From the Department of Physiology, University of Melbourne, Parkville
(F.J.C., S.B.H.), and Bone Marrow Transplant Unit, Alfred Hospital, Prahran
(M.K.K.), Victoria, Australia.
Correspondence to Professor S.B. Harrap, Department of Physiology, University of Melbourne, Parkville, Victoria 3052, Australia. E-mail s.harrap{at}physiology.unimelb.edu.au
There are several possible explanations for the elevated NGF mRNA in
SHR, including (1) DNA sequence differences in or near the NGF gene
resulting in increased transcription or stability of mRNA, (2) genetic
differences in other genes that influence NGF gene transcription, or
(3) the physiological influence of increased BP on
NGF gene expression in the SHR. Given the demonstrated linkage between
the NGF locus and SHR hypertension,3 we studied
the role of the NGF gene locus in determination of abnormal NGF mRNA
levels.
The concentration of NGF mRNA in blood vessels at ages 2, 10, and 43
days4 and kidneys at ages 10 and 43
days7 was greater in SHR than in Wistar-Kyoto
rats (WKY), but increased NGF gene expression seems to be developmental
stage–specific. At 3 weeks of age the level of NGF in the spleen, the
sciatic nerve, and the mesenteric arteries was higher in SHR than in
WKY, but not at 12 weeks of age.8 Higher levels
of NGF peptide levels in mesenteric arteries and aortas from young
(20-day-old) SHR were not observed in adult (6-month-old)
SHR.9 10
Renal sympathetic nerves in SHR are increased in
density,11 and renal denervation results in a
reduction in BP.12 Therefore, in the present
study we examined the levels of NGF mRNA in the kidney of SHR at
different ages. In addition, we performed a cross-breeding study to
determine whether inheritance of the SHR NGF allele was associated
with any significant effect on renal NGF mRNA levels in young
animals.
Quantification of Kidney NGF mRNA
Genotyping of the NGF Locus
Statistical Analysis
Relative Quantification of NGF mRNA
Renal NGF mRNA
The mean NGF mRNA levels in F2 rats were similar
to those in F1 animals. There was a significant
difference (P<0.05) in the mean NGF mRNA relative abundance
between the 3 possible NGF genotypes (Figure 3
At 4 weeks of age, the mean NGF mRNA abundance of the SHR parents was
significantly higher than that of the DRY parents
(P<0.0001), with F1 values
intermediate (Table 1
In F2 rats, those homozygous for the S
allele had the highest levels of NGF mRNA at 4, 10, or 20 weeks of
age (Table 1
Blood Pressure
However, the new finding in the present study is that this
increased level of NGF mRNA is associated with the inheritance of the
NGF locus from SHR in a genetically segregating
F2 population. The importance of this observation
is that it provides a link in vivo between the SHR NGF gene and NGF
gene expression. In our previous study,3 the NGF
locus could only be considered a linkage marker of hypertension,
despite the strong circumstantial evidence in favor of its etiologic
role in SHR. The important implication of the present study is that
a mutation in or near the NGF gene contributes to increased NGF gene
expression in young animals and therefore to high BP.
The elevated NGF in the SHR at young ages is important because it is a
likely explanation of the sympathetic hyperinnervation implicated in
this model of genetic hypertension.7 16 17 18 These
early increases in NGF mRNA may augment levels of sympathetic
innervation for the life of the animal.5 6 7 We
have also recently shown that brief treatment of young SHR with an
angiotensin-converting enzyme inhibitor causes
a persistent reduction in both renal NGF mRNA and
BP.14 In addition, increased NGF may contribute
to the structural hypertrophy of resistance
arteries19 that are characteristic of SHR
strain.
