(Hypertension. 1995;26:208-212.)
© 1995 American Heart Association, Inc.
Articles |
Influence of Androgen on Tyrosine Hydroxylase mRNA in Adrenal Medulla of Spontaneously Hypertensive Rats
From the Department of Pharmacology, St Marianna University School of Medicine, Kawasaki, Japan.
Correspondence to Toshio Kumai, Department of Pharmacology, St Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki, 216 Japan.
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Abstract |
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Abstract We investigated the effects of castration and testosterone propionate on tyrosine hydroxylase mRNA, its activity, and catecholamine synthesis in the adrenal medulla of spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Four-week-old male rats were castrated. Testosterone propionate (500 µg per rat) was administered subcutaneously twice a week to castrated rats (between 14 and 25 weeks of age). Systolic pressure was measured at the age of 25 weeks, and rats were decapitated. The systolic pressure of castrated SHR was significantly lower than that of control and testosterone-replaced SHR. Epinephrine and norepinephrine levels, tyrosine hydroxylase activity, and tyrosine hydroxylase mRNA in the adrenal medulla of castrated SHR were significantly lower than in control and testosterone-replaced SHR. Systolic pressure and epinephrine and norepinephrine levels, tyrosine hydroxylase activity, and tyrosine hydroxylase mRNA levels in the adrenal medulla of WKY showed no significant differences among the control, castrated, and testosterone-replaced groups. These results suggest that androgens contribute to the development and maintenance of hypertension in SHR via sustained enhancement of tyrosine hydroxylase synthesis in the adrenal medulla, leading to increased epinephrine and norepinephrine levels.
Key Words: tyrosine 3-monooxygenase • RNA, messenger • androgens • adrenal medulla • epinephrine • norepinephrine • rats, inbred SHR
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Introduction |
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The sympathetic nervous system is thought to play an important role in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR).1 Ozaki et al2 reported that epinephrine and norepinephrine contents in the adrenal medulla of SHR were significantly higher than those of Wistar-Kyoto rats (WKY). The adrenal medulla plays an important role in epinephrine and norepinephrine synthesis and release.3 4 Tyrosine hydroxylase (TH) is a rate-limiting enzyme in the synthetic pathway of catecholamines. We recently reported that the expression of TH mRNA in adrenal medulla was increased in SHR.5 Therefore, the increased TH mRNA expression in the adrenal medulla may contribute to hypertension in SHR.
Epidemiological and experimental evidence suggests that androgen contributes to hypertension.6 7 We have reported that castration retards the development of hypertension in SHR.8 Cambotti et al9 demonstrated that neonatally androgenized female SHR exhibited a pattern of blood pressure (BP) increase similar to that of male SHR during maturation. Furthermore, Ganten et al10 demonstrated that chemical castration with cyproterone and flutamide, which are androgen receptor antagonists, attenuated the development of hypertension in male SHR. Thus, androgen appears to be involved in the development and maintenance of hypertension in SHR, but its mechanisms are not yet clear.
Hamill and Schroeder11 reported that androgen facilitates neuronal activity. Testosterone has been shown to modulate norepinephrine in sympathetic fibers innervating the rat vas deferens.12 Chen and Meng13 have shown that posterior hypothalamic norepinephrine stores increase in response to an androgen stimulus in SHR. Furthermore, we recently reported that TH activities and norepinephrine concentration in the abdominal aorta and mesenteric artery of SHR were decreased by castration and recovered to control levels by testosterone replacement treatments, but this did not occur in WKY.14 Therefore, it is expected that androgen might elevate BP in SHR via potentiation of sympathetic nerve activities.
To evaluate this assumption, in the present study we investigated the effects of castration and testosterone replacement on epinephrine and norepinephrine levels, TH activity, and TH mRNA level as indexes of sympathetic nerve activities in the adrenal medulla of SHR.
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Methods |
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Animals
Studies were performed according to the "Guiding Principles for the Care and Use of Laboratory Animals" of the Japanese Pharmacological Society. Male SHR and WKY rats were bred at the St Marianna University School of Medicine from stock originally supplied by Dr K. Okamoto (Kinki University). Four-week-old male SHR and WKY were castrated under light ether anesthesia. The rats were housed in a semibarrier system in a room where temperature (23±1°C), humidity (55±5%), and lighting (light from 6 AM to 6 PM) were controlled.
