1521-0103/358/3/528–536$25.00 http://dx.doi.org/10.1124/jpet.116.234005THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 358:528–536, September 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

A Novel Model of Dexamethasone-Induced Hypertension: Use inInvestigating the Role of Tyrosine Hydroxylase

Alexandra E. Soto-Piña, Cynthia Franklin, C.S. Sheela Rani, Helmut Gottlieb,Carmen Hinojosa-Laborde, and Randy StrongUniversidad Autónoma del Estado de México, Facultad de Medicina, Toluca, México (A.E.S.-P.); Department of Pharmacology,University of Texas Health Science Center at San Antonio, San Antonio, Texas (C.S.S.R., C.H.-L., R.S.); Feik School ofPharmacy, University of the Incarnate Word, San Antonio, Texas (H.G., C.F.); and Audie L. Murphy Division, South TexasVeterans Health Care System, San Antonio, Texas (R.S.)

Received March 28, 2016; accepted July 8, 2016

ABSTRACTOur objective was to study hypertension induced by chronicadministration of synthetic glucocorticoid, dexamethasone (DEX),under nonstressful conditions and examine the role of catechol-amine biosynthesis. To achieve this, we did the following: 1) usedradiotelemetry to record mean arterial pressure (MAP) and heartrate (HR) in freely moving rats, and 2) administered different dosesof DEX in drinking water. To evaluate the involvement of tyrosinehydroxylase (TH), the rate-limiting step in catecholamine bio-synthesis, we treated rats with the TH inhibitor, a-methyl-para-tyrosine (a-MPT), for 3 days prior to administration of DEX andassessed TH mRNA and protein expression by quantitative real-time polymerase chain reaction and Western blot in the adrenalmedulla. We observed a dose-dependent elevation in bloodpressure with a DEX dose of 0.3 mg/kg administered for 10 days,

significantly increasing MAP by 115.0 6 1.1 mm Hg, whileconcomitantly reducing HR. Although this DEX treatment alsosignificantly decreasedbodyweight, pair-fed animals that showedsimilar decreases in body weight due to lowered food intake werenot hypertensive, suggesting that body weight changes may notaccount for DEX-induced hypertension. Chronic DEX treatmentsignificantly increased the TH mRNA and protein levels in theadrenal medulla, and a-MPT administration not only reduced DEXpressor effects, but also inhibited TH (serine40) phosphorylation.Our study thus validates a novel model to study hypertensioninduced by chronic intake of DEX in freely moving rats not subjectto the confounding factors of previous models and establishes itsdependence on concomitant activation of peripheral catechol-amine biosynthesis.

IntroductionSynthetic glucocorticoids are used in the chronic treatment

of inflammatory diseases (Kirwan, 1995; Saag, 2002; vanEverdingen et al., 2002). Dexamethasone (DEX) is one of themost commonly used synthetic glucocorticoids because of itspotency (Kari et al., 1994; Kaal and Vecht, 2004; Piette et al.,2006). However, cumulative exposure to glucocorticoids in-creases the risk of cardiovascular outcomes, including hy-pertension (Souverein et al., 2004; Davis et al., 2007).Approximately 70% of the patients undergoing chronic gluco-corticoid therapy become hypertensive, the severity beingdependent on the dose and duration of the treatment(Souverein et al., 2004; Panoulas et al., 2008). Nevertheless,the mechanisms by which chronic glucocorticoid therapyinduces hypertension are not well understood.Cortisol, the endogenous glucocorticoid, can produce hy-

pertension associated with increased sodium and water reten-tion and cause renal impairment via interaction with both

mineralocorticoid and glucocorticoid receptors (Whitworthet al., 1989, 2000; Mangos et al., 2003). DEX, in contrast,affects renal function (Bia et al., 1982) specifically via theglucocorticoid receptor (GR) (Reul et al., 1987). However,studies evaluating the kidney-specific homozygous deletionof GR in mice concluded that the GR in the distal nephron isnot necessary for the development or maintenance of DEX-induced hypertension (Goodwin et al., 2010). A potentialalternative site would be a central effect. However, DEX doesnot readily cross the blood brain barrier (De Kloet, 1997;Meijer et al., 1998), precluding direct activation of the centralnervous system. Thus, the mechanism by which DEX raisesblood pressure is most likely peripheral, possibly throughactivation of the peripheral sympathoadrenal system. It islong known that sympathetic hyperactivation can result inelevated cardiac output and total peripheral resistance(Ulrych et al., 1968; Fletcher et al., 1976). Increased sympa-thetic nerve activity and elevated circulating catecholamineconcentrations are common characteristics of essential hyper-tension (Esler et al., 1980), as well as in deoxycorticosterone-salt hypertension and in spontaneously hypertensive rats(Sakaguchi et al., 1983; Lange et al., 1998). Despite thisknowledge on the involvement of sympathetic nervous system

This work was supported by the Department of Veteran Affairs (to R.S.),Consejo Nacional de Ciencia y Technología (to A.E.S.-P.), and a predoctoralaward from the American Heart Association (to A.E.S.-P.).

dx.doi.org/10.1124/jpet.116.234005.

ABBREVIATIONS: a-MPT, a-methyl-para-tyrosine; BPM, beats per minute; DEX, dexamethasone; GR, glucocorticoid receptor; HR, heart rate;MAP, mean arterial pressure; SCG, superior cervical ganglion; TH, tyrosine hydroxylase; VEH, vehicle.

528

at ASPE

T Journals on D

ecember 22, 2021

jpet.aspetjournals.orgD

ownloaded from

in blood pressure development (Esler et al., 2008; DiBona andEsler, 2010), how chronic glucocorticoid exposure affects it islargely unknown.Tyrosine hydroxylase (TH) is the first and rate-limiting

enzyme in catecholamine biosynthesis and is highly expressedin the adrenal medulla and sympathetic neuronal terminals.At the molecular level, our laboratory identified an atypicalglucocorticoid response element in the promoter region of ratand human TH gene that is stimulated by DEX (Rani et al.,2009; Sheela Rani et al., 2013). In the present study, wehypothesized that an underlying mechanism for DEX-inducedhypertension involves increases in TH gene expression in theadrenalmedulla, and perhaps in other sympathetic terminals,upon chronic DEX treatment; this in turn leads to a sustainedincrease in catecholamine synthesis, higher enzyme activity,and finally to hypertension. Indeed, DEX administration isknown to increase plasma catecholamine levels in hyper-tensive patients (Watanabe et al., 1995). We chose to studyadrenal medulla as a representative of peripheral sympa-thetic ganglia because medullary cells are, indeed, modifiedpostganglionic cells of the autonomic nervous system thatreceive innervation from corresponding preganglionic fibers(Perlman and Chalfie, 1977).Furthermore, we used a-methyl-para-tyrosine (a-MPT), an