The difference in NGF mRNA between F2 SS
and F2 DD rats seemed to disappear between
2 and 4 weeks of age. We are cautious about using observations from
relatively modest numbers of rats from the segregating
F2 population to determine the timing of abnormal
NGF gene expression. The absence of a statistically significant
difference in NGF mRNA between the genotypic groups at 4 and 10 weeks
of age in part reflects insufficient statistical power to detect
differences that diminish as the animals age. In this respect it is
relevant that when all F2 rats were combined, a
significant association between NGF genotype and NGF mRNA was
observed. However, it is clear that the difference in NGF mRNA between
the SHR and DRY is larger and persists longer than that between
F2 SS and F2 DD
rats. This contrast is likely to reflect the fact that
F2 rats are on average 50% SHR and 50% DRY in
genetic origin. Other genes unlinked to the NGF gene locus may reduce
both the magnitude and the duration of NGF mRNA differences in the
F2 population.
The NGF gene consists of 4 exons (IA, IB, II, IIIA, IIIB, IV) separated
by introns of varying sizes, including a putative transcription unit of
>45 kb.20 Alternative splicing gives rise to 4
types of cDNA clones with different sequences preceding the region
coding for the NGF protein (exon IV).20
Differential expression of RNA might be explained either by specific
tissue activation of a number of promoters or by the presence of
regulatory sequences within a promoter recognized by tissue-specific
transcriptional factors. The NGF gene transcription is thought to be
mainly regulated by cis-elements in the region immediately upstream to
exon IB.20 The cis-regulatory elements in the
area preceding exon IB consist of 2 putative TATA boxes, 2 putative
CAAT boxes, and a high content of G and C residues, which indicates the
presence of binding sites for the regulatory protein
SP1.21 Studies by D'Mello and
Heinrich22 located regions in a 1-kb region close
to exon IB that affect transcription of NGF in L929 cells, which
include a proximal activator region containing several
SP1-like elements known to increase the number of
transcription-initiation complexes. They also identified in that region
a transcription suppression recognition site and an AP1 binding site
for the transcription factors c-fos and c-jun at
the junction of exon IB and the second intron. The activation of
c-fos and c-jun genes and their interaction with
AP1 binding site may facilitate the expression of the NGF gene by a
variety of stimuli such as angiotensin
II.14 23 Therefore, the NsiI
polymorphic site in SHR may be in linkage disequilibrium with a
functional mutation in a region that alters tissue and developmental
stage–specific gene expression.
Another mechanism by which the genotype of the NGF SHR locus
may exert its influence on the NGF mRNA levels is by its effects on
transcript stability. The half-life of NGF mRNA in astrocytes, Schwann
cells, and fibroblasts has been shown to be 30 to 60 minutes. The
half-life appears to be influenced by AU nucleotide–rich
sequences including repetitive AUUUA sequences, present in the
3'-untranslated region (UTR) of the mRNA. The half-life of wild-type
NGF mRNA with the 3'-UTR region averages 3 hours, whereas NGF mRNA
without the AU-rich 3'-UTR region had a half-life of
In summary, this study shows that the NGF locus implicated in BP
regulation is linked to age-specific expression of NGF in the kidney of
SHR. Comparisons of sympathetic nerve density of the cross-bred animals
should provide further clues to the physiological
importance of the NGF gene in SHR.
Received March 30, 1998;
first decision April 27, 1998;
accepted June 18, 1998.
2.
Judy WV, Watanabe AM, Murphy WR, Aprison BS, Yu PL.
Sympathetic nerve activity and blood pressure in normotensive backcross
rats genetically related to the spontaneously hypertensive rat.
Hypertension. 1979;1:598–604.
3.
Kapuscinski M, Charchar F, Mitchell GA, Innes B,
Harrap SB. Nerve growth factor gene and genetic hypertension in
spontaneously hypertensive rats. J Hypertens. 1996;14:191–197.[Medline]
[Order article via Infotrieve]
4.
Falckh P, Harkin L, Head R. Resistance vessel gene
expression in young spontaneously hypertensive rats. J
Hypertens. 1992;10:913–918.[Medline]
[Order article via Infotrieve]
5.
Heumann R, Korching S, Scott J, Theonen H.
