Effect of Clonidine on Tyrosine Hydroxylase Activity in the Adrenal Medulla and Brain of Spontaneously Hypertensive Rats

© 2008 The Authors

Doi: 10.1111/j.1742-7843.2008.00339.x

Journal compilation

© 2008 Nordic Pharmacological Society

. Basic & Clinical Pharmacology & Toxicology

,

104

, 113–121

Blackwell Publishing Ltd

Effect of Clonidine on Tyrosine Hydroxylase Activity in the Adrenal Medulla and Brain of Spontaneously Hypertensive Rats

Eduardo Moura

1,2

, Joana Afonso

1

, Maria Paula Serrão

1

and Maria Augusta Vieira-Coelho

1,2

1

Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal, and

2

Institute for Molecular and Cell Biology, University of Porto, Porto, Portugal

(Received May 12, 2008; Accepted August 26, 2008)

Abstract:

In the present study, we evaluated the effect of the

α

2

-adrenoceptor agonist clonidine on tyrosine hydroxylaseactivity in adrenal medulla and brain of spontaneously hypertensive rats and Wistar Kyoto rats. Six-week-old animals weretreated with clonidine (100

μ

g/kg body weight, daily, i.p.) for 4 weeks. Treatment with clonidine significantly reduced meanarterial blood pressure in spontaneously hypertensive rats to values similar to normotensive Wistar Kyoto rats. In the adrenalmedulla of spontaneously hypertensive rats, clonidine treatment produced a significant increase in tyrosine hydroxylaseactivity with higher V

max

values and no changes in K

M

values. This effect was accompanied by a significant increase in tyrosinehydroxylase protein expression and of noradrenaline and adrenaline levels. In the brain of spontaneously hypertensiverats, treatment with clonidine produced a significant decrease in tyrosine hydroxylase activity with lower V

max

values andno changes in K

M

values accompanied by a significant decrease in tyrosine hydroxylase protein expression and of dopamineand noradrenaline levels. In Wistar Kyoto rats, clonidine treatment had no effect on tyrosine hydroxylase activity andprotein expression or catecholamine levels in adrenal medulla or brain. Clonidine treatment significantly reducednoradrenaline and adrenaline plasma levels in spontaneously hypertensive rats and Wistar Kyoto rats. In conclusion, treatmentwith the

α

2

-adrenoceptor agonist clonidine prevented the increase in mean arterial blood pressure in young spontaneouslyhypertensive rats. This effect was accompanied by opposite effects on tyrosine hydroxylase activity in spontaneouslyhypertensive rat adrenal medulla and brain: an increase in adrenal medulla and a decrease in brain, bringing sympathetic

function to a similar profile found in normotensive Wistar Kyoto rats.

The endogenous catecholamines noradrenaline and adrena-line mediate sympathetic regulation of blood pressure [1].These effectors of the sympathetic nervous system are syn-thesized within sympathetic neurons and chromaffin cells ofthe adrenal medulla by a well-defined series of reactionsthat start with the conversion of tyrosine to 3,4-dihydroxy-phenylalanine mediated by tyrosine hydroxylase, the initialand rate-limiting enzyme of the pathway [2]. Noradrenalineis the principal neurotransmitter of sympathetic neurons,whereas adrenaline is secreted from the chromaffin cells ofthe adrenal medulla into the circulation. Whereas the actionsof noradrenaline are mostly restricted to the sites of releasefrom sympathetic nerves, adrenaline acts as a neurohormoneto activate cardiovascular and metabolic responses via theblood stream.

Increased sympathetic nervous system activity with highertyrosine hydroxylase activity and catecholamine levels arecommon features of hypertension in human beings and inthe spontaneously hypertensive rat [3,4], an animal modelused to investigate the mechanisms of essential hypertension[5]. It is noteworthy that in spontaneously hypertensiverats subjected to neonatal sympathectomy alone [6], theextent of hypertension development is attenuated but not

prevented, whereas in spontaneously hypertensive ratssubjected to neonatal sympathectomy combined with removalof the adrenal medulla, the development of hypertension isabolished [7,8]. Therefore, the adrenal medulla in combina-tion with the sympathetic nervous system plays a fundamentalrole in the epigenesis of hypertension in spontaneously hyper-tensive rats [7–11]. However, contrary to what is found inthe sympathetic nervous system, tyrosine hydroxylase activityand expression as well as catecholamine levels are decreasedin the adrenal medulla of spontaneously hypertensive rats [12].This reduction is not dependent on changes in blood pressure,as it is already present in the pre-hypertensive phase of sponta-neously hypertensive rats (6 weeks of age) and stays low afterdevelopment of high blood pressure (12 and 22 weeks of age).