Drug Treatment
Testosterone propionate (500 µg per rat, Wako) dissolved
in sesame oil (500 µg per 0.1 mL) was administered subcutaneously
twice a week to castrated rats between 11 and 12 weeks of age.
BP Measurement
Systolic BP was measured in four to six rats from each
group by the tail-cuff method (PS-100, Riken Kaihatsu Co) in conscious
rats placed on a hot plate (37°C) at the age of 25 weeks. Six to
seven BP readings were obtained for each rat and averaged.
Epinephrine and Norepinephrine
Analysis
Six to 10 rats from each group were decapitated at the age of 25
weeks without anesthesia (24 hours after the final
administration of testosterone propionate), the adrenal surface was cut
with a surgical blade, and the nuclear tissues were collected and
rinsed with cold saline. We used these tissues as the adrenal medulla.
The tissues were homogenized in a glass tissue grinder in
1.05 mL ice-cold 0.05 mol/L perchloric acid with 5 ng
dihydroxybenzylamine as an internal standard.
The homogenate was centrifuged at 15 000g for 20 minutes at 4°C, and the supernatant was used for assay of epinephrine and norepinephrine. Epinephrine and norepinephrine were extracted with aluminum oxide. The supernatant was mixed with 10 mg aluminum oxide and 100 µL of 2 mol/L Tris-EDTA (pH 8.7) for 15 minutes. The precipitate was washed with 1 mL of 16.5 mmol/L Tris-EDTA (pH 8.1) and then dried and mixed with 200 µL of solvent medium (acetic acid/10% sodium metabisulfite/5% EDTA/water [0.1:0.05:0.05:9.8]) for 15 minutes followed by centrifugation at 1800g for 1 minute. The supernatant was passed through a 0.22-µm filter, and an aliquot (10 µL) was injected into a high-performance liquid chromatograph (Waters 510 pump) with an electrochemical detector (Waters 460 detector) and a Cosmosil 5C18-AR packed column (4.6x150 mm, Nakarai Tesque Co). The mobile phase consisted of the following components (mmol/L): sodium acetate 50, citric acid 20, sodium octyl sulfate 3.75, di-n-butylamine 1, and EDTA 0.134 as well as 5% (vol/vol) methanol. All separations were performed isocratically at a flow rate of 0.9 mL/min at 35°C. The detector potential was maintained at +0.65 V.
TH Activity Analysis
TH activity was measured in four to seven rats from each group
by a modification of the method of Nagatsu et al.15
Tissues were homogenized with 0.25 mol/L sucrose (50 vol)
in a glass tissue grinder. The standard incubation medium consisted of
the following components in a total volume of 250 µL: 100 µL of
tissue homogenate, 40 µL of 1 mol/L sodium acetate buffer
(pH 6.0), 40 µL of 1 mmol/L L-tyrosine, 20 µL of 1
mmol/L 6-methyl-5,6,7,8-tetrahydropterine in 1 mol/L 2-mercaptoethanol,
20 µL of 20 mmol/L catalase, and 30 µL of water. For blank
incubation, D-tyrosine was used as the substrate instead
of L-tyrosine. The medium was incubated at 37°C for 30
minutes, and the reaction was interrupted with 200 µL of 1 mol/L
perchloric acid containing 0.1 µg/mL dihydroxybenzylamine as an
internal standard and 50 µL of 0.2 mol/L EDTA in an ice bath. After
10 minutes, 150 µL of 1 mol/L potassium carbonate and 1 mL of 0.2
mol/L Tris-HCl (pH 8.5) containing 1% EDTA were added and
centrifuged at 1800g for 5 minutes. For
extraction of 3,4-dihydroxyphenylalanine (DOPA), the supernatant was
mixed with 10 mg aluminum oxide and 100 µL of 2 mol/L Tris-EDTA (pH
8.7) for 15 minutes. The precipitate was washed with 1 mL of 16.5
mmol/L Tris-EDTA (pH 8.1), dried, and then mixed with 200 µL of
solvent medium (acetic acid/10% sodium metabisulfite/5% EDTA/water
[0.1:0.05:0.05:9.8]) for 15 minutes. This medium was
centrifuged at 1800g for 1 minute. Forty microliters
of supernatant was transferred to a new tube and mixed with 17 µL of
0.1N NaOH and 10 mg of Amberlite CG-50 (Aldrich Chemical Co) for 15
minutes. The supernatant was passed through a 0.22-µm filter, and an
aliquot (20 µL) was injected into the same
chromatographic system as mentioned above. The mobile phase
consisted of the following components (mmol/L): sodium acetate 50,
citric acid 20, sodium octyl sulfate 12.5, di-n-butylamine
1, and EDTA 0.134. All separations were performed isocratically at a
flow rate of 0.6 mL/min at 28°C. The detector potential was
maintained at +0.65 V.
