© 1999 American Heart Association, Inc.
Molecular Medicine |
Altered Structure, Regulation, and Function of the Gene Encoding the Atrial Natriuretic Peptide in the Stroke-Prone Spontaneously Hypertensive Rat
From the Division of Cardiovascular Diseases (S.R., M.A.L.-K., B.G., K.L.), Department of Medicine, Brigham and Women’s Hospital, and the Department of Cardiology (K.L.), Children’s Hospital, Harvard Medical School, Boston, Mass; Istituto Neurologico Mediterraneo Neuromed, Pozzilli (S.R., P.D., R.G., A.L., M.V.), and the Department of Experimental Medicine and Pathology, La Sapienza University (M.V., S.R.), Rome, Italy; and the Max Delbruch Centre for Molecular Medicine (K.L.), Berlin, Germany. Dr Lee-Kirsch is currently affiliated with the Universitaetskinderklinik, Technische Universittaet t Dresden, Dresden, Germany.
Correspondence to Speranza Rubattu, MD, Istituto Neurologico Mediterraneo Neuromed (Parco Tecnologico), Localita Camerelle (Zona Industriale). 86077 Pozzilli (Is), Italy. E-mail rubattu.speranza{at}neuromed.it
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Abstract |
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Abstract—Through the genotype/phenotype cosegregation analysis of an F2 intercross, from the crossbreeding of stroke-prone spontaneously hypertensive rats (SHRSP) and stroke-resistant spontaneously hypertensive rats (SHR), we previously identified a quantitative trait locus for stroke on rat chromosome 5 (STR2) that colocalized with the genes encoding atrial and brain natriuretic peptides (ANP and BNP) and conferred a stroke-delaying effect. To further characterize ANP and BNP as candidates for stroke, we performed additional studies. Comparative sequence analysis revealed point mutations in both the coding and regulatory regions of ANP, whereas no interstrain differences were found for BNP. In in vitro studies in COS-7 and AtT-20 cells that were performed to test the relevance of a G
A substitution at position 1125, a GlySer transposition
in the SHRSP pro-ANP peptide resulted in different posttranslational
processing of the SHRSP ANP gene product that was also associated
with higher cGMP production (P<0.05).
Furthermore, an analysis of a 5' end mutation affecting a PEA2
regulatory binding site in the 5' untranslated regulatory
sequence of SHRSP ANP demonstrated a significantly lower
ANP promoter activation in endothelial
cells (P<0.05 versus the SHR ANP). In addition, the
expression of ANP was significantly reduced in the
brain, but not in the atria, of SHRSP compared with SHR
(P<0.0001). No differences were detected with regard to
BNP expression. The present results reveal
substantial differences in ANP, but not
BNP, structure and product among SHR and SHRSP,
which supports a role of ANP in the pathogenesis of
stroke in the SHRSP animal model.
Key Words: genetics • atrial natriuretic peptide • stroke • natriuretic peptide
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Introduction |
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Stroke remains an area of substantial, unmet medical need, in part because of the rather sketchy understanding we have of its cause and responsible pathogenic mechanisms. Although hypertension is recognized as the most significant risk factor for stroke, twin and family studies, as well as epidemiological surveys, have consistently provided evidence for an important genetic component to its cause.1 2 3 4 5 6
The genetic dissection of a complex trait like stroke has been recently brought into our reach through the development of new molecular genetic methods and techniques, combined with the use of highly inbred animal models for this phenotype. In this regard, the stroke-prone spontaneously hypertensive rat (SHRSP), obtained through selective breeding from the stroke-resistant spontaneously hypertensive rat (SHR), represents a suitable model for stroke.7 Interestingly, a number of characteristics associated with stroke in this model are also found in human stroke, such as high blood pressure and dietary factors (eg, high Na+, low K+, low protein diets), all of which play a critical, permissive role in the development of stroke in rats8 9 and in humans.10 Furthermore, the histopathologic aspects of the cerebrovascular lesions in SHRSP resemble those observed in a rare human form of inherited stroke, called CADASIL.11
To elucidate the genetic underpinnings of stroke, we recently performed a genotype/phenotype cosegregation analysis by using an F2 cohort obtained through the intercrossing of SHRSP and SHR strains. We used latency until the manifestation of stroke under a stroke-permissive dietary regimen ("Japanese-style diet": low protein, low K+, high Na+) as the quantitative phenotype, and we found clear evidence for the existence of genes that specifically and independently of blood pressure levels contributed to the pathogenesis of stroke.12 In particular, we were able to detect 3 major stroke-relevant loci or quantitative trait loci on rat chromosomes 1, 4, and 5 that when combined accounted for about one third of the overall genetic variance.