Normal sympathetic nervous system regulation and func-tion occurs via

α

2

-adrenoceptors, and almost all presynapticinhibitory autoreceptor function in the central sympatheticnervous system is exerted by the

α

2A

subtype [13]. Thissubtype is responsible for lowering sympathetic outflow andblood pressure in response to

α

2

-adrenoceptor agonist drugssuch as clonidine [14–16]. Moreover,

α

2

-adrenoceptors aresolely responsible for autocrine feedback inhibition ofcatecholamine secretion from the adrenal gland, which isnormally induced by activation of nicotinic cholinergic recep-tors [17]. Increased catecholamine release from the adrenalmedulla due to down-regulation of the inhibitory

α

2

-adrenoceptors has been implicated in the development ofheart failure in two different heart failure models [18].

Author for correspondence: Maria Augusta Vieira-Coelho, Instituteof Pharmacology and Therapeutics, Medical Faculty, University ofPorto, Alameda Prof. Hernani Monteiro, 4200-319, Porto, Portugal(fax +351-225516343, e-mail [email protected]).

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© 2008 The AuthorsJournal compilation

© 2008 Nordic Pharmacological Society.

Basic & Clinical Pharmacology & Toxicology

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, 113–121

Despite the importance of adrenal

α

2

-adrenoceptors forsympathetic nervous system function, their role in thedevelopment of hypertension is yet to be tested.

The purpose of this study was to evaluate the effect ofprolonged treatment with the

α

2

-adrenoceptor agonist clonidine,an antihypertensive drug, on catecholamine synthesis in theadrenal medulla and in the brain of spontaneously hyper-tensive rats, using the Wistar Kyoto rats as normotensivecontrol strain. Treatment was initiated in pre-hypertensivespontaneously hypertensive rats in order to test the effect ofsympathetic inhibition on development of hypertension inthis strain. The clonidine dose selected for this study (100

μ

g/kg)is at the lower end of the spectrum of doses (30

μ

g/kg to50 mg/kg) that have previously been used to investigate

α

2

-adrenoceptor-mediated functions in rodents [19–21].

Materials and Methods

Animals and tissues.

Three-week-old male spontaneously hypertensiverats and Wistar Kyoto rats were obtained from Charles River(Barcelona, Spain). The health reports of the animals were inaccordance with Federation of European Laboratory Animal Sci-ence Associations recommendations. The animals were kept undercontrolled environmental conditions (a 12-hr light:dark cycle androom temperature 22 ± 2

°

). All animals were fed

ad libitum

through-out the study with standard rat chow (PANLAB, Barcelona, Spain).Animals were selected after a 5-day period of stabilization andadaptation to blood pressure measurements. Blood pressure (systo-lic and diastolic) and heart rate were measured in consciousrestrained animals between 7:00 and 10:00 a.m., using a photoelectrictail-cuff pulse detector (LE 5000, Letica, Barcelona, Spain). Fivedeterminations were made each time and the mean was used forfurther calculation. We calculated mean arterial blood pressure (MAP)as follows: MAP = DBP + [(SBP – DBP)/3], where DBP and SBPare diastolic and systolic blood pressures. From the age of 6 weeks,clonidine (100

μ

g/kg body weight) or saline (placebo) was adminis-tered (intraperitoneally) daily to spontaneously hypertensive ratsand Wistar Kyoto rats for a period of 4 weeks. Having reached4 weeks of treatment (10 weeks of age), rats were anaesthetized withsodium pentobarbital (60 mg/kg body weight, intraperitoneally).Blood from the vena cava was collected into tubes containingheparin for plasma catecholamine determination. The right and leftadrenal gland were rapidly removed through an abdominal midlineincision and rinsed free of blood with saline. The right gland wasplaced in a vial containing 1 ml of 0.2 mol/l perchloric acid forcatecholamine assay and the left gland was immediately frozen at–80

°

for evaluation of tyrosine hydroxylase activity and expression.The brain was also removed and placed in a vial containing 1.0 mlof 0.2 mol/l perchloric acid or immediately frozen at –80

°

for evalu-ation of tyrosine hydroxylase activity and expression.