Relationship between the level of nerve growth factor (NGF) and its
messenger RNA in sympathetic ganglia and peripheral target
tissues. EMBO J. 1984;3:3183–3189.[Medline]
[Order article via Infotrieve]
6.
Shelton DL, Reichardt LF. Expression of the nerve
growth factor correlates with the density of sympathetic innervation in
effector organs. Proc Natl Acad Sci U S A. 1984;81:7951–7955.
7.
Falckh PH, Harkin LA, Head RJ. Nerve growth
factor mRNA content parallels altered sympathetic innervation in the
spontaneously hypertensive rats. Clin Exp Pharmacol Physiol. 1992;19:541–545.[Medline]
[Order article via Infotrieve]
8.
Ueyama T, Hamada M, Hano T, Nishio I, Masuyama Y,
Furukawa S. Increased nerve growth factor levels in spontaneously
hypertensive rats. J Hypertens. 1992;10:215–219.[Medline]
[Order article via Infotrieve]
9.
Donohue SJ, Head RJ, Stitzel RE. Elevated nerve growth
factor levels in young spontaneously hypertensive rats.
Hypertension. 1989;14:421–426.
10.
Zettler C, Rush RA. Elevated concentration of nerve
growth factor in heart and mesenteric arteries of spontaneously
hypertensive rats. Brain Res. 1993;614:15–20.[Medline]
[Order article via Infotrieve]
11.
De Michele M, Amenta F. Increase in perivascular
noradrenergic nerve density and decrease in
acetylcholinesterase-positive sympathetic nerve density in the kidneys
of spontaneously hypertensive rats. Clin Exp Hypertens.
1988;A10:1031–1049.
12.
Winterwitz SR, Katholi RE, Oparil S. Role of the renal
sympathetic nerves in the development and maintenance of
hypertension in the spontaneously hypertensive rat. J Clin
Invest. 1980;66:971–978.
13.
Chomczynski CN, Sacchi N. Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem. 1987;162:156–159.[Medline]
[Order article via Infotrieve]
14.
Charchar FJ, Kapuscinski M, Harrap SB. Persistent
reduction in renal nerve growth factor mRNA after perindopril treatment
of young spontaneously hypertensive rats. Hypertension. 1998;31:678–683.
15.
Zettler C, Zhou X-F, Bridges D, Rush RA. Increased
concentration of nerve growth factor detected in tissue extracts with
an improved extraction procedure. J Neurosci Res. 1996;46:581–594.[Medline]
[Order article via Infotrieve]
16.
Rush RA, Chie E, Liu D, Tafreshi A, Zettler C, Zhou XF.
Neurotrophic factors are required by mature sympathetic neurons for
survival, transmission and connectivity. Clin Exp Pharamcol
Physiol. 1997;24:549–555.
17.
Lewin GR, Barde YA. Physiology of the neurotrophins.
Ann Rev Neurosci. 1996;19:289–317.[Medline]
[Order article via Infotrieve]
18.
Hoyle GW, Mercer EH, Palmiter RD, Brinster RL.
Expression of NGF in sympathetic neurones leads to excessive axon
outgrowth from ganglia but decreased terminal innervation within
tissues. Neuron. 1993;10:1019–1034.[Medline]
[Order article via Infotrieve]
19.
Zettler C, Head RJ, Rush RA. Chronic nerve growth
factor treatment of normotensive rats. Brain Res. 1991;538:251–262.[Medline]
[Order article via Infotrieve]
20.
Selby MJ, Edwards R, Sharp F, Rutter WJ. Mouse nerve
growth factor gene: structure and expression. Mol Cell Biol. 1987;7:3057–3064.
21.
Zheng M, Heinrich G. Structural and functional
analysis of the promoter region of the nerve growth factor
gene. Mol Brain Res. 1988;3:133–140.
22.
