enzyme gene regulation and confirms the involvement of the cGMP-dependent signaling pathway. Our studies further demonstrate that NO is capable of modulating both the glucocorticoid and cholinergic activation of adrenal CA biosynthetic enzymes, via interaction of the glucocorticoid, PKA and PKC signaling with the cGMP/ PKG signaling system.
This study was supported by funding from the Natural Sciences and Engineering Council of Canada and the Ontario Graduate Scholarship.