TH activity was calculated as the amount of DOPA formed from tyrosine per gram tissue per hour.
RNA Isolation
RNA isolation was performed in four to five rats from each group
by a modification of the method of Glisin et al.16 The
total RNA fraction was extracted from homogenized adrenal
medulla into 3 mL of guanidine thiocyanate solution (50% guanidine
thiocyanate, 100 mmol/L sodium acetate, 5 mmol/L EDTA). The
homogenate was centrifuged at 20°C for 15 hours
at 100 000g with 4 mL of 83% cesium chloride, and the
precipitate obtained was dissolved in 10 mmol/L Tris-EDTA and
10% sodium dodecyl sulfate (SDS). The protein in this solution
was removed by phenol-chloroform extraction. After the total RNA
fraction was purified by ethanol, it was dissolved in sterilized water
and measured for total RNA by absorbance at 260/280 nm. The ratio of
absorbance at 260 nm and 280 nm in samples used for analysis in
these studies was 1.8 to 2.0.
Dot Blot Analysis
TH mRNA levels were determined by dot blot
analysis.17 The rat TH cDNA (containing the region
from +14 to +1165 bp) was a gift from Dr D.M. Chikaraishi (Tufts
University School of Medicine, Boston, Mass).18 Human
ß-actin cDNA (0.4 kb, Wako Junyaku Co) was used as
control.19 The TH cDNA and ß-actin cDNA probes were
labeled with a random priming kit (Megaprime DNA labeling system
RPN-1607, Amersham) with the use of [
-32P]dCTP
(Dai-ichi Kagaku Yakuhin Co). The specific activities of the TH cDNA
probe and ß-actin cDNA probe were 1.3x1011 and
2.2x1010 Bq/µg, respectively. Dot blotting was carried
out as follows: Total RNA (2 µg) was incubated with 6 mol/L glyoxal
and dimethyl sulfoxide at 50°C for 1 hour, and then this solution was
applied to a nitrocellulose membrane. This membrane was baked at 80°C
for 60 minutes, prehybridized in hybridization buffer (50% formamide,
5% SDS, 25x Denhardt's solution, 5x SSPE, and 0.25 mg/mL salmon
sperm DNA) for 6 hours and then hybridized in hybridization buffer with
each radiolabeled probe (TH cDNA, 6.5x108 Bq/mL; ß-actin
cDNA, 1.1x108 Bq/mL) overnight. The membranes were washed
at 40°C with two changes of 5x SSPE for 15 minutes each and then
with two changes of 1x SSPE at 65°C for 30 minutes each. The
membrane was exposed to scientific imaging film (Eastman Kodak Co) with
an intensifying screen at -80°C for 1 day. The radioactivity of the
membrane was counted by a scintillation counter (LSC-1000, Aloka Co).
TH mRNA level was calculated as the ratio of the radioactivity of TH
mRNA to that of ß-actin.
Statistical Analysis
Data are presented as mean±SEM. The statistical
difference between mean values was analyzed by Student's
t test. A probability value less than .05 was considered
statistically significant.
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Results |
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Effects of Castration and Testosterone Replacement on Systolic BP in SHR and WKY
The Table shows the effects of castration and testosterone replacement on systolic BP, body weight, and adrenal medulla weight in SHR and WKY. Systolic BP of control SHR was significantly higher than that of control WKY (P<.01). Systolic BP of castrated SHR was significantly lower than that of control SHR (P<.01) and testosterone-replaced SHR (P<.01). Systolic BP of WKY showed no significant difference among the castrated, testosterone-replaced, and control groups.
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The body weights of castrated SHR and WKY were significantly lower than those of control SHR and WKY (P<.01 and P<.05, respectively). The adrenal medulla weight of control WKY was significantly higher than that of control SHR (P<.01). The adrenal medulla weight of castrated WKY was significantly higher than that of control WKY (P<.05). The ratio of adrenal medulla weight to body weight of castrated SHR and WKY was significantly higher than that of control SHR and WKY each (P<.01 and P<.05, respectively). The ratio of adrenal medulla weight to body weight of control SHR was significantly lower than that of control WKY (P<.05).