D'Mello S, Heinrich G. Structural and functional
identification of regulatory regions and cis elements surrounding the
nerve growth factor gene promoter. Mol Brain Res. 1991;11:255–264.[Medline]
[Order article via Infotrieve]
23.
Tuttle JB, Etheridge R, Creedon DJ. Receptor mediated
stimulation and inhibition of nerve growth factor secretion by vascular
smooth muscle. Exp Cell Res. 1993;538:350–361.
24.
Tang B, Wang M, Wise BC. Nerve growth factor mRNA
stability is controlled by a cis-acting instability determinant in the
3'-untranslated region. Mol Brain Res. 1997;46:118–126.[Medline]
[Order article via Infotrieve]
25.
Whittmore SR, Ebendal T, Larkfors L, Olsen L, Seiger A,
Stromberg I, Persson H. Developmental and regional expression of
ß-nerve growth factor messenger RNA and protein in the rat central
nervous system. Proc Natl Acad Sci U S A. 1986;83:817–821.
26.
Whittmore SR, Freiedman PL, Larhammar D, Persson H,
Gonazalez-Carvajal M, Holets VR. Rat ß-nerve growth factor
sequence and site of synthesis in the adult hippocampus. J
Neurosci Res. 1988;20:403–410.[Medline]
[Order article via Infotrieve]
© 1998 American Heart Association, Inc.
Scientific Contributions
Nerve Growth Factor Gene Locus Explains Elevated Renal Nerve Growth Factor mRNA in Young Spontaneously Hypertensive Rats
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Abstract—Nerve growth factor
(NGF) controls the growth of sympathetic nerves and is increased in
young spontaneously hypertensive rats (SHR). The NGF gene has been
linked genetically with hypertension in the SHR strain and may explain
high NGF mRNA levels. To test for genetic linkage between the NGF gene
and its expression in vivo, we examined renal NGF mRNA levels in male
SHR, control Donryu rats (DRY), and F2 rats derived from
SHR and DRY at ages 2, 4, 10, and 20 weeks. Tail-cuff blood pressure
was measured at 4, 10, and 20 weeks of age. NGF mRNA levels in SHR (NGF
genotype: SS) were higher than those in DRY (NGF
genotype: DD) at 2, 4, and 10 weeks of age
(P<0.0001) but the same at 20 weeks of age. In the
F2 generation, the S allele was
associated with significantly (P=0.01) higher renal NGF
mRNA levels at 2 weeks of age. Mean NGF mRNA levels fell
(P=0.01) with age in F2 rats, and the
difference between SS and DD
genotype F2 rats diminished at older ages and was
not significant. In F2 rats there was a positive
correlation between the number of NGF S alleles
inherited and tail-cuff pressure (P<0.007). Our
findings indicate that the NGF locus is an important regulator of NGF
mRNA levels. It is likely that mutations in or near the NGF gene
explain in part high early NGF gene expression in SHR.
Key Words: growth substances • hypertension • rats, inbred SHR • gene expression • kidney
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
In both humans and the spontaneously hypertensive rat
(SHR) animal model, there is convincing evidence that the sympathetic
nervous system plays an important role in etiology of
hypertension.1 2 During development, nerve growth
factor (NGF) is required for the survival of sympathetic and some
sensory neurons. We have reported previously a genetic linkage of the
NGF gene of the SHR with an increment in blood pressure (BP) in
segregating F2 rats derived from a cross of the
SHR and the normotensive Donryu (DRY) strains.3
Studies of NGF mRNA by Northern analysis reveal higher
abundance of NGF mRNA in SHR tissues than in age-matched normotensive
rats.3 4 Increased NGF mRNA has been associated
with increased NGF peptide levels and higher densities of sympathetic
innervation5 6 and is likely to reflect enhanced
NGF gene expression.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Animals
Male SHR and female DRY were bred and crossed as described
previously.3 Male SHR (n=5), DRY (n=5),
F1 (n=4), and F2 rats were
studied at the age of 2, 4, 10, and 20 weeks (F2:
n=20, 23, 20, and 28, respectively). The inbred status of parental
strains has been tested and reported previously.3
Systolic BP was measured twice a week in conscious animals
between 9 and 11 AM with a photoelectric tail-cuff pulse
detection system (IITC Inc, Life Science Instrumentation). The
size of the restrainer cylinders and cuffs was matched to the size of
growing animals. Before the experimental period, the rats were
conditioned to the restraining cylinders and BP measurement. Rats were
prewarmed at 37°C for 8 to 10 minutes to facilitate tail blood flow
before BP was measured. The mean of 3 readings of systolic tail
blood pressure was used as the BP value for each rat. Readings were not
made in 2-week-old rats because their small size precluded accurate
measurements. These experiments were approved by the University of
Melbourne Animal Experimentation Ethics Committee.