irreversible TH inhibitor (Udenfriend et al., 1965; Ankenmanand Salvatore, 2007), to explore the possible role of adrenal THin DEX-induced hypertension, because earlier studies indi-cated that this drug is effective in treating certain types ofhypertension involving increased serum catecholamine levels(Perry et al., 1990; Chiou-Tan et al., 1994; Shimizu et al.,2001). In addition to adrenalmedulla, a-MPTwould also allowus to assess the role of sympathetic nerve terminals because ithas been shown to cause catecholamine depletion in terminalsas well (Nagatsu et al., 1964; Spector et al., 1965).The primary aim of this study was to generate a novel in vivo

model to study hypertension following chronic DEX admin-istration without the involvement of stressors due to themethodologies used for drug administration and blood pres-sure recording. To accomplish this, we delivered DEX indrinking water and used a radiotelemetry approach to contin-uously record blood pressure. This method has the advantageof measuring blood pressure directly in freely moving/restinganimals, thus avoiding handling-related changes in theirarousal state (Kurtz et al., 2005). We evaluated the dose-response relationship between chronic administration of DEXin drinking water and changes in blood pressure, and alsoexamined TH expression and phosphorylation at serine40

(pSer40) as possible mechanisms in the generation of DEX-induced hypertension by a-MPT systemic administration.

Materials and MethodsAnimals. Male Fisher 344 rats, 8–10 weeks old and weighing

250–300 g, were purchased from Harlan (Houston, TX). They weremaintained on a 14/10-hour light/dark cycle and fed ad libitum for thewhole study. Rats were allowed to acclimate to housing conditions for3 days and then underwent telemetric implant surgery. After thesurgery, the rats were individually housed in plastic cages withcontinuous access to rat chow (Harlan Teklad 7912) and water. Theexperiments were approved and performed under the guidelines of theInstitutional Animal Care and Use Committee of the University ofTexas Health Science Center at San Antonio.

Radiotelemetry Implantation and Measurement of BloodPressure and Heart Rate. Implantation surgery was done underisoflurane anesthesia. A catheter attached to a CA11PA-C40 radio-telemetry transmitter (Data Science International, St. Paul, MN) wasimplanted in the peritoneal aorta and secured to the abdominalmuscle. The animals were allowed to recover for 8 days. Each animalwas housed in a plastic cage, placed on top of a RPC-1 receiver thatwasattached to a Data Exchange Matrix (Data Science International).After recovery, systolic blood pressure, diastolic blood pressure, andmean arterial pressure (MAP), as well as heart rate (HR), weremonitored for 3–5 days to establish the baseline. Recordings wereperformed every 10 minutes; data were collected using DataquestA.R.T. 4.1 software (Data Science International). Because systolicblood pressure and diastolic blood pressure responded similarly toMAP, we present our data only asMAP. Results of change in (D) MAP,DHR, and Dbody weight were calculated by subtracting the 24-hourmean values from the average of the mean of 3 days of baselinerecording for DEX dose-response curves and pair-feeding experi-ments, and 5 days of baseline recording for the a-MPT experiment.Day 1 indicates the first day of recording to establish the baseline, andthe numeration continues until the last day of recording/treatment onday 13 for DEX dose-response and pair-feeding experiments, and onday 17 for the a-MPT study.

Dexamethasone Treatment. To evaluate the dose of DEX thatproduces a significant increase in blood pressure, animals receivedeither regular drinking water (controls) or a water-soluble form ofsynthetic glucocorticoid (GC), dexamethasone 21-phosphate disodiumsalt (Sigma Aldrich, St. Louis, MO), in drinking water. Three differentgroups of rats received DEX in drinking water for 10 days: low(0.03 mg/kg/d), medium (0.3 mg/kg/d), and high (3 mg/kg/d). Waterintake was measured every day and to calculate daily DEX doses wasadjusted to it as well. Body weight was monitored daily to determinepossible catabolic DEX effects.

Pair-Feeding. To determine the role of body weight loss on theblood pressure effects of DEX, we treated a group of rats with DEX(0.16 mg/kg/d) for 10 days and performed pair feeding of a group ofuntreated rats with the same amount of food previously determined tobe consumedby theDEX-treated group.Weused this singlemid-rangedose (0.16 mg/kg/d) in this control study because both 0.03 and0.3 mg/kg/d had earlier produced elevations in blood pressure. Foodintake was calculated as the difference of weight of the food containerbetween the daywhen the containerwas filled and 24 hours after that.

Measurement of TH mRNA Content in the Adrenal Medullaand Brainstem. After 10 days of DEX treatment, the animals wereeuthanized, and the adrenalmedullaewere removed, flash frozen, andstored at 280°C. Brains were removed, and the brainstem wasseparated from the encephalon. A 4-mm section of the medullaoblongata was cut. The left adrenal medullae and the brainstemregions were homogenized in 1 ml TRizol reagent (Life Technologies/Invitrogen, Carlsbad, CA). Total RNA was isolated and treated withDNase and used for synthesizing cDNA using the High CapacitycDNA synthesis kit (Life Technologies/Applied Biosystems, FosterCity, CA), according to manufacturer’s instructions. cDNA (10 ng) wasused for quantitative real-time polymerase chain reaction, as de-scribed previously (Green et al., 2011), using a TaqMan gene ex-pression master mix and 6-carboxyfluorescein–labeled probe for ratTH (Gene Expression Assay ID Rn00562500_m1; Life Technologies/Applied Biosystems) and 18S rRNA, which was used as the endoge-nous normalizer and amplified simultaneously using TaqManEukaryotic 18S rRNA endogenous control VIC MGB probe (primerlimited, 4319413E). The assays were performed in duplicate usingthe ABI PRISM 7900 Sequence Detection System. The relative ex-pression was calculated from the average difference in cycle thresholdbetween the vehicle (VEH) control and DEX-treated samples using theDD cycle threshold method (Livak and Schmittgen, 2001).

Pharmacological Inhibition of TH. To determine the role of THin DEX hypertension, separate groups of rats were treated withthe TH inhibitor, a-methyl-DL-tyrosine methyl ester hydrochloride

A Novel Model of Dexamethasone-Induced Hypertension 529

at ASPE

T Journals on D

ecember 22, 2021

jpet.aspetjournals.orgD

ownloaded from

(Sigma-Aldrich), prior to induction of hypertension. After 5 days ofbaseline recording, one group of rats was treated with VEH (0.9%NaCl, i.p.), and the second was treated with a-MPT (50 mg/kg, i.p.) ondays 6, 8, and 10 of recording (Fig. 1). Both groupswere further dividedinto two groups, as follows: one received water and the other receivedDEX in drinking water (0.3 mg/kg/d) for 7 days (Fig. 1). Blood pressureandHRwere recorded continuously, as described above.We computedDMAP and DHR based on the average of 5 days of baseline recording.