Effects of Castration and Testosterone Replacement on
Epinephrine and Norepinephrine Levels in the
Adrenal Medulla of SHR and WKY
Fig 1 shows the effects of castration and
testosterone replacement on epinephrine and
norepinephrine levels in the adrenal medulla of SHR and
WKY. Epinephrine levels of control SHR (5309.5±78.1 nmol/g
tissue) were significantly higher than those of control WKY
(2883.7±384.3 nmol/g tissue, P<.01). Epinephrine
levels of castrated SHR (3733.1±286.0 nmol/g tissue) were
significantly lower than those of control SHR (P<.01).
Epinephrine levels of testosterone-replaced SHR (4801.3±203.1
nmol/g tissue) recovered to the level of control SHR.
Epinephrine levels of WKY showed no significant difference
among the castrated (2976.5±515.3 nmol/g tissue),
testosterone-replaced (2548.6±127.7 nmol/g tissue), and control
groups.
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Norepinephrine levels of control SHR (1346.9±111.1 nmol/g tissue) were significantly higher than those of control WKY (521.9±89.2 nmol/g tissue, P<.01). Norepinephrine levels of castrated SHR (1011.2±76.2 nmol/g tissue) were significantly lower than those of control SHR (P<.05). Norepinephrine levels of testosterone-replaced SHR (1263.6±53.2 nmol/g tissue) recovered to the level of control SHR. Norepinephrine levels of WKY showed no significant difference among the castrated (614.1±108.2 nmol/g tissue), testosterone-replaced (487.6±23.0 nmol/g tissue), and control groups.
Effects of Castration and Testosterone Replacement on TH Activities
in the Adrenal Medulla of SHR and WKY
Fig 2 shows the effects of castration and
testosterone replacement on TH activities in the adrenal medulla of SHR
and WKY. The TH activity of control SHR (2057.6±211.4 nmol/g tissue
per hour) was significantly higher than that of control WKY
(949.6±89.7 nmol/g tissue per hour, P<.01). The TH
activity of castrated SHR (1052.7±335.9 nmol/g tissue per hour) was
significantly lower than that of control SHR (P<.05). The
TH activity of testosterone-replaced SHR (2003.3±617.9 nmol/g tissue
per hour) recovered to the level of control SHR. The TH activity of WKY
showed no significant difference among the castrated (1114.1±198.4
nmol/g tissue per hour), testosterone-replaced (1494.5±475.7 nmol/g
tissue per hour), and control groups.
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Effects of Castration and Testosterone Replacement on the Ratio of
TH mRNA to ß-Actin mRNA in the Adrenal Medulla of SHR and WKY
We previously confirmed that TH mRNA expression in the adrenal
medulla was detected by Northern blot analysis as a single band
of 1.8 kb.5
Fig 3 shows the effects of castration and testosterone replacement on the ratio of TH mRNA to ß-actin mRNA in the adrenal medulla of SHR and WKY. The ratio of TH mRNA to ß-actin mRNA of control SHR (1.63±0.36) was significantly higher than that of control WKY (0.47±0.11, P<.05). The ratio of TH mRNA to ß-actin mRNA of castrated SHR (0.59±0.15) was significantly lower than that of control SHR (P<.05). The ratio of TH mRNA to ß-actin mRNA of testosterone-replaced SHR (1.62±0.93) recovered to the level of control SHR. The ratio of TH mRNA to ß-actin mRNA of WKY showed no significant differences among the castrated (0.47±0.15), testosterone-replaced (0.53±0.30), and control groups.
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Discussion |
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The major findings of the present study were that systolic BP, epinephrine and norepinephrine levels, TH activity, and TH mRNA level in the adrenal medulla of SHR may be related to androgen, being significantly higher in control and testosterone-replaced male SHR than in castrated male SHR. On the other hand, those parameters of male WKY showed no significant differences among the control, castrated, and testosterone-replaced groups.