Total RNA was isolated from 100 to 200 mg of whole kidneys
according to the acid phenol method of Chomczynski and
Sacchi.13 Reverse transcription–polymerase chain
reaction (RT-PCR) was used to provide relative quantification of the
amount of renal NGF mRNA, as reported
previously.14 GAPDH amplification was performed
to provide internal standards for relative quantification of NGF
transcripts in the isolated total RNA.
Genomic DNA was extracted with the use of standard methods, as
previously reported.3 Aliquots (20 µg) of
genomic DNA were digested with NsiI restriction
endonuclease (New England Biolabs) according to the manufacturer's
instructions, and 10 µg of digested DNA was electrophoresed in 0.8%
agarose gel with the use of Tris-borate-EDTA buffer. DNA was
transferred onto a charged nylon membrane (Hybond
N+, Amersham) and hybridized to mouse NGF cDNA
probe.3
All data appearing in text, figures, and tables are reported as
means with 95% CI for the mean unless stated otherwise. When we tested
for the effects of genotype on the NGF mRNA or BP, groups were
compared using 1-way ANOVA. Differences between individual group means
were assessed with Tukey's honestly significant difference test. To
assess overall relationships between NGF genotype, BP, NGF
mRNA, and age, we undertook a multiple regression analysis in
the F2 animals.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Genotypes
Southern blot analysis of genomic DNA digested with
NsiI revealed single distinct bands in the parental strains.
The SHR allele (S) restriction fragment was 4.8 kb, and
the DRY allele (D) restriction fragment was 2.8 kb. A
typical autoradiogram from F2
rats showed the 3 possible genotypes after NsiI
digestion of DNA and hybridization with NGF cDNA (Figure 1
).
View larger version (126K):
[in a new window]
Figure 1. NGF alleles in segregating F2 rats
revealed by Southern blot analysis with NGF cDNA probe. Only
the 4.8-kb DNA fragment is detected in rats homozygous for SHR NGF
alleles (SS: lanes 2 and 4), whereas animals
homozygous for DRY NGF allele exhibit only the 2.8-kb band
(DD: lane 1). Heterozygotes possessing both bands
(SD) are shown in lanes 3, 5, and 6.
The relative NGF mRNA expression was examined in the kidneys of
SHR, DRY, F1, and 3 possible genotypes in
the F2 population at 2, 4, 10, and 20 weeks of
age. Figure 2 shows
representative images of NGF and GAPDH PCR products
from kidney cDNA at 4 weeks of age in the SHR,
F2, DRY, and F1 rats. The
sizes of PCR products were 440 base pairs for NGF and 220 base
pairs for the GAPDH product. Southern blot analysis with
oligonucleotide probes internal to the PCR primers
confirmed the identity of these products (data not shown).
View larger version (29K):
[in a new window]
Figure 2. Representative images of NGF and
GAPDH PCR products from kidney cDNA as scanned by the
fluorescence imaging system. The sizes of PCR products were
440 base pairs for NGF and 220 base pairs for the GAPDH product.
The figure shows NGF and GAPDH mRNA levels from the kidneys of
4-week-old SHR, F2, DRY, and F1 rats.