Measurement of Adrenal TH Protein after a-MPT byWestern Blotting. Rat adrenal medulla samples kept frozen at280°C after euthanasia were thawed on ice and homogenized in radio-immunoprecipitation assay buffer: 50 mM Tris-HCl (pH 8), 150 mMNaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS withSigma-Aldrich protease inhibitor cocktail (P8340, 1:100 dilution) and1 mM phenylmethylsulfonyl fluoride. Protein samples (10 mg) wereheat-denatured in lithium dodecyl sulfate (LDS) buffer containingdithiothrietol reducing agent (Invitrogen) and then electrophoresed on4–12% NuPage Bis-Tris gel and transferred to a polyvinylidenedifluoride membrane using the iBlot system (Life Technologies/ThermoFischer Scientific, Carlsbad, CA). The membrane was probed simulta-neously with monoclonal antibodies to TH (1:1000, T1299 mousemonoclonal Ab; Sigma-Aldrich) and b-actin (1:1000, ab8226; AbCam,Cambridge, MA) as well as polyclonal anti-pSer40 TH (1:500 AB5935;Millipore, Billerica, MA). The TH and b-actin signals were detectedusing a secondary antibody IRDye 800CW goat anti-mouse IgG(1:10,000, 925-32210; LI-CORBiosciences, Lincoln, NE), and pSer40 THwas detected by IRDye 680RD goat anti-rabbit IgG (1:10,000, 926-68071; LI-COR Biosciences). The fluorescent bands were identified at60 kDa for TH and pSer40 TH and at 42 kDa for b-actin. Detection anddensitometry quantificationwere performedusing theOdyssey infraredimaging system (LI-COR Biosciences).

Statistical Analysis. To analyze ΔMAP, ΔHR, and Δbody weight,we used two-way analysis of variance with repeated measures usingSigmaStat 4.0 (Systat Software, San Jose, CA) statistical package.The Holm-Sidak test was used as post hoc analyses. TH expressionanalyses were performed using the Prism v7 (Graph Pad Software, LaJolla, CA). THmRNAwas analyzed by theMann–WhitneyU test, andTH protein was analyzed by one-way analysis of variance withDunnett’s multiple comparison test; P , 0.05 (two-sided comparison)was accepted as a statistically significant level.

ResultsBlood Pressure Responses to Oral DEX Treatment in

Drinking Water. With administration of DEX, MAP in-creased significantly above baseline values in all groups(Fig. 2A). The average MAP at baseline (days 1–3) in thegroup with the lowest dose, 0.03 mg/kg/d (n5 3), was 104.364.0 mmHg. After the administration of DEX, this group

showed an elevation of MAP by day 11 (19.4 6 3.5 mmHg)with respect to the VEH control group (#P , 0.01) andcontinued increasing to 13.5 6 3.8 mmHg by day 13 (***P ,0.001 versus day 3 and %P , 0.001 versus VEH). The groupthat received the medium dose of 0.3 mg/kg/d (n 5 5) showedan averageMAP at baseline of 102.26 2.7, and, at day 9, MAPincreased 11.06 1.4mmHg, reaching earlier a high significantlevel (***P, 0.001 versus day 3 and %P , 0.001 versus VEH)than the group with lowest dose. This level was maintaineduntil day 13 (115.0 6 1.1 mmHg, ***P , 0.001 versus day3 and %P , 0.001 versus VEH). Animals treated with thehighest dose of DEX, 3 mg/kg/d (n 5 4), presented an averagebaseline of 101.1 6 0.9 mmHg. Nonetheless, they did notsurvive beyond 7 days of DEX treatment. By day 10, therewere three animals alive. They also showed significantincreases in MAP from days 8 to 10 that ranged from 10.2 61.8 mmHg (***P , 0.001 versus day 3) to 17.9 6 8.3 mmHg(***P , 0.01 versus day 3 and #P , 0.01 versus VEH),respectively.With regard to HR, there were significant dose-dependent

decreases (Fig. 2B). The group with the lowest dose,0.03 mg/kg/d, showed an average baseline of 366.9 6 9.7beats per minute (BPM); upon DEX treatment, there weresignificant decreases (***P , 0.01 versus day 3) on days 6(29.26 4.2 BPM), 8 (210.26 2.7 BPM), 9 (211.56 4.0 BPM),and 10 (212.0 6 4.4 BPM). Only day 10 was different fromthe VEH group ($P , 0.05). The group that received themedium dose had a HR average at baseline of 368.8 6 5.2BPM. Specifically, at this dose, HR decreased significantly ondays 8 (222.7 6 4.1 BPM) to 13 (225.3 6 3.1 BPM) withrespect to day 3 of baseline recording (***P, 0.001) and fromday 10 (226.0 6 3.2 BPM, $P , 0.05) to day 12 (224.5 6 1.2BPM, $P , 0.05) with respect to the VEH group. The thirdgroup with the highest dose (3 mg/kg/d) had a baseline HRaverage of 373.9 6 5.0 BPM, and it started decreasingsignificantly (***P , 0.01 versus day 3 and #P , 0.01 versusVEH) from day 6 (243.8 6 4.6 BPM) until day 10 (254.2 69.3 BPM), being that latter the last day of survival in thisgroup.Moreover, body weight decreased upon DEX administration

in the three groups (Fig. 2C). The VEH control group showedsignificant increases (***P , 0.001 versus day 3) from day4 (118.4 6 6.4 g) to 13 (145.2 6 5.2 g). Animals treated withthe lowest dose had significant reductions (***P, 0.001 versusday 3) on days 12 (213.96 1.3 g) and 13 (218.16 0.8 g). It wasalso significantly reduced from the VEH group from day 5 to13 ($P , 0.05). Furthermore, the group with the medium doseshowed significant reductions on body weight by day 8 (225.76 1.1 g, ***P , 0.001 versus day 3) and continued until day13 (246.3 6 7.3 g, ***P , 0.001 versus day 3). It was alsosignificantly different from the VEH control group from day4 to 13 (#P , 0.01). Finally, the third group receiving thehighest dose of DEX showed significant decreases from day7 (225.96 0.9 g, ***P, 0.001 versus day 3) to 10 (270.06 3.2 g,***P , 0.001 versus day 3) and from day 5 to 10 with respectto the VEH group (#P , 0.01).Reduction in Body Weight Is Not Responsible for

DEX-Induced Hypertension. To isolate the effect of re-duced body weight from blood pressure, we used pair feedingwith reduced food intake and compared it with the effects ofDEX administration (n 5 3/group). In Fig. 3A, DEX-treatedrats showed a significantly reduced food intake on days

Fig. 1. Timeline of experimental procedures. After telemeter implantsurgery and recovery, blood pressure and HR were monitored through therest of study, until the end of the different pharmacological treatments.Baseline was recorded for 5 days; then, animals received treatments witha-MPT on days 6, 8, and 10 and DEX or their respective VEH on days11–17.

