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Department of Biology, The University of Akron, Akron, Ohio 44325-3908
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The objective of this study
was to compare strain and gender differences in kidney and heart
norepinephrine (NE) content and turnover rate in normotensive
Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR,
SHR/a, and SHR/y). Our laboratory has shown that the Y chromosome has a
significant effect on blood pressure in the SHR model of hypertension
through the use of two new rat stains, SHR/a and SHR/y, to study the Y
chromosome. SHR/a have a SHR autosomal genetic background with a WKY Y
chromosome, whereas the SHR/y rats have a WKY autosomal genetic
background with a SHR Y chromosome. Tissues were homogenized after
-methyl-DL-p-tyrosine injection and analyzed
for NE. The male kidney NE content was significantly lower in the WKY
compared with the SHR, SHR/y, and SHR/a. Kidney and heart NE content
was significantly higher in females compared with males in all strains
except the SHR/y. The WKY and SHR/y females had significantly lower
kidney NE turnover rates, and the SHR and SHR/a females had
significantly higher kidney NE turnover rates than strain-matched
males. This study suggests both a strain and gender difference in
sympathetic nervous system activity through noradrenergic neurotransmission.
Y chromosome; borderline hypertension; sympathetic nervous system
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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NUMEROUS STUDIES HAVE SHOWN that increased activity of the sympathetic nervous system (SNS) plays a role in the pathogenesis of essential hypertension (10, 20, 26, 29). SNS activity can be estimated by measurement of plasma catecholamine levels (13), norepinephrine (NE) turnover (9) or spillover rate (8), microneurography (33), and heart rate variability (28).
Our laboratory has shown that the Y chromosome from a spontaneously hypertensive rat (SHR) father when backcrossed to a normotensive Wistar-Kyoto rat (WKY) mother increased SNS indexes (4) and maintained an increase in blood pressure (BP) of ~15-20 mmHg even after 11 generations (5). The Y chromosome effect accelerated the pubertal rise of androgen levels (6) and required the androgen receptor for full effect (7). In addition, testosterone and the SHR Y chromosome increased the storage and release of NE in the isolated kidney (19).
Additionally, gender differences in hypertension have been demonstrated. The incidence and severity of hypertension have been shown to be lower in women than men (30). Because gender differences in SNS regulation have been implicated in many models of hypertension (2, 14-17, 24), it is possible that one mechanism of protection in females may be reduced activation or enhanced inhibition of the SNS. For instance, Li and Duckles (25) have shown that sensitivity to adrenergic nerve stimulation in rat tail arteries was greater in males compared with females. The amount of NE available to modulate an end-organ response can be affected by neurotransmitter synthesis, release, and reuptake.
NE stores are maintained in equilibrium by balanced processes of
synthesis and removal. Brodie et al. (1) have shown that catecholamine levels decline exponentially after pharmacological blockade of tyrosine hydroxylase (TH), the rate-limiting enzyme in the
NE synthesis pathway. TH activity is one technique to assess catecholamine synthesis. In the work of Kohler et al.
(21), no gender differences were observed in TH activity
in vascular tissue, and its activity was not affected by gonadectomy.
However, more recently Kumai and co-workers demonstrated that
castration lowered vascular (23) and adrenal medullary
(22) TH activities in SHR. TH activities in
testosterone-replaced SHR recovered to the levels obtained in intact
SHR. Gender differences in presynaptic 2-adrenoceptor
number have been observed (18, 27), and, when blocked, the
increased release of NE to nerve stimulation was greater in isolated
hearts from females compared with males and was abolished by
ovariectomy (3).