The relative abundance of renal NGF mRNA after correction for
GAPDH at 2 weeks of age is shown in Figure 3. The SHR showed significantly greater
amounts of renal NGF mRNA compared with DRY. The mean mRNA levels in
the F1 rats were intermediate and significantly
different from both parental strains (P<0.05).
View larger version (36K):
[in a new window]
Figure 3. Level of NGF mRNA in the kidney in 2-week-old (A)
SHR (n=5), DRY (n=5), and F1 (n=4) and (B) F2
(3 possible genotypes: DD: n=4,
SD: n=9, SS: n=7). Mean abundance of NGF
is expressed as the ratio of NGF to GAPDH mRNA. *P<0.05
by Tukey's honestly significant difference range test. 
). The rats
homozygous for the SHR allele (SS) showed a higher level
of NGF mRNA than F2 rats homozygous for the DRY
allele (DD). The F2 animals
heterozygous for the NGF allele showed an intermediate level of
mRNA, and there was a significant (P<0.01) linear
association between NGF mRNA levels and the number of NGF S
alleles. ). The
relative abundance of NGF mRNA (Table 1) was also significantly higher
in the 10-week-old SHR than in DRY and F1.
However, at 20 weeks of age the renal NGF mRNA in SHR was less than at
younger ages and was not significantly different from that in DRY
(Table 1).
View this table:
[in a new window]
Table 1. NGF mRNA Levels in the Kidney of SHR, DRY, and
F1 Rats and the 3 Genotype Groups in F2
Rats ). Although these weekly differences were not significant
alone by ANOVA, regression analysis of all
F2 rats between 2 and 20 weeks of age revealed
significant univariate correlations of higher NGF mRNA
levels with increasing numbers of S alleles
(r=0.23, P=0.01). In addition, there was a
significant association between increasing age and a reduction in NGF
mRNA (r=-0.24, P=0.01). When all variables
were entered in multiple regression analysis, both age
(P=0.03) and NGF genotype (P=0.04) showed
independent and significant associations with NGF mRNA.
Mean tail-cuff BP values are shown in Table 2 for ages 4, 10, and 20 weeks. At 10 and
20 weeks of age, the average BP of SHR was the highest of all groups
(P<0.0001) (Table 2). At 4, 10, or 20 weeks of age,
F2 rats homozygous for the S
allele had the highest BP values (Table 2), and although not
significant by ANOVA at each week, multiple regression analysis
incorporating all F2 animals aged between 4 and
20 weeks showed significant independent association between BP and the
number of NGF S alleles inherited by each animal
(P=0.007).
View this table:
[in a new window]
Table 2. Systolic BP in SHR, DRY, and F1 Rats and
the 3 Genotype Groups in F2 Rats
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
The present study indicates that levels of NGF mRNA are
elevated from as early as 2 weeks of age in the kidneys of SHR compared
with DRY. High renal NGF mRNA in SHR persists until 10 weeks of age.
Between 10 and 20 weeks of age, the level of NGF in SHR falls and
becomes similar to that in the DRY strain. These results are
consistent with a number of independent published findings of
high NGF in the SHR strain.3 4 7 8 9 10 15 
9
hours.24 The rapid turnover of NGF mRNA, which
acts to maintain a low level of production of the protein, may
be retarded in the SHR. The finding that there is some
heterogeneity of NGF transcript
size6 25 26 raises the possibility that the
difference in steady state mRNA levels may reflect differences in
stability between the alternatively spliced transcripts. Sequencing of
the NGF gene is required to determine the presence or absence of this
region or its size in the SHR. It is also possible that the AU-rich
3'-UTR area interacts with other abnormal proteins in the SHR, which
may affect the stability of NGF mRNA.
Acknowledgments
This study was supported in part by the National Health and
Medical Research Council of Australia.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Folkow B. Physiological
aspects of primary hypertension. Physiol Rev. 1982;62:347–504.
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