Tyrosine hydroxylase activity.

Tyrosine hydroxylase activity wasmeasured as previously described [12]. In brief, the left adrenalgland or the brain was homogenized in phosphate buffer solution(50 mmol/l, pH 7.0, containing 0.2% Triton X-100 and 0.35% Pefabloc– Boehringer Mannheim GmbH, Mannheim, Germany) and centri-fuged at 12,000

×

g for 20 min. (4

°

), recovering the supernatant.Protein concentration was measured as previously described [22].An aliquot of 40

μ

l was used for each tyrosine hydroxylase assay.The reaction mixture for the tyrosine hydroxylase assay containedsodium acetate buffer (0.1 M, pH = 6.0), 6-methyl-5,6,7,8-tetra-hydropterine in a solution of 2-mercaptoethanol (1 M) and ferroussulphate heptahydrate (0.50 mM). In the standard assay, 10-min.incubation at 37

°

was performed either with a saturating concentrationof the substrate (L-tyrosine, 1000

μ

M) and varying concentrations

of the cofactor (6-methyl-5,6,7,8-tetrahydropterine, 10–1000

μ

M),or with a saturating concentration of the cofactor (6-methyl-5,6,7,8-tetrahydropterine, 1000

μ

M) and varying concentrations of substrate(L-tyrosine, 10–1000

μ

M). The reaction was stopped with 300

μ

l ofperchloric acid (0.5 M). In the blank incubation, 3-iodo-L-tyrosine(100

μ

M), a tyrosine hydroxylase inhibitor, was present and L-tyrosinewas replaced by D-tyrosine (10–1000

μ

M). The 3,4-dihydroxy-phenylanine formed in the reaction was measured by high-performanceliquid chromatography with electrochemical detection. Tyrosinehydroxylase activity was expressed as the amount of 3,4-dihydroxy-phenylanine formed per milligram protein per hour.

Tyrosine hydroxylase Western blot analysis.

An aliquot (20

μ

g) of thehomogenates used for tyrosine hydroxylase activity assay was frac-tioned by 10% sodium dodecyl sulphate-polyacrylamide gel electro-phoresis as previously described [23] and transferred to supportednitrocellulose membranes (Sigma, St Louis, CA, USA) using asemi-dry Transphor system (Hoeffer TE 70 Series, AmershamPharmacia Biotech, Piscataway, NJ, USA) as previously described[24]. For the assay of tyrosine hydroxylase expression, the mem-branes were blocked in 3% non-fat dry milk in phosphate-bufferedsaline (PBS)-Tween (PBS, pH 7.4 containing 0.05% Tween-20) for20 min. and then incubated with the rabbit anti-tyrosine hydroxy-lase affinity purified monoclonal antibody (Chemicon InternationalInc., Temecula, CA, USA) for 2 hr (diluted 1:1000). For the assay of

β

-actin abundance, membranes were incubated with the anti-

β

-actinprimary antibody (Santa Cruz Biotechnology Inc., Santa Cruz,CA, USA) for 1 hr (diluted 1:15,000). The immunoblots were sub-sequently washed and incubated with the respective fluorescentlylabelled secondary antibody (goat anti-rabbit from Rockland) for1 hr at room temperature and protected from light. Membraneswere washed and imaged by scanning at 700 nm or 800 nm withthe Odyssey Infrared System (LICOR Biosciences, Lincoln, NE,USA). Data were normalized to the expression of

β

-actin and areshown as percentage of the mean density of the control group.

Assay of catecholamines.

The assay of the catecholamines dopamine,noradrenaline and adrenaline and of 3,4-dihydroxyphenylanine, inthe right adrenal gland, brain and plasma was performed by high-performance liquid chromatography with electrochemical detectionas previously described [25]. In brief, aliquots of 300

μ

l of perchloricacid where tissues had been kept or 1000

μ

l of the plasma supernatantwere placed in 5-ml conical base glass vials containing 50 mg ofalumina, and the pH of the samples was adjusted to 8.6 by additionof Tris buffer. 3,4-Dihydroxybenzylamine hydrobromide was usedas internal standard. The adsorbed catecholamines were then elutedfrom the alumina with 200

μ

l of 0.2 mol/l perchloric acid on CostarSpin-X microfilter tubes; 50

μ

l of the eluate was injected into ahigh-performance liquid chromatography with electrochemical detec-tion system (Gilson Model 141, Gilson Medical Electronics, Villiers,Le Bel, France). The lower limit of detection of catecholamines and3,4-dihydroxyphenylanine ranged from 350 to 1000 fmol.