Therefore the objective of this study was to compare gender differences in NE content and turnover rate in normotensive and hypertensive rats. We hypothesized that if the Y chromosome was responsible for SNS hyperactivity, then the strains with the SHR Y chromosome would have the highest kidney and heart NE content and/or turnover compared with the strains with the Y chromosome for the normotensive WKY. In addition, we predicted that the females will have lower kidney and heart NE content and turnover rate than the strain-matched males.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Rat strains. The parental WKY/hsd and SHR/hsd strains were originally obtained from Harlan Sprague Dawley (Indianapolis, IN) and have been inbred in our laboratory since 1981. In the following studies, we also used the consomic strain (SHR/y ua), developed in our laboratory, which has the SHR Y chromosome backcrossed into the WKY background for 17 generations (32). Briefly, a WKY female was mated with a SHR male. The males of the F1 generation were mated with a WKY female. This protocol was continued for 17 generations. As a result, 99.9% of the autosomes of the SHR/y strain are from the WKY strain and only the Y chromosome is from the SHR strain. A second congenic was developed concurrently from a SHR × WKY cross, and sons were backcrossed to a SHR. This strain is designated SHR/a, because it has the SHR autosomal, X chromosomal, and pseudoautosomal regions of SHR with a WKY Y chromosome. The SHR/y and SHR/a male strains are hypertensive. Females in all strains have lower BP than the strain-matched males. The SHR/y and SHR/a females are borderline hypertensive (32).
Rats were acclimated from birth to a 12-h light (0600-1800)-dark (1800-0600) cycle, and this was continued throughout the entire experimental procedure with a controlled temperature (27-29°C) and humidity (50-70%). All animals were treated in a humane manner according to the National Institutes of Health guidelines, and all experiments were approved by the University of Akron Institutional Animal Use and Care Committee.Catecholamine content and turnover.
-Methyl tyrosine inhibits TH and prevents reaccumulation of
NE, which is released in response to neural stimuli. The endogenous tissue levels then decline at a rate proportional to the initial NE
concentration (31). When the log of NE is plotted vs.
time, the slope of the straight line is 0.434 times the fractional
turnover rate (k). The NE turnover rate is calculated as the
product of k times the endogenous concentration of NE at
time 0 (1). The reciprocal of k is
the turnover time (1) or the time interval required for
the biosynthesis of an amount of NE equal to that stored in the tissue.
-methyl-DL-p-tyrosine (Sigma Chemical; an
initial dose of 250 mg/kg ip at time 0 and booster
injections of 125 mg/kg every 3 h) until termination
(12). Control rats were injected with saline at time
0. Zero, 3, 6 and 15 h after saline or
-methyl-DL-p-tyrosine injection, rats were
anesthetized with sodium brevital (50 mg/kg ip, Lilly, Indianapolis,
IN) and decapitated, and heart and kidney were rapidly removed. Tissues
were weighed, frozen in liquid nitrogen, and stored at 
80°C until
NE assay. Tissues were homogenized in ice-cold mobile phase. After
removal of protein by centrifugation, NE was assayed by using an HPLC
system with an electrochemical detector (11).
Statistics. All values are expressed as means ± SE. The k of NE was calculated by least-square linear regression of the log NE content vs. time. Multiple comparisons were performed by two-way ANOVA and pairwise t-tests. Significance was assumed if P < 0.05. All statistics were performed using SigmaStat (Jandel Scientific).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Male kidney strain comparisons (Fig.
1A) showed a significantly
lower NE content in the WKY compared with the other strains with no
significant differences between the SHR, SHR/y, and SHR/a strains.
Female kidney strain comparisons (Fig. 1A) showed a
significantly lower NE content in the WKY and SHR/y compared with the
SHR and SHR/a with no significant difference between the WKY and SHR/y or between the SHR and SHR/a. Kidney NE content showed a significant interaction between strain and gender.
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The NE content was significantly higher in females compared with males in both kidney (Fig. 1A) and heart (Fig. 1B) in all but the SHR/y strain, in which there was no significant gender difference.
Heart NE content (Fig. 1B) showed a significant interaction between strain and gender, implying an effect of strain that is influenced by gender (and vice versa).
Figure 2 shows a typical example of NE
turnover in the kidney. There was a significantly lower kidney NE
turnover in the WKY males compared with the SHR/y males (Fig.