530 Soto-Piña et al.

at ASPE

T Journals on D

ecember 22, 2021

jpet.aspetjournals.orgD

ownloaded from

6 (13.3 6 0.5 g, ***P , 0.001) to 12 (13.2 6 0.8 g, ***P ,0.001) and 13 (14.2 6 0.5 g, **P , 0.01), with respect to day2 of baseline. It was also significantly reduced on days 7, 8,11, and 12 with respect to the VEH control group (aP , 0.05versus VEH). Moreover, the pair-fed group also had adecreased food intake on days 9 (10.8 6 1.6 g, %P , 0.01versus day 3) to 13 (12.8 6 0.8 g, %P , 0.01 versus day 3) aswell as on days 8, 9, 11, 12, and 13 with respect to the VEHgroup (#P , 0.05). Regarding body weight (Fig. 3B), it wasreduced (**P , 0.01 and ***P , 0.001 versus day 3) in bothgroups: the pair-fed (days 8–13) and DEX-treated animals(days 6–13). These were also significantly different from theVEH group, bP , 0.01 and gP , 0.001, respectively. Finally,MAP increased only in DEX group, on days 6 to 8 (**P, 0.01)and 9 to 13 (***P , 0.001) with respect to day 3 of baselineand on days 11 to 13 (gP , 0.001) with respect to the VEHgroup (Fig. 3C), whereas MAP in the pair-fed group wasnot significantly different from the VEH control group orbaseline.DEX Increased TH Expression in the Adrenal Me-

dulla, but Not in Brainstem. We tested the effect of DEX atthemedium dose of 0.3mg/kg/d on TH expression because thisdose was the one that produced a significant elevation in bloodpressure required by our statistical power analysis. Further-more, to identify alterations of TH expression between theperipheral and central nervous system, TH mRNA wasmeasured in two of its main locations: the adrenal medullaand brainstem. There was a 2.5-fold increase in TH mRNA

levels in the adrenalmedulla (**P, 0.01), whereas THmRNAin the brainstem was not modified (Fig. 4).Effect of a-MPT on DEX-Induced Hypertension. In

Fig. 5A, MAP increased in VEH/DEX on days 12–17 (***P ,0.001 versus day 5) and 14–17 with respect to the VEH/VEHgroup (#P , 0.01). After the administration of a-MPT, therewere no changes in MAP baseline in any of the groups.Nonetheless, the a-MPT/DEX group exhibited significantlydiminished pressor effect on days 14–17 (15.9 6 1.6, 19.1 61.4, 111.1 6 1.2, and 111.9 6 1.8 mmHg, respectively; aP ,0.025) compared with the responses in the group treated withDEXalone on the same days (112.06 1.2,114.86 1.4,117.561.3, and 118.8 6 1.7 mmHg, respectively). Despite that,there were increases in the a-MPT/DEX group against theVEH/VEH group on days 13–17 ($P , 0.05). Furthermore, acomparison of DMAP of the a-MPT/DEX and VEH/DEXgroups, normalized to their respective control group (Fig.5B), was performed to discard possible interferences withbaseline, and significant differences were observed (bP ,0.05) between these groups on days 12–17. Finally, theadministration of a-MPT did not change HR during thebaseline recording, nor did it influence DEX-induced de-creases in HR (Fig. 5C). It decreased in VEH/DEX group ondays 12–17 compared with day 5 of baseline (***P , 0.001)and days 13–17 compared with the VEH/VEH group (#P ,0.01). The a-MPT/DEX group had decreases on days 13–17against day 5 of baseline (***P , 0.01) and VEH/VEHgroup (#P , 0.01). No significant differences were observed

Fig. 2. Dose-response curves for chronic DEX administration in drinking water. Each point represents the difference of 24-hour mean 6 S.E.M.responses in DMAP (A), DHR (B), and Dbody weight (C) between the average of 3 days of baseline and each day of recording in VEH (n = 5) and three DEXdoses: 0.03 mg/kg/d (n = 3), 0.3 mg/kg/d (n = 5), and 3 mg/kg/d (n = 4). Two-way analysis of variance with repeated measurements and Holm-Sidak posthoc analysis were used to compare responses between groups with respect to day 3 of baseline (***P, 0.001) and VEH group ($P, 0.05, #P, 0.01, and%P , 0.001).

A Novel Model of Dexamethasone-Induced Hypertension 531

at ASPE

T Journals on D

ecember 22, 2021

jpet.aspetjournals.orgD

ownloaded from

when normalizing HR to the respective VEH control groups(Fig. 5D).At the end of telemetric recordings, animals were eutha-

nized and the adrenal medullae were isolated to measure THprotein. Therewas a significant increase in TH, 1.3-fold (**P,0.01), after 7 days of DEX treatment compared with the VEH-treated group. Moreover, a-MPT pretreatment inhibitedDEX-induced increase in TH protein (Fig. 6A). TH phosphor-ylation also increased in the VEH/DEX group, as shown byan elevation in the level of pSer40-TH (*P , 0.05 versusVEH/VEH), and this increase was completely inhibited bypretreatment with a-MPT (Fig. 6B).

DiscussionOur study describes the development of an animal model to

study hypertension induced by the chronic administration ofDEX. Mainly, by choosing radiotelemetry for recording bloodpressure and an oral route for chronic administration of DEXin rats, our model circumvents the confounding factors seen inearlier studies (Iijima and Malik, 1988; Wallwork et al., 2003;Mondo et al., 2006; Ong et al., 2007). These include stress fromrestraint that was used for blood pressure recording andadministration of DEX, which may have had an effect on thearousal state of the animal, possibly altering blood pressure

Fig. 3. Effects of pair feeding (n = 3) with respect to VEH (n = 3)- and DEX-treated rats (0.16 mg/kg/d, n = 3) on food intake (A), Dbody weight (B), andDMAP (C). Data for body weight and MAP are presented as the difference between the average of 3 days baseline and each day of recording for bodyweight and MAP (24-hour mean 6 S.E.M.). The administration of drugs is indicated with arrows below the x-axis. Two-way analysis of variance withrepeated measurements and Holm-Sidak post hoc analysis revealed significant differences between the groups. (A) shows differences within DEXtreatment and day 2 of baseline (***P , 0.001) as well as VEH group (aP , 0.05). Also, it shows differences within pair feeding and day 3 of baselinerecording (%P, 0.01) as well as VEH group (#P, 0.05). In (B) and (C), differences are indicated against day 3 of baseline (***P, 0.001) and VEH group(bP , 0.01 and gP , 0.001).