Data analysis.

Results are expressed as mean ± standard error ofmeans (S.E.M.). The kinetic parameters, V

max

and K

M

values for theactivity of tyrosine hydroxylase were calculated from non-linearregression analysis by using the Graphpad Prism Software package(version 5.0). This enzyme kinetic study was performed usingtriplicate samples from each experimental group of six animals.The significance of differences between means was evaluatedusing Student’s t-test or two-way analysis of variance. Values wereconsidered statistically different when P < 0.05.

Results

Cardiovascular parameters.

In the beginning of the study (6-week-old animals), meanarterial blood pressure and heart rate values were similar

INFLUENCE OF CLONIDINE ON SHRTH ACTIVITY

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between Wistar Kyoto rats and spontaneously hypertensiverats (fig. 1). In spontaneously hypertensive rats treated withplacebo, mean arterial blood pressure and heart rate valuesprogressively increased with age. After 4 weeks (10-week-oldanimals), mean arterial blood pressure values were about50 mmHg higher in spontaneously hypertensive ratscompared to Wistar Kyoto rats treated with placebo (fig. 1).This hypertensive profile was prevented by clonidine. At theend of 4 weeks of treatment with clonidine, spontaneouslyhypertensive rats presented mean arterial blood pressureand heart rate values similar to normotensive Wistar Kyoto

rats (fig. 1). In Wistar Kyoto rats, clonidine treatment pro-duced a significant reduction in mean arterial blood pressurevalues from the first to the third week of the study, but bythe fourth week this hypotensive effect was no longer present.

Plasma catecholamine levels.

At the end of the study, spontaneously hypertensive ratstreated with placebo had significantly higher noradrenalineplasma levels (fig. 2A) compared to Wistar Kyoto rats treatedwith placebo, but similar adrenaline plasma levels (fig. 2B).Treatment with clonidine significantly reduced noradrenaline

Fig. 1. Mean arterial blood pressure (A) (mmHg) and heart rate (B) (b.p.m.) in Wistar Kyoto (WKY) rats and spontaneously hypertensiverats (SHR) during treatment with placebo or clonidine. Symbols represent mean and vertical lines show S.E.M. (n = 6). *P < 0.05 versusWistar Kyoto rats. #P < 0.05 versus corresponding values for placebo.

Fig. 2. Plasma levels of noradrenaline (A) and adrenaline (B) from Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR)after 4-week treatment with placebo or clonidine. Columns represent mean and vertical lines show S.E.M. (n = 6). *P < 0.05 versus WistarKyoto rats. #P < 0.05 versus corresponding values for placebo.

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and adrenaline plasma levels in both spontaneously hyper-tensive rats and Wistar Kyoto rats. In spontaneously hypertensiverats, noradrenaline plasma levels dropped to values similarto Wistar Kyoto rats treated with placebo, but did not reachthe noradrenaline plasma values of Wistar Kyoto rats treatedwith clonidine.

Adrenal and brain catecholamine content.

Noradrenaline and adrenaline tissue levels in the adrenalmedulla of spontaneously hypertensive rats treated withplacebo were significantly lower compared to Wistar Kyotorats treated with placebo (fig. 3A,B). Treatment with clonidineincreased the noradrenaline and adrenaline adrenal tissuelevels in spontaneously hypertensive rats, but did not produceany significant changes in Wistar Kyoto rats (fig. 3A,B).In the brain, noradrenaline and dopamine tissue levels inspontaneously hypertensive rats were significantly higher

compared to Wistar Kyoto rats (fig. 4A,B). Treatment withclonidine significantly reduced noradrenaline and dopaminetissue levels in the brain of spontaneously hypertensive rats,without significant changes in Wistar Kyoto rats (fig. 4A,B).

Tyrosine hydroxylase activity and expression.

