A Novel Model of Dexamethasone-Induced Hypertension: Use in Investigating the Role of Tyrosine Hydroxylase.

Our objective was to study hypertension induced by chronic administration of synthetic glucocorticoid, dexamethasone (DEX), under nonstressful conditions and examine the role of catecholamine biosynthesis. To achieve this, we did the following: 1) used radiotelemetry to record mean arterial pressure (MAP) and heart rate (HR) in freely moving rats, and 2) administered different doses of DEX in drinking water. To evaluate the involvement of tyrosine hydroxylase (TH), the rate-limiting step in catecholamine biosynthesis, we treated rats with the TH inhibitor, α-methyl-para-tyrosine (α-MPT), for 3 days prior to administration of DEX and assessed TH mRNA and protein expression by quantitative real-time polymerase chain reaction and Western blot in the adrenal medulla. We observed a dose-dependent elevation in blood pressure with a DEX dose of 0.3 mg/kg administered for 10 days, significantly increasing MAP by +15.0 ± 1.1 mm Hg, while concomitantly reducing HR. Although this DEX treatment also significantly decreased body weight, pair-fed animals that showed similar decreases in body weight due to lowered food intake were not hypertensive, suggesting that body weight changes may not account for DEX-induced hypertension. Chronic DEX treatment significantly increased the TH mRNA and protein levels in the adrenal medulla, and α-MPT administration not only reduced DEX pressor effects, but also inhibited TH (serine(40)) phosphorylation. Our study thus validates a novel model to study hypertension induced by chronic intake of DEX in freely moving rats not subject to the confounding factors of previous models and establishes its dependence on concomitant activation of peripheral catecholamine biosynthesis.

Identification of an activator protein-1-like sequence as the glucocorticoid response element in the rat tyrosine hydroxylase gene.

Glucocorticoids (GCs) generally stimulate gene transcription via consensus glucocorticoid response elements (GREs) located in the promoter region. To identify the GRE in the rat tyrosine hydroxylase (TH) gene promoter, we transiently transfected PC12 cells with a 9-kilobase (kb) TH promoter-luciferase (Luc) construct. Dexamethasone (Dex) stimulated Luc activity, which was abolished by mifepristone (RU486). Serial deletion mutations revealed a Dex-responsive 7-base pair (bp) sequence, TGACTAA, located at -5734 to -5728. Deletion of just these seven nucleotides from the 9-kb promoter completely abolished the Dex response and partially reduced the response to phorbol ester but not to forskolin. The Dex response was fully retained in a construct in which most of the 9-kb promoter was deleted, except for 100 bp around the -5.7-kb region, clearly identifying this 7-bp sequence as solely responsible for GC responsiveness. Conversely, deletion of the proximal cAMP-response element (-45/-38) or activator protein-1 (AP-1) (-207/-201) sites in the 9-kb promoter did not affect Dex and phorbol ester responses. A radiolabeled 25-bp promoter fragment bearing the 7-bp TH-GRE/AP-1 showed specific binding to PC12 nuclear proteins. Using antibodies against the glucocorticoid receptors and AP-1 family of proteins and primers for the TH-GRE/AP-1 region, we detected a specific DNA amplicon in a chromatin immunoprecipitation assay. This 7-bp TH-GRE/AP-1 sequence (TGACTAA) does not bear similarity to any known GRE but closely resembles the consensus AP-1 binding site, TGACTCA. Our studies describe for the first time a novel GRE/AP-1 site present in the TH gene promoter that is critical for glucocorticoid regulation of the TH gene.

Dexamethasone Causes Hypertension in Rats Even Under Chemical Blockade of Peripheral Sympathetic Nerves.