Fig. 4. THmRNA in the adrenal medulla (A) and brainstem(B) of animals treated with VEH or DEX medium dose of0.3 mg/kg/d after 10 days of DEX treatment (**P , 0.01).Results are shown as the relative expression calculated fromthe average difference in cycle threshold between the VEHand DEX groups in the adrenal medulla (n = 5 and 6/group,respectively) and brainstem (n = 3 and 4/group, respectively).Data were analyzed using Mann–Whitney U test.

532 Soto-Piña et al.

at ASPE

T Journals on D

ecember 22, 2021

jpet.aspetjournals.orgD

ownloaded from

results. We found that MAP increases in a cumulative anddose-dependent manner over the period of continuous oralDEX administration (Fig. 2A), with significant increases(111 mmHg) seen after 6 days of treatment with a mediumdose of DEX (0.3 mg/kg/d). In an attempt to relate our exper-imental protocol to a clinically relevant dose/time frame,we cansay our treatment paradigm would be approximately equiva-lent to 6 months of medium-dose GC therapy in humans(Quinn, 2005; Andreollo et al., 2012). Interestingly, patientsaffected by rheumatoid arthritis receiving long-term GC treat-ment (.6 months) are more prone to develop hypertensionwhen using a medium dose of prednisone (0.1–4 mg/kg/d) thanthose with limited GC exposure (,0.1 mg/kg/d for ,6 months)(Panoulas et al., 2008).Concomitant with an increase in MAP, a 10-day continuous

oral administration of DEX reduces food intake, but not waterintake (data not shown), and is accompanied by a decrease inbody weight in rats. However, the pair-fed group, which has

similar reduction in body weight as in the DEX-treated group,does not show any change in MAP. This suggests food intakeand body weight reductions due to DEX may not be directlyrelated to its pressor effect through the sympathoadrenalsystem. Studies in the literature point to a complex interplaybetweenGCs and other hormonal factors that regulate feedingbehavior and subsequent body weight changes (Jahng et al.,2008; Cummings et al., 2013). For instance, DEXwas shown toincrease insulin secretion via improvement of b cell function(Cummings et al., 2013), in addition to affecting leptin releaseand its hypothalamic actions (Jahng et al., 2008), enhancingenergy expenditure and anorexia. In contrast, GC therapy inhumans is often associated with weight gain, in addition toincrease in blood pressure (Whitworth et al., 2005).Many factors, including diet, exercise, stress, and aging

itself, affect adrenal expression of TH (Fluharty et al., 1983;Tumer et al., 1992; Kvetnansky et al., 2003; Patel et al., 2005).Several studies have explored to understand the relationship

Fig. 5. Top panel shows the time course of responses in (A) DMAP and (B) DMAP normalized to VEH/VEH or a-MPT/VEH groups before and aftera-methyl-para-tyrosine (a-MPT, 50 mg/kg, i.p.) and DEX administration (0.3 mg/kg/d). Bottom panel shows (C) DHR and (D) DHR normalized toVEH/VEH or a-MPT/VEH groups. The administration of drugs is indicated with arrows below the x-axis. Two-way analysis of variance with repeatedmeasurements and Holm-Sidak post hoc analysis was used to compare responses between groups (***P , 0.001 versus day 5, $P , 0.05 and #P , 0.01versus VEH/VEH, aP , 0.025 versus VEH/DEX, bP , 0.05 versus VEH/DEX normalized to baseline; n = 6/group).

A Novel Model of Dexamethasone-Induced Hypertension 533

at ASPE

T Journals on D

ecember 22, 2021

jpet.aspetjournals.orgD

ownloaded from

between peripheral TH expression and hypertension (Guoet al., 2005; Burgi et al., 2011; Congo Carbajosa et al., 2015).Recent studies have uncovered natural polymorphic varia-tions in the human TH gene that influence transcription,autonomic function, and hypertension (Rao et al., 2007, 2010).A causal relationship was revealed when acute DEX treat-ment in rats (1 mg/kg for 2 days) was shown to upregulate THmRNA in the adrenal medulla and cause hypertension in rats(Kumai et al., 2000), and antisense TH gene therapy in ratswas effective in causing hypotension in the SHRmodel (Kumaiet al., 2001).Because our model allows monitoring long-term effects of

DEX treatment on blood pressure and assessing the un-derlying mechanism, we find that, similar to acute DEXtreatment, chronic DEX exposure also causes an increase inTHmRNAand protein levels, most likely viaGR in chromaffinand ganglionic cells of the adrenal medulla (Phillips et al.,2001). This increase in TH transcription, perhaps, leads tosynthesis of new enzyme molecules that in turn enhance THactivity and catecholamine synthesis. The exact medullarycell type involved in TH transcription in this model system isnot known andwill need to be determined in future studies. Ofnote, we did not find an increase in TH mRNA in thebrainstem. This indicates that the long-term genomic effectof DEX on TH occurs mainly in the periphery, includingadrenal medulla and other sympathetic nerve terminals. Had

DEX crossed into the brain, its effects on blood pressure mayhave been opposite, based on the report that intracerebroven-tricular injection of DEX reduces blood pressure (Tonolo et al.,1993). We never observed a decrease in blood pressure duringthe entire period of DEX administration, consistent withperipheral, not central effects.We did not explore the role of TH in superior cervical

ganglion (SCG) and carotid body, although they could also bepotential peripheral sites for DEX effects; indeed, TH mRNAat these sites increases in response to stressful conditions(Chen et al., 1995). However, the duration and robustness ofthese increases are different, and, more importantly, theyentail different signaling pathways (Nankova et al., 1999). Forexample, activator protein-1 has a lower binding to the THpromoter motif upon immobilization in the SCG than in theadrenal medulla (Sabban et al., 2004). The genomic effect ofDEX on TH via the atypical glucocorticoid response elementmight not occur at the SCG, and was less likely to be ofstimulatory nature that we saw in vitro (Rani et al., 2009;Sheela Rani et al., 2013). Consistent with this, systemica-MPT administration generates a greater decrease in bloodpressure than guanethidine, an inhibitor of the norepineph-rine uptake transporter (Chiou-Tan et al., 1994). This sug-gests that noradrenergic chromaffin cells that contain TH,but lack this transporter (Phillips et al., 2001), contribute toblood pressure regulation to a greater extent than adrenergic

Fig. 6. TH protein and pSer40-TH in the adrenal medulla after DEX and a-MPT. (A) shows TH protein Western blot and densitometry percentage fromcontrol. (B) presents pSer40 THWestern blot and pSer40-TH/TH:b-actin ratio. There were significant differences between VEH/VEH and VEH/DEX, butnot with a-MPT–treated groups. Results were analyzed using one-way analysis of variance with Dunnett’s multiple comparison test (*P , 0.05 and**P , 0.01 versus VEH/VEH, n = 4/group).