In agreement with catecholamine content, adrenal tyrosinehydroxylase activity increased in spontaneously hypertensiverats treated with clonidine with no significant changes inWistar Kyoto rats (table 1). Also in good agreement with thecatecholamine content, brain tyrosine hydroxylase activitywas significantly reduced in spontaneously hypertensive ratstreated with clonidine with no changes in Wistar Kyoto rats(table 2). Tyrosine hydroxylase protein expression accom-panies the changes in enzyme kinetics, as in the adrenals ofspontaneously hypertensive rats, tyrosine hydroxylasetotal protein expression significantly increases with clonidine

Fig. 3. Adrenal tissue levels of noradrenaline (A) and adrenaline (B) from Wistar Kyoto (WKY) rats and spontaneously hypertensive rats(SHR) after 4-week treatment with placebo or clonidine. Columns represent mean and vertical lines show S.E.M. (n = 6). *P < 0.05 versusWistar Kyoto rats. #P < 0.05 versus corresponding values for placebo.

Fig. 4. Brain tissue levels of noradrenaline (A) and dopamine (B) from Wistar Kyoto (WKY) rats and spontaneously hypertensive rats(SHR) after 4-week treatment with placebo or clonidine. Columns represent mean and vertical lines show S.E.M. (n = 6). *P < 0.05 versusWistar Kyoto rats. #P < 0.05 versus corresponding values for placebo.


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treatment (fig. 5A), while in the brain tyrosine hydroxylasetotal protein decreases with clonidine treatment (fig. 5B).In Wistar Kyoto rats, no significant differences were observedin total tyrosine hydroxylase protein expression after clonidinetreatment, both in adrenals and in the brain (fig. 5A,B).

Discussion

In this study, we have shown that in spontaneously hyper-tensive rats, prolonged treatment with the

α

2

-adrenoceptoragonist clonidine increased tyrosine hydroxylase activityand catecholamine tissue levels in adrenal medulla while havingthe opposite effect in the same parameters in the brain, adecrease. These changes are accompanied by a reduction inthe release of noradrenaline from sympathetic nerves andadrenaline from chromaffin cells of the adrenal medulla, asreflected by the plasma levels of these catecholamines.

In a previous study, our group showed that in the adrenalmedulla of spontaneously hypertensive rats compared toage-matched normotensive Wistar Kyoto rats, tyrosinehydroxylase activity and catecholamine levels are reducedbefore and after development of high blood pressure [12].We have broadened the scope of the present study to includethe central nervous system and evaluated tyrosine hydroxy-lase activity and catecholamine levels in the brain. In agree-ment with our previous data, here we show that in theadrenal medulla of spontaneously hypertensive rats withestablished hypertension (10 weeks old), tyrosine hydroxylaseactivity and expression along with noradrenaline and adren-aline tissue content are decreased compared to age-matchedWistar Kyoto rats, whereas in the central nervous system,tyrosine hydroxylase activity and expression as well as cate-cholamine tissue levels are increased.

In adrenal medullary cells, tyrosine hydroxylase is therate-limiting enzyme involved in the biosynthesis of catecho-lamines. Tyrosine hydroxylase activity can be regulated by alarge array of physiological regulatory mechanisms that canbe divided into short-term and long-term regulation mecha-nisms. Short-term regulation of enzyme activity occurs atthe posttranscriptional level where phosphorylation oftyrosine hydroxylase leads to its activation. Long-termregulation occurs after tyrosine hydroxylase gene transcriptionat the level of protein synthesis [2]. Long-term regulation oftyrosine hydroxylase can also occur through feedback inhi-bition by catecholamines via two inhibitory pathways: oneis a classic kinetic-mediated and readily reversible inhibitionof the enzyme that acts as a sensor for local concentrationsof catecholamine product; a second involves a long-termsequestration of the enzyme in a less active but more stableform [2].

The fact that spontaneously hypertensive rats have areduction in tyrosine hydroxylase activity and catecholaminelevels in the adrenal medulla could appear controversial, giventhat this tissue plays an important role in the epigenesis ofspontaneously hypertensive rats hypertension. In fact, spon-taneously hypertensive rats have increased noradrenalineplasma levels before and after development of high bloodpressure, but adrenaline levels remain similar to what isobserved in Wistar Kyoto rats [12]. A possible explanation isthat the adrenal medulla responds to changes in noradrena-line plasma levels by increasing or decreasing catecholaminesynthesis in order to balance out the changes in sympatheticdrive. In this view, the reduced expression of tyrosine hydroxy-lase and of catecholamine levels in the adrenal medulla ofspontaneously hypertensive rats may be peripheral down-regulation due to increased central stimulation. The decreased

Table 1.