2A). There was no significant difference in turnover between
the WKY and the SHR/y females (Fig. 2B). There was no
significant kidney difference in NE turnover between the male SHR and
SHR/a or the female SHR and SHR/a.
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Figure 3A compares the kidney
NE turnover rate in both genders of all four strains. WKY males and
SHR/y males were significantly higher than strain-matched females. By
contrast, SHR females and SHR/a females were significantly higher than
strain-matched males. There was a significantly lower NE kidney
turnover rate in the WKY males compared with the other male strains.
There was no significant difference in NE kidney turnover between the
female WKY and SHR/y. Figure 3B shows no significant
difference in heart NE turnover between the WKY and SHR/y males.
However, there was a significantly higher NE turnover in the SHR/y
females compared with the WKY females. Figure 3B shows no
significant heart NE turnover difference between the male SHR and SHR/a
or female SHR and SHR/a. Although there is a trend toward gender
differences in the heart NE turnover (Fig. 3B), there is
only significance in the WKY strain.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Because of the breeding protocol that produced the SHR/y and SHR/a strains, the female SHR/y has a similar genotype to the WKY female. Similarly, the SHR/a female should be genotypically similar to the SHR female. Any male strain differences between the WKY and SHR/y would be due to the Y chromosome. Kidney NE content and turnover showed a similarity in SHR and SHR/y males. This suggests that there is a locus on the Y chromosome that either directly or indirectly influences the SNS through catecholamine content and turnover rates. In addition, the similarity observed in the WKY and SHR/y females supports this hypothesis. However, there were no strain differences in the heart NE content or turnover, suggesting that there may be NE gender differences that are tissue specific.
An interesting observation is the higher kidney NE content in the
females compared with the males in all strains except the SHR/y. This
finding was not predicted because in all four strains females have
lower BP than strain-matched males (32). If we compare the
turnover rates in Table 1
(ng · g
1 · h1) of all the
groups, even though the WKY and SHR/y females have a higher NE baseline
content than the WKY and SHR/y males, the rate of synthesis (if we
accept the premise that NE levels are maintained in a steady state then
the rate of decline should equal the rate of synthesis) in the females
is actually significantly lower than in the males. In addition, the two
normotensive groups (male and female WKY) have the lowest turnover
rates of all the groups. The heart follows a similar pattern in NE
content and turnover rate, although significance was not observed. A
comparison of heart NE content (WKY male = 330 ng; SHR/y male = 403 ng) and turnover rate (WKY male = 3.43 ng · g1 · h1; SHR/y = 24.56 ng · g1 · h1) clearly
shows the same trend as was observed in the kidney NE content and
turnover rates. Evidence for the Y chromosome effect is supported by
the observation that the SHR/y males have a significantly higher
turnover rate compared with the WKY males, with whom the SHR/y are
genotypically similar except for the Y chromosome. In fact, there was
no significant difference in turnover rates among the SHR/y, SHR, and
SHR/a males, all of which are phenotypically hypertensive.
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In conclusion, the hypertensive rats had higher kidney NE turnover rates compared with the normotensive rats. Females had higher baseline NE content in both the kidney and heart compared with strain-matched males. However, only the hypertensive females had higher turnover rates than strain-matched males. In addition, the strain with the Y chromosome from the SHR had higher kidney NE content and turnover rates compared with the WKY strain. This study suggests both a strain and gender difference in SNS activity through noradrenergic neurotransmission.
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ACKNOWLEDGEMENTS |
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We express appreciation for the technical support of Fieke Bryson.
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FOOTNOTES |
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This research was supported by National Heart, Lung, and Blood Institute Grant 48072-07.
Address for reprint requests and other correspondence: D. Ely, Dept. of Biology, The Univ. of Akron, Akron, OH 44325-3908 (E-mail: ely1{at}uakron.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/japplphysiol.00557.2001
Received 1 June 2001; accepted in final form 28 September 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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