534 Soto-Piña et al.

at ASPE

T Journals on D

ecember 22, 2021

jpet.aspetjournals.orgD

ownloaded from

chromaffin cells, ganglion cells, or nerve fibers that have thetransporter.We found that TH protein in the adrenal medulla did not

change in the a-MPT/VEH group, indicating that basal levelsof catecholamine synthesis in the adrenal medulla may not beaffected by a-MPT. This is in keeping with the stability ofbaselineMAPduring treatmentwith this drug. However, totalTHprotein and pSer40-THdecreased in thea-MPT/DEX groupcompared with the VEH/DEX group, suggesting that a-MPTmight reduce only DEX-induced TH expression and phosphor-ylation. Indeed, this is the first study to show such an effect ofthis drug, a-MPT. It is thus possible that DEX affects THenzyme not only at a transcriptional level, but also post-translationally, perhaps involving the N terminus of thecatalytic domain (Nakashima et al., 2009).However, the interpretation of these results is complicated

due to the ability of a-MPT to cross into the blood brain barrierand inhibit endogenous TH, both peripherally and centrally. Inthat case, our results could resemble studies in which thereduction of central catecholamine concentrations alters sym-pathetic outflow and elevates blood pressure (Doba and Reis,1974; Fujino, 1984). In our study, such a consequence of a-MPTaccess into the brain may contribute to the maintenance of anormal baseline MAP and HR during the administration ofa-MPT (Fig. 5A) on days 6–10 and also result in a side effectthat prevents a complete reversal of DEX pressor response.We did not observe significant alteration inHR upon a-MPT

administration, althoughwe had expected it to be reduced dueto a decrease in cardiac sympathetic activity. As mentionedabove, this could also be associated with a-MPT centraleffects. More importantly, HR maintenance could be due toopposing responses of the parasympathetic nervous system toreduced sympathetic activity (Dickson et al., 1981; Levy, 1984;Lacroix et al., 1990), in addition to potential cardiovascularsupersensitivity to catecholamines that may occur immedi-ately after a-MPT administration (Brus et al., 1970; Tripovicet al., 2010). In contrast, decreased HR upon DEX adminis-tration is probably a reflex central response to the elevatedblood pressure.In summary, in this study we developed an animal model to

study chronic DEX-induced hypertension in which the cata-bolic effects of DEX are independent of its effects on bloodpressure. The chronic administration of DEX modified theperipheral catecholamine biosynthetic pathway at the level ofTH in the adrenal medulla. Furthermore, the levels of TH insympathetic nerve terminals and ganglia, although challeng-ing to measure due to small sample, may be necessary to teaseout their relative contributions in DEX-induced hypertension.In particular, the adrenalmedulla and sympathetic nerves areimportant sources of excessive catecholamine levels that leadto cardiovascular alterations and hence hypertension. Becauseof their repercussion on blood pressure, they are potentialtargets of intervention to prevent cardiovascular side effectsof glucocorticoid therapy.

Acknowledgments

We thank Teresa Craig, Vanessa Martinez, and Paul AnthonyMartinez for excellent technical assistance.

Authorship Contributions

Participated in research design: Soto-Piña, Strong, Hinojosa-Laborde, Sheela Rani.

Conducted experiments: Soto-Piña, Franklin, Strong, Hinojosa-Laborde, Sheela Rani.

Performed data analysis: Soto-Piña, Franklin, Strong, Hinojosa-Laborde, Gottlieb, Sheela Rani.

Wrote and contributed to the writing of the manuscript: Soto-Piña,Sheela Rani, Franklin, Strong, Hinojosa-Laborde, Gottlieb.

References

Andreollo NA, Santos EF, Araújo MR, and Lopes LR (2012) Rat’s age versus human’sage: what is the relationship? Arq Bras Cir Dig 25:49–51.

Ankenman R and Salvatore MF (2007) Low dose alpha-methyl-para-tyrosine (AMPT)in the treatment of dystonia and dyskinesia. J Neuropsychiatry Clin Neurosci 19:65–69.

Bia JM, Tyler K, and DeFronzo RA (1982) The effect of dexamethasone on renalelectrolyte excretion in the adrenalectomized rat. Endocrinology 111:882–888.

Brus R, Hess ME, and Jacobowitz D (1970) Effect of 6-hydroxydopamine and thy-roxine on chronotropic response to norepinephrine. Eur J Pharmacol 10:323–327.

Burgi K, Cavalleri MT, Alves AS, Britto LR, Antunes VR, and Michelini LC (2011)Tyrosine hydroxylase immunoreactivity as indicator of sympathetic activity: si-multaneous evaluation in different tissues of hypertensive rats. Am J PhysiolRegul Integr Comp Physiol 300:R264–R271.

Chen J, Dinger B, and Fidone SJ (1995) Second messenger regulation of tyrosinehydroxylase gene expression in rat carotid body. Biol Signals 4:277–285.

Chiou-Tan FY, Lenz ML, Robertson CS, and Grabois M (1994) Pharmacologictreatment of autonomic dysreflexia in the rat. Am J Phys Med Rehabil 73:251–255.

Congo Carbajosa NA, Corradi G, Verrilli MA, Guil MJ, Vatta MS, and Gironacci MM(2015) Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasomesystem in PC12 cells and hypothalamic and brainstem neurons from spontaneouslyhypertensive rats: possible implications in hypertension. PLoS One 10:e0116597.

Cummings BP, Bremer AA, Kieffer TJ, D’Alessio D, and Havel PJ (2013) In-vestigation of the mechanisms contributing to the compensatory increase in insulinsecretion during dexamethasone-induced insulin resistance in rhesus macaques. JEndocrinol 216:207–215.

Davis 3rd, JM, Maradit Kremers H, Crowson CS, Nicola PJ, Ballman KV, TherneauTM, Roger VL, and Gabriel SE (2007) Glucocorticoids and cardiovascular events inrheumatoid arthritis: a population-based cohort study. Arthritis Rheum 56:820–830.

De Kloet ER (1997) Why dexamethasone poorly penetrates in brain. Stress 2:13–20.DiBona GF and Esler M (2010) Translational medicine: the antihypertensive ef-fect of renal denervation. Am J Physiol Regul Integr Comp Physiol 298:R245–R253.

Dickson DW, Lund DD, Subieta AR, Prall JL, Schmid PG, and Roskoski R, Jr (1981)Regional distribution of tyrosine hydroxylase and dopamine beta-hydroxylase ac-tivities in guinea pig heart. J Auton Nerv Syst 4:319–326.

Doba N and Reis DJ (1974) Role of central and peripheral adrenergic mechanisms inneurogenic hypertension produced by brainstem lesions in rat. Circ Res 34:293–301.

Esler M, Eikelis N, Schlaich M, Lambert G, Alvarenga M, Kaye D, El-Osta A, Guo L,Barton D, Pier C, et al. (2008) Human sympathetic nerve biology: parallel influ-ences of stress and epigenetics in essential hypertension and panic disorder. Ann NY Acad Sci 1148:338–348.