Kinetic parameters maximum velocity (Vmax) and Michaelis constant (KM) of tyrosine hydroxylase activity for the substrate (L-tyrosine) andthe cofactor 6-methyl-5,6,7,8-tetrahydropterine 6-BH4.

L-tyrosine 6-BH4

WKY-P WKY-C SHR-P SHR-C WKY-P WKY-C SHR-P SHR-C

Vmax (nmol/mg protein/hr) 22.78 ± 0.90 22.81 ± 0.70 18.26 ± 0.931 23.32 ± 1.132 15.11 ± 0.71 14.40 ± 1.25 10.29 ± 0.941 14.78 ± 0.692

KM (μM) 51 ± 8 41 ± 5 51 ± 10 51 ± 10 77 ± 13 62 ± 19 65 ± 19 51 ± 10

Enzyme assay was performed with adrenal homogenates obtained from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) after 4-weektreatment with placebo (P) or clonidine (C). Values are presented as mean ± S.E.M. (n = 6). 1P < 0.05 versus Wistar Kyoto rats. 2P < 0.05 versuscorresponding values for placebo.

Table 2.

Kinetic parameters maximum velocity (Vmax) and Michaelis constant (KM) of tyrosine hydroxylase activity for the substrate (L-tyrosine) andthe cofactor 6-methyl-5,6,7,8-tetrahydropterine 6-BH4.

L-tyrosine 6-BH4

WKY-P WKY-C SHR-P SHR-C WKY-P WKY-C SHR-P SHR-C

Vmax (nmol/mg protein/hr) 1.38 ± 0.07 1.47 ± 0.11 1.83 ± 0.081 1.37 ± 0.152 1.09 ± 0.06 1.09 ± 0.14 1.56 ± 0.091 1.03 ± 0.132

KM (μM) 73 ± 11 87 ± 19 66 ± 9 54 ± 9 21 ± 6 27 ± 7 42 ± 10 19 ± 6

Enzyme assay was performed with brain homogenates obtained from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) after 4-weektreatment with placebo (P) or clonidine (C). Values are presented as mean ± S.E.M. (n = 6). 1P < 0.05 versus Wistar Kyoto rats. 2P < 0.05 versuscorresponding values for placebo.

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tyrosine hydroxylase activity/expression and catecholaminelevels in the adrenal medulla of spontaneously hypertensiverats would be end-product feedback inhibition by the increasedplasma noradrenaline. In support of this notion, our resultsshow that treatment with clonidine produced a reduction innoradrenaline plasma levels in spontaneously hypertensiverats to values close to those of the normotensive WistarKyoto rats and this was accompanied by an increase intyrosine hydroxylase activity and catecholamine levels in theadrenal medulla of spontaneously hypertensive rats.

Consistent with the idea that catecholamine synthesis inthe central nervous system and in the periphery is differentlyregulated and in agreement with previous reports [26–28],we also show that the activity of tyrosine hydroxylase andcatecholamine levels in the brain of spontaneously hyper-tensive rats was reduced after clonidine treatment. Catecho-lamine synthesis in spontaneously hypertensive rat brain hasbeen shown to be decreased in the pre-hypertensive stageand increased in hypertensive animals [29]. Interestingly,clonidine treatment has little or no effect on brain tyrosinehydroxylase in adult spontaneously hypertensive rats [26,30,31].One may raise the possibility that by abolishing the increasein catecholamine synthesis in the central nervous system,the development of high blood pressure may have beenprevented. Given that the mechanism responsible for thereduction in tyrosine hydroxylase in the central nervoussystem appears to be desensitized and unresponsive totherapy after development of hypertension, changes in thismechanism may provide clues for the trigger that leads tothe development of hypertension in spontaneously hyper-tensive rat. In this view, an interesting finding was that thedecrease in catecholamine synthesis by the reduced tyrosinehydroxylase activity had a more marked effect on dopaminelevels than on noradrenaline levels. Altered dopaminergic

function may be involved in the development of hyperten-sion and that increased central dopaminergic activity is oneof the causes of hypertension in spontaneously hypertensiverat [32]. Although a study of the dopaminergic system isbeyond the scope of this work, it would be interesting tounderstand if changes in central dopamine levels may some-how contribute to the observed changes in blood pressure.