Synthetic glucocorticoids (GCs) are widely used to treat inflammatory conditions. However, chronic use of GCs can lead to hypertension. The cause of this undesired side effect remains unclear. Previously, we developed an in vivo rat model to study the mechanisms underlying hypertension induced by the chronic administration of the potent synthetic GC, dexamethasone (DEX) and found that the catecholamine biosynthetic pathway plays an important role. In the current study, we used this model to investigate the role of the adrenal medulla, renal nerves, and other peripheral sympathetic nerves in DEX-induced hypertension. After 5 days of baseline telemetric recording of mean arterial pressure (MAP) and heart rate (HR), rats were subjected to one of the following treatments: renal denervation (RDNX), adrenal medullectomy (ADMX), 6-hydroxydopamine (6-OHDA, 20 mg/kg, i.p.) to induce chemical sympathectomy, or a combination of ADMX and 6-OHDA. On day 11, the animals received vehicle (VEH) or DEX in drinking water for 7 days, with the latter causing an increase in MAP in control animals. ADMX and RDNX by themselves exacerbated the pressor effect of DEX. In the chemical sympathectomy group, DEX still caused a rise in MAP but the response was lower (ΔMAP of 6-OHDA/DEX < VEH/DEX, p = 0.039). However, when ΔMAP was normalized to day 10, 6-OHDA + DEX did not show any difference from VEH + DEX, certainly not an increase as observed in DEX + ADMX or RDNX groups. This indicates that sympathetic nerves do not modulate the pressor effect of DEX. TH mRNA levels increased in the adrenal medulla in both VEH/DEX (p = 0.009) and 6-OHDA/DEX (p = 0.031) groups. In the 6-OHDA group, DEX also increased plasma levels of norepinephrine (NE) (p = 0.016). Our results suggest that the activation of catecholamine synthetic pathway could be involved in the pressor response to DEX in animals even under chemical sympathectomy with 6-OHDA.

Potential role of cAMP response element-binding protein in ethanol-induced N-methyl-D-aspartate receptor 2B subunit gene transcription in fetal mouse cortical cells.

We have shown previously that long-term ethanol treatment causes an up-regulation of N-methyl-D-aspartate (NMDA) receptor 2B subunit (NR2B) number and function in cultured fetal mouse cortical neurons. To examine the intracellular signaling pathways involved in this NR2B gene transcription, we have subjected fetal cortical neurons to long-term treatment with ethanol and studied its effect on cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK) levels by Western blot and enzyme-linked immunosorbent assay. We find a significant increase in phosphorylated CREB, without change in total CREB protein, in cells treated with ethanol for 5 days. Long-term ethanol treatment did not increase levels of both total and phospho-ERK in serum-free medium, whereas it did increase ERK phosphorylation in medium containing serum, without affecting total ERK levels. CREB phosphorylation was increased by ethanol treatment in both media, irrespective of the presence of serum. Electrophoretic mobility shift assay, using a 25-base pair (bp) double-stranded DNA fragment containing the cyclic AMP response element (CRE)-like sequence of the NR2B promoter as (32)P-labeled probe, showed an increase in specific CRE binding to nuclear proteins isolated from cells undergoing long-term ethanol treatment. A 467-bp DNA fragment of the NR2B promoter containing the CRE sequence cloned into the luciferase vector exhibited high reporter activity in transient cotransfection assay of mouse cortical neurons, and ethanol treatment increased this activity. Introducing site-directed mutation in the CRE sequence significantly reduced the reporter activity relative to the wild-type construct, and it also abolished the stimulatory effect by ethanol. Our results indicate that CREB is probably involved in mediating ethanol-induced up-regulation of NR2B gene.

Neuron-restrictive silencer factor regulates the N-methyl-D-aspartate receptor 2B subunit gene in basal and ethanol-induced gene expression in fetal cortical neurons.

Neuron-restrictive silencer factor (NRSF) is a transcriptional repressor of multiple neuronal genes. This study addressed the role of NRSF in N-methyl-D-aspartate (NMDA) receptor NR2B promoter activity and the molecular mechanisms of ethanol-induced NR2B up-regulation in fetal cortical neurons. The 5'-flanking region of the NR2B gene contains five NRSE-like elements. Functional analysis of the upstream regions of the NR2B gene by transient transfection of neurons revealed that neuron-restrictive silencer element (NRSE) motifs located between base pair -1407 and -2741 represses transcription of the gene. Analysis by electrophoretic mobility shift assay and reporter gene assay identified NRSE2 and 3 as responsible for repressing NR2B gene transcription. The identity of NRSF as the functional binding factor is suggested by the specific binding of in vitro synthesized NRSF or cell lysate to the labeled probes and the specific antibody-induced supershift. Furthermore, whereas mutations of NRSE2 and 3 motifs increased the promoter activity, overexpression of NRSF reduced it significantly. The pattern of NRSF expression during development was investigated and demonstrated that the highest expression is on embryonic day 14 with moderate expression on postnatal day 0, reflecting a possible role of NRSF as a regulator during development. Treatment of cultured cortical neurons with 100 mM ethanol for 5 days caused a significant decrease in the NRSF mRNA and protein levels, NRSF/NRSE binding activity, and an increase in the promoter activity. Therefore, our studies suggest that NRSF is a negative regulator of NR2B expression and may contribute to the ethanol-induced up-regulation of the NR2B gene in fetal cortical neurons.