Esler M, Jackman G, Leonard P, Bobik A, Skews H, Jennings G, Kelleher D,and Korner P (1980) Determination of noradrenaline uptake, spillover to plasmaand plasma concentration in patients with essential hypertension. Clin Sci 59(Suppl 6):311s–313s.

Fletcher PJ, Korner PI, Angus JA, and Oliver JR (1976) Changes in cardiac outputand total peripheral resistance during development of renal hypertension in therabbit: lack of conformity with the autoregulation theory. Circ Res 39:633–639.

Fluharty SJ, Snyder GL, Stricker EM, and Zigmond MJ (1983) Short- and long-termchanges in adrenal tyrosine hydroxylase activity during insulin-induced hypogly-cemia and cold stress. Brain Res 267:384–387.

Fujino K (1984) Brain catecholamines in spontaneously hypertensive and DOCA-salthypertensive rats. Acta Med Okayama 38:325–340.

Goodwin JE, Zhang J, Velazquez H, and Geller DS (2010) The glucocorticoid receptorin the distal nephron is not necessary for the development or maintenance ofdexamethasone-induced hypertension. Biochem Biophys Res Commun 394:266–271.

Green MK, Rani CS, Joshi A, Soto-Piña AE, Martinez PA, Frazer A, Strong R,and Morilak DA (2011) Prenatal stress induces long term stress vulnerability,compromising stress response systems in the brain and impairing extinction ofconditioned fear after adult stress. Neuroscience 192:438–451.

Guo H, Brewer JM, Champhekar A, Harris RB, and Bittman EL (2005) Differentialcontrol of peripheral circadian rhythms by suprachiasmatic-dependent neuralsignals. Proc Natl Acad Sci USA 102:3111–3116.

Iijima F and Malik KU (1988) Contribution of vasopressin in dexamethasone-inducedhypertension in rats. Hypertension 11:I42–I46.

Jahng JW, Kim NY, Ryu V, Yoo SB, Kim BT, Kang DW, and Lee JH (2008) Dexa-methasone reduces food intake, weight gain and the hypothalamic 5-HT concen-tration and increases plasma leptin in rats. Eur J Pharmacol 581:64–70.

Kaal EC and Vecht CJ (2004) The management of brain edema in brain tumors. CurrOpin Oncol 16:593–600.

Kari MA, Hallman M, Eronen M, Teramo K, Virtanen M, Koivisto M, and Ikonen RS(1994) Prenatal dexamethasone treatment in conjunction with rescue therapy ofhuman surfactant: a randomized placebo-controlled multicenter study. Pediatrics93:730–736.

Kirwan JR; The Arthritis and Rheumatism Council Low-Dose Glucocorticoid StudyGroup (1995) The effect of glucocorticoids on joint destruction in rheumatoid ar-thritis. N Engl J Med 333:142–146.

A Novel Model of Dexamethasone-Induced Hypertension 535

at ASPE

T Journals on D

ecember 22, 2021

jpet.aspetjournals.orgD

ownloaded from

Kumai T, Asoh K, Tateishi T, Tanaka M, Watanabe M, Shimizu H, and Kobayashi S(2000) Involvement of tyrosine hydroxylase up regulation in dexamethasone-induced hypertension of rats. Life Sci 67:1993–1999.

Kumai T, Tateishi T, Tanaka M, Watanabe M, Shimizu H, and Kobayashi S (2001)Tyrosine hydroxylase antisense gene therapy causes hypotensive effects in thespontaneously hypertensive rats. J Hypertens 19:1769–1773.

Kurtz TW, Griffin KA, Bidani AK, Davisson RL, and Hall JE; AHA Council on HighBlood Pressure Research, Professional and Public Education Subcommittee (2005)Recommendations for blood pressure measurement in animals: summary of anAHA scientific statement from the Council on High Blood Pressure Research,Professional and Public Education Subcommittee. Arterioscler Thromb Vasc Biol25:478–479.

Kvetnansky R, Rusnak M, Dronjak S, Krizanova O, and Sabban EL (2003) Effect ofnovel stressors on tyrosine hydroxylase gene expression in the adrenal medulla ofrepeatedly immobilized rats. Neurochem Res 28:625–630.

Lacroix JS, Anggård A, Hökfelt T, O’Hare MM, Fahrenkrug J, and Lundberg JM(1990) Neuropeptide Y: presence in sympathetic and parasympathetic innervationof the nasal mucosa. Cell Tissue Res 259:119–128.

Lange DL, Haywood JR, and Hinojosa-Laborde C (1998) Role of the adrenal medullaein male and female DOCA-salt hypertensive rats. Hypertension 31:403–408.

Levy MN (1984) Cardiac sympathetic-parasympathetic interactions. Fed Proc 43:2598–2602.

Livak KJ and Schmittgen TD (2001) Analysis of relative gene expression data usingreal-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25:402–408.

Mangos GJ, Whitworth JA, Williamson PM, and Kelly JJ (2003) Glucocorticoids andthe kidney. Nephrology 8:267–273.

Meijer OC, de Lange EC, Breimer DD, de Boer AG, Workel JO, and de Kloet ER(1998) Penetration of dexamethasone into brain glucocorticoid targets is enhancedin mdr1A P-glycoprotein knockout mice. Endocrinology 139:1789–1793.

Mondo CK, Yang WS, Su JZ, and Huang TG (2006) Atorvastatin prevented andreversed dexamethasone-induced hypertension in the rat. Clin Exp Hypertens 28:499–509.

Nagatsu T, Levitt M, and Udenfriend S (1964) Tyrosine hydroxylase: the initial stepin norepinephrine biosynthesis. J Biol Chem 239:2910–2917.

Nakashima A, Hayashi N, Kaneko YS, Mori K, Sabban EL, Nagatsu T, and Ota A(2009) Role of N-terminus of tyrosine hydroxylase in the biosynthesis of catechol-amines. J Neural Transm 116:1355–1362.

Nankova BB, Tank AW, and Sabban EL (1999) Transient or sustained transcrip-tional activation of the genes encoding rat adrenomedullary catecholamine bio-synthetic enzymes by different durations of immobilization stress.Neuroscience 94:803–808.

Ong SL, Vickers JJ, Zhang Y, McKenzie KU, Walsh CE, and Whitworth JA (2007)Role of xanthine oxidase in dexamethasone-induced hypertension in rats. Clin ExpPharmacol Physiol 34:517–519.

Panoulas VF, Douglas KM, Stavropoulos-Kalinoglou A, Metsios GS, Nightingale P,Kita MD, Elisaf MS, and Kitas GD (2008) Long-term exposure to medium-doseglucocorticoid therapy associates with hypertension in patients with rheumatoidarthritis. Rheumatology 47:72–75.