Interestingly, clonidine treatment also reduced noradren-aline levels in Wistar Kyoto rats, but did not have an effecton tyrosine hydroxylase activity or catecholamine levels inthe adrenal medulla and in the brain of these animals.Modulation of tyrosine hydroxylase activity and expressionby changes in noradrenaline levels from sympatheticnervous system activity may therefore be physiologicallylimited. There are other mechanisms controlling catecholaminesynthesis in brain and the adrenal medulla besides changesin noradrenaline levels that could signal changes in sympa-thetic activity. One other example for differential regulationof tyrosine hydroxylase could be attributed to angiotensin II.Whereas activation of the angiotensin II type 1 receptor inthe brain increases tyrosine hydroxylase activity andexpression [33] in the adrenal medulla activation of the pre-dominant angiotensin II type 2 receptor significantlyreduces the net intracellular content of catecholamine. Thisprobably occurs through both inhibition of catecholaminesynthesis and stimulation of catecholamine secretion, subse-quently leading to catecholamine depletion and sym-pathoadrenergic inhibition [34]. Spontaneously hypertensiverats have a global up-regulation of angiotensin II type 1receptor gene expression [35], whereas angiotensin II type 2receptor remains unaltered [36]. Nevertheless, given thatangiotensin II plasma levels are unaltered in spontaneouslyhypertensive rats [37] and there are no significant differencesin angiotensin II type 2 receptor in the adrenal medulla, it is

Fig. 5. Bottom, representative Western blots for tyrosine hydroxylase, including β-actin as normalization control, performed on proteinextracts from whole adrenal medulla (A) and whole brain (B) of Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) after4-week treatment with placebo or clonidine. Top, densitometric analysis (normalized to actin levels) of six independent experiments performedin each group. Columns indicate relative density and represent mean of four separate experiments; vertical lines show SEM (n = 6). *P < 0.05versus Wistar Kyoto rats. #P < 0.05 versus corresponding values for placebo.


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not likely that changes in adrenal of spontaneously hypertensiverats could be explained by this mechanism.

It should be pointed out that this feature of the adrenalmedulla is not limited to spontaneously hypertensive rathypertension. The same has also been observed in an animalmodel of secondary hypertension, the transgenic hypertensiveTGR (mREN2) 27 rats: tyrosine hydroxylase activity/expressionand catecholamine tissue levels are increased in the centralnervous system (hypothalamus) and reduced in the adrenalmedulla [38]. Furthermore, like what we report for the spon-taneously hypertensive rats, this difference is present in pre-hypertensive animals (4 weeks of age) and in animals withhigh blood pressure (10 weeks of age). Taken together, thisstudy and our data provide support to the notion that theincreased sympathetic central nervous system activity inhypertension is accompanied by a reduction in adrenalmedulla sympathetic activity.

The sympathetic nervous system plays an important rolein the regulation of blood pressure. Plasma catecholamineconcentrations are considered to be reliable indices ofsympatho-neuronal (noradrenaline) and sympatho-adrenal(adrenaline) activity and reactivity. The striking efficacy ofcomplete sympatho-adrenal ablation indicates importantroles for both circulating noradrenaline and adrenaline.Targeted inhibition of the sympatho-adrenal system by reduc-ing sympathetic activity therefore represents a therapeuticstrategy for spontaneously hypertensive rat hypertension.

Clonidine is a sympatholytic drug with central and peripheraleffects [39]. Clonidine may act at two anatomic sites to lowerblood pressure [40]. In addition, clonidine may activatepresynaptic inhibitory

α

2

-adrenoceptors on postganglionicsympathetic fibres to lower sympathetic noradrenaline release.Clonidine has also been shown to reduce adrenaline releasefrom the adrenal medulla not only via central mechanism,but also by direct activation of receptors in the adrenalmedulla [41]. Recently, it was shown that

α

2

-adrenoceptorsmediate an inhibitory feedback mechanism by a similarmechanism to that present in the sympathetic nerve termi-nals, but for adrenaline and noradrenaline release from theadrenal medulla [16].