In this study, we observed that castration of male SHR retarded the development of hypertension and that testosterone replacement reversed this retardation. However, neither castration nor testosterone replacement had any effect on the systolic BP of WKY. These results were consistent with our previous findings.14 Iam and Wexler20 have demonstrated that gonadectomy at an early age (30 days) retards the development of hypertension. We have previously reported that late gonadectomy of SHR (17 weeks) also significantly reduced BP.8 Ganten et al,10 Lengsfeld et al,21 Chen and Meng,13 and Chen et al22 suggested that androgen is important in producing the male pattern of hypertension in SHR. These findings suggested that androgen may be involved in the hypertension of male SHR.
Colby et al23 demonstrated that testosterone inhibits 11ß-hydroxylase in adrenal cortices and testosterone-induced hypertension in the Sprague-Dawley rat. Gallant et al24 demonstrated that testosterone decreased the expression of cytochrome P-450 11ß mRNA. However, we could not observe the effects of testosterone on systolic BP in castrated WKY. The discrepancy in these data may be due to differences in the testosterone doses administered (Colby et al and Gallant et al used 10 mg/d, and we used 500 µg twice a week). Further study will be needed to assess the effect of our testosterone doses on 11ß-hydroxylase in the adrenal of WKY.
The adrenal medulla is known to contain large amounts of epinephrine and norepinephrine. Circulating norepinephrine originates from the adrenal medulla and postganglionic sympathetic nerve endings, and circulating epinephrine originates almost exclusively from the adrenal medulla.25 Epinephrine and norepinephrine levels in the adrenal medulla were decreased by castration of SHR, and testosterone replacement reversed this decrease. Moreover, alterations in TH activities by these treatments were consistent with those of the epinephrine and norepinephrine levels of SHR. On the other hand, neither castration nor testosterone replacement had any effect on the TH activity or epinephrine and norepinephrine levels of WKY. Kohler et al26 have reported that gonadectomy of normotensive male Sprague-Dawley rats did not alter TH activity in the mesenteric artery and that testosterone had no effect on this enzyme activity. These findings suggest that the effects of testosterone on the epinephrine and norepinephrine synthetic pathway were specific to SHR.
The TH mRNA level of SHR decreased with castration to the level seen in the SHR control group with testosterone replacement, along with alterations in epinephrine and norepinephrine levels and TH activity of SHR with these treatments. On the other hand, neither castration nor testosterone replacement had any effects on TH mRNA levels of WKY. Mueller et al27 have suggested that sympathoadrenal hyperactivity can increase the amount of TH in adrenergic areas of the central and peripheral nervous systems. Also, TH synthesis is influenced by many factors, some of which appear to act at the level of mRNA transcription. Lewis et al18 have reported that glucocorticoid and cAMP alter the transcription rate of the TH gene. Testosterone is known to play an important role in gene transcription.28 Regulation of specific gene transcription by steroid hormones is mediated by binding of the hormone receptor to steroid-responsive elements.29 Therefore, it is possible to produce alterations in TH mRNA level with castration, and testosterone replacement may interact with these gene transcription–regulating mechanisms in SHR.
Turner et al30 have reported that the BP increase shown by male SHR was related to the Y chromosome. Ely et al31 have recently shown further that the testes and androgen receptor play important roles in mediating the effects of the hypertensinogenic Y chromosome. Furthermore, Ganten et al10 have reported that BP is higher in male than in female hypertensive rats and that this sexual dimorphism is not present in normotensive rats; they further suggested that this sexual dimorphism in SHR is linked to the "hypertensive genes." These findings, coupled with our observations that testosterone increases TH mRNA levels in adrenal medulla of SHR, suggest that the hypertension seen in the control and testosterone-replaced SHR groups may be related to the catecholamine synthetic pathway of adrenal medulla, which is activated by androgen. Further studies will be needed for assessment of the mechanism of this high TH mRNA expression by testosterone in the SHR adrenal medulla.
In conclusion, our results suggest that androgen may contribute to hypertension in SHR, which increased epinephrine and norepinephrine levels because of elevated TH activity caused by high levels of TH mRNA in the adrenal medulla. Whether the transcription rate of genomic TH DNA is accelerated by androgen in the adrenal medulla of SHR still remains to be studied.
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Acknowledgments |
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We are grateful to Dr Dona M. Chikaraishi for the generous gift of rat TH cDNA. We also thank Dr Shoji Watabe and Tomoko Hiroi for their helpful discussions.
Received November 15, 1994; first decision December 16, 1994; accepted March 10, 1995.
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