Patel P, Nankova BB, and LaGamma EF (2005) Butyrate, a gut-derived environ-mental signal, regulates tyrosine hydroxylase gene expression via a novel promoterelement. Brain Res Dev Brain Res 160:53–62.

Perlman RL and Chalfie M (1977) Catecholamine release from the adrenal medulla.Clin Endocrinol Metab 6:551–576.

Perry RR, Keiser HR, Norton JA, Wall RT, Robertson CN, Travis W, Pass HI,Walther MM, and LinehanWM (1990) Surgical management of pheochromocytomawith the use of metyrosine. Ann Surg 212:621–628.

Phillips JK, Dubey R, Sesiashvilvi E, Takeda M, Christie DL, and Lipski J (2001)Differential expression of the noradrenaline transporter in adrenergic chromaffincells, ganglion cells and nerve fibres of the rat adrenal medulla. J Chem Neuroanat21:95–104.

Piette C, Munaut C, Foidart JM, and Deprez M (2006) Treating gliomas with glu-cocorticoids: from bedside to bench. Acta Neuropathol 112:651–664.

Quinn R (2005) Comparing rat’s to human’s age: how old is my rat in people years?Nutrition 21:775–777.

Rani CS, Elango N, Wang SS, Kobayashi K, and Strong R (2009) Identification of anactivator protein-1-like sequence as the glucocorticoid response element in the rattyrosine hydroxylase gene. Mol Pharmacol 75:589–598.

Rao F, Zhang K, Zhang L, Rana BK, Wessel J, Fung MM, Rodriguez-Flores JL,Taupenot L, Ziegler MG, and O’Connor DT (2010) Human tyrosine hydroxylasenatural allelic variation: influence on autonomic function and hypertension. CellMol Neurobiol 30:1391–1394.

Rao F, Zhang L, Wessel J, Zhang K, Wen G, Kennedy BP, Rana BK, Das M,Rodriguez-Flores JL, Smith DW, et al. (2007) Tyrosine hydroxylase, therate-limiting enzyme in catecholamine biosynthesis: discovery of common humangenetic variants governing transcription, autonomic activity, and blood pressurein vivo. Circulation 116:993–1006.

Reul JM, van den Bosch FR, and de Kloet ER (1987) Relative occupation of type-I andtype-II corticosteroid receptors in rat brain following stress and dexamethasonetreatment: functional implications. J Endocrinol 115:459–467.

Saag KG (2002) Glucocorticoid use in rheumatoid arthritis. Curr Rheumatol Rep 4:218–225.

Sabban EL, Nankova BB, Serova LI, Kvetnansky R, and Liu X (2004) Molecularregulation of gene expression of catecholamine biosynthetic enzymes by stress:sympathetic ganglia versus adrenal medulla. Ann N Y Acad Sci 1018:370–377.

Sakaguchi A, LeDoux JE, and Reis DJ (1983) Sympathetic nerves and adrenal me-dulla: contributions to cardiovascular-conditioned emotional responses in sponta-neously hypertensive rats. Hypertension 5:728–738.

Sheela Rani CS, Soto-Pina A, Iacovitti L, and Strong R (2013) Evolutionary conser-vation of an atypical glucocorticoid-responsive element in the human tyrosinehydroxylase gene. J Neurochem 126:19–28.

Shimizu H, Kumai T, and Kobayashi S (2001) Involvement of tyrosine hydroxylaseupregulation in cyclosporine-induced hypertension. Jpn J Pharmacol 85:306–312.

Souverein PC, Berard A, Van Staa TP, Cooper C, Egberts AC, Leufkens HG,and Walker BR (2004) Use of oral glucocorticoids and risk of cardiovascular andcerebrovascular disease in a population based case-control study. Heart 90:859–865.

Spector S, Sjoerdsma A, and Udenfriend S (1965) Blockade of endogenous norepi-nephrine synthesis by alpha-methyl-tyrosine, an inhibitor of tyrosine hydroxylase.J Pharmacol Exp Ther 147:86–95.

Tonolo G, Soro A, Madeddu P, Troffa C, Melis MG, Patteri G, Parpaglia PP, Sabino G,Maioli M, and Glorioso N (1993) Effect of chronic intracerebroventricular dexa-methasone on blood pressure in normotensive rats. Am J Physiol 264:E843–E847.

Tripovic D, Pianova S, McLachlan EM, and Brock JA (2010) Transient supersensi-tivity to alpha-adrenoceptor agonists, and distinct hyper-reactivity to vasopressinand angiotensin II after denervation of rat tail artery. Br J Pharmacol 159:142–153.

Tümer N, Hale C, Lawler J, and Strong R (1992) Modulation of tyrosine hydroxylasegene expression in the rat adrenal gland by exercise: effects of age. Brain Res MolBrain Res 14:51–56.

Udenfriend S, Zaltzman-Nirenberg P, and Nagatsu T (1965) Inhibitors of purifiedbeef adrenal tyrosine hydroxylase. Biochem Pharmacol 14:837–845.

Ulrych M, Frohlich ED, Dustan HP, and Page IH (1968) Immediate hemodynamiceffects of beta-adrenergic blockade with propranolol in normotensive and hyper-tensive man. Circulation 37:411–416.

van Everdingen AA, Jacobs JW, Siewertsz Van Reesema DR, and Bijlsma JW (2002)Low-dose prednisone therapy for patients with early active rheumatoid arthritis:clinical efficacy, disease-modifying properties, and side effects: a randomized,double-blind, placebo-controlled clinical trial. Ann Intern Med 136:1–12.

Wallwork CJ, Parks DA, and Schmid-Schönbein GW (2003) Xanthine oxidase activityin the dexamethasone-induced hypertensive rat. Microvasc Res 66:30–37.

Watanabe T, Noshiro T, Akama H, Kusakari T, Shibukawa S, Miura W, Abe K,and Miura Y (1995) Effect of dexamethasone on plasma free dopamine: dopami-nergic modulation in hypertensive patients. Hypertens Res 18 (Suppl 1):S197–S198.

Whitworth JA, Gordon D, Andrews J, and Scoggins BA (1989) The hypertensive effectof synthetic glucocorticoids in man: role of sodium and volume. J Hypertens 7:537–549.

Whitworth JA, Mangos GJ, and Kelly JJ (2000) Cushing, cortisol, and cardiovasculardisease. Hypertension 36:912–916.

Whitworth JA, Williamson PM, Mangos G, and Kelly JJ (2005) Cardiovascularconsequences of cortisol excess. Vasc Health Risk Manag 1:291–299.

Address correspondence to: Dr. Alexandra E. Soto-Piña, Paseo Tollocanesq. Jesús Carranza, Toluca, Estado de México, México, Z.C. 50180. E-mail:aesotop@uaemex.mx

536 Soto-Piña et al.

at ASPE

T Journals on D

ecember 22, 2021

jpet.aspetjournals.orgD

ownloaded from