Although treatment with clonidine produced a significantreduction in both noradrenaline and adrenaline plasmalevels of Wistar Kyoto rats and spontaneously hypertensiverats, noradrenaline levels remained significantly higher inspontaneously hypertensive rats compared to Wistar Kyotorats. However, the reduction in adrenaline levels afterclonidine treatment was similar between spontaneously hyper-tensive rats and Wistar Kyoto rats. One possible explanationfor this difference is that control of adrenaline release fromthe adrenal medulla remains functional, despite the bluntedcontrol of noradrenaline from sympathetic terminals. Themain

α

2

-adrenoceptor subtype regulating noradrenalinerelease from sympathetic nerve terminals,

α

2A

, has beenreported to be down-regulated in spontaneously hyper-tensive rats [42], whereas there is yet no evidence for changes in

α

2

-adrenceptors of the adrenal medulla. The idea that theinhibitory mechanism of the adrenal medulla of spontane-

ously hypertensive rats is functional has some support fromstudies in which the response to stress was evaluated in theserats [43,44]. Spontaneously hypertensive rats have a fasteradaptation to stress than Wistar Kyoto rats, resulting in partfrom a reduction of the evoked release of catecholaminesinto blood. Although further studies need to be carried outto assess the inhibitory function of

α

2

-adrenoceptors in theadrenal medulla of spontaneously hypertensive rats, thisstudy provides further support to the idea that adrenal func-tion is not blunted during development of hypertension inspontaneously hypertensive rats and may respond to changesin sympathetic drive.

The feedback inhibitory mechanism of

α

2

-adrenoceptorsin the adrenal medulla has been shown to be important forcardiovascular function. Mice lacking this receptor haveincreased adrenaline plasma levels and are more prone todevelopment of heart failure [18,45]. Furthermore, in miceand in rats that develop heart failure in association toincrease adrenergic drive from the adrenal medulla, recoveryof the inhibitory adrenal function rescues heart function[18]. Spontaneously hypertensive rats do not develop heartfailure before 18 months of age. It would be interesting toinvestigate if changes in this mechanism play a role in thisfunctional change in these animals.

One may also speculate about the role of the adrenalmedulla in the development of hypertension. Given that theadrenal medulla is the major source of adrenaline and ourstudy shows that the control of adrenaline synthesis andrelease is functional in spontaneously hypertensive rats, it ismore likely that changes in targeted organs of this aminecontribute to the observed effect on blood pressure.

Clonidine suppressed the increase in blood pressure thatis characteristic in spontaneously hypertensive rats from6 weeks of age and onwards. This was achieved by dailyadministration of this drug during the 4 weeks. This givesfurther support to the notion that regulation of catecho-lamine release plays a major role in spontaneously hyper-tensive rat hypertension, via

α

2

-adrenoceptors. However, in thisstudy, the effect of clonidine withdrawal after 4-weektreatment was not addressed. Atkinson and coworkers havepreviously shown that although chronic clonidine treatmentimproved cardiovascular parameters in 3-month-old spon-taneously hypertensive rats, withdrawal from clonidine wasmarked by tachycardia and an increase in mean arterialblood pressure and in heart rate [26]. Given that in thisstudy, we began treatment with clonidine in young normo-tensive spontaneously hypertensive rats, rather than hyper-tensive rats, the question of whether or not this treatmentabolishes or attenuates the development of hypertension inspontaneously hypertensive rats remains open. In a previousstudy, pre-weaning treatment with the

α

1

/

β

-adrenoceptorantagonist carvedilol attenuated, but did not prevent thedevelopment of hypertension in spontaneously hypertensiverats [46]. It would also be interesting to see if a similartreatment targeting of

α

2

-adrenoceptors would be able toabolish the development of hypertension in spontaneouslyhypertensive rats.

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In conclusion, clonidine treatment had opposite effectson tyrosine hydroxylase activity and catecholamine levelson brain and adrenal medulla of spontaneously hypertensiverats, a decrease in the brain, and an increase in adrenal medulla.Treatment with clonidine produces a decrease in noradrena-line and adrenaline release in both Wistar Kyoto rats andspontaneously hypertensive rats, and prevented hypertensiondevelopment in the latter. Targeted inhibition of the sympatho-adrenal system by activation of

α

2

-adrenoceptors may providefurther insight to spontaneously hypertensive rat hypertension.

AcknowledgementsThis work was supported by grants from FCT, Programa

Ciência, Tecnologia e Inovação do Quadro Comunitáio deApoio and FEDER (PTDC/SAU-FCF/66502/2006).

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Effect of Clonidine on Tyrosine Hydroxylase Activity in the Adrenal Medulla and Brain of Spontaneously Hypertensive Rats