The locus identified on rat chromosome 5 was found to confer a significant protective effect against stroke in the presence of the SHRSP allele. Its peak LOD score colocalized with the genes encoding atrial and brain natriuretic peptides (ANP and BNP, respectively). These peptides have important cardiovascular functions (they induce vasorelaxation, diuresis, and natriuresis13 14 15 ) and may therefore be viewed as legitimate candidate genes for cardiovascular diseases, including stroke. In addition, both ANP and BNP are expressed in cardiac and cerebral tissues, although to different degrees.16 17 ANP is mainly produced by the atria, whereas BNP is secreted primarily by the ventricle. Although BNP is widely distributed in the brain, ANP is mainly localized in hypothalamic and brain stem areas, which are involved in body fluid volume and blood pressure regulation.18 19 20
The aim of the present study was to perform a detailed comparative analysis of ANP and BNP in our SHR and SHRSP strains, with particular regard to structure, regulation, and function, in an attempt to further support the candidacy of these genes as representing the substrate of the previously characterized locus STR2.
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Materials and Methods |
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The SHR and SHRSP strains used for the present study have been described previously.12
Sequence Analysis
The complete ANP and BNP coding sequences
of SHR and SHRSP and a region of 641 bp upstream from the CAP site for
ANP were amplified through the use of primers generated on
the basis of the published sequence information. Direct sequencing was
performed with an ABI377 sequencing apparatus
(Perkin–Elmer Cetus).
Genotyping Assay and Single-Strand Conformational
Polymorphism
A single–base pair mutation, found at position 1125 of
ANP in SHRSP, was analyzed with the use of a
BstUI polymerase chain reaction (PCR)–restriction fragment
length polymorphism (RFLP) assay. Another base pair mutation,
detected at position -463 (within the 5' untranslated regulatory
sequence [URS] of ANP) in SHRSP, was analyzed with
the use of single-strand conformational polymorphism (SSCP).
ANP cDNA Subcloning and Transfection Studies: Western and Northern
Blotting Analyses of Transfected Cells
The full-length SHR and SHRSP ANP cDNAs were amplified from
reverse transcribed mRNA, inserted into the PCR TM3 vector
(InVitrogen), and used to transfect COS-7 and AtT-20 cells. At 2 days
after transfection, cellular extracts were obtained for protein
analysis, and total RNA was extracted for Northern
blotting.
Analysis of ANP on Tissues: Reverse Transcription–PCR and
Northern and Western Blotting
Total RNA was used to obtain the ANP cDNA and for Northern
blotting analysis. Total proteins were extracted from atria and
from hypothalami of both strains. To test the ex vivo processing of
atrial pro-ANP, 20 µg of atrial extract from both strains was
processed as described previously21 and analyzed
through Western blotting. Protein extracts from hypothalami were also
analyzed with Western blotting.
In Vitro Functional Studies of ANP Coding Mutation
AtT-20 cells were transfected with the ANP GC-A cDNA
construct.22 Two days after transfection, they were
stimulated with supernatants from cells transfected in parallel with
either the ANP SHR or SHRSP construct. Thus, cGMP was collected as
described previously 23 and measured with a
radioimmunoassay kit (Amersham Corp). The ANP levels of the
supernatants used as stimulants were measured as reported
previously.24
Functional Characterization of ANP 5' End Mutation
A 737-bp fragment of the 5' URS of ANP
(position -683 to +54) was amplified from genomic DNA of both strains,
inserted into the pCAT enhancer vector (Promega), and used for
transient transfection of primary rat endothelial cells
along with the pSV2 ß-galactosidase construct
(Promega). At 30 hours after transfection, the chloramphenicol
acetyltransferase (CAT) assay was performed as described
previously.25
In Vivo Regulation of ANP and
BNP Genes
Total RNA was extracted from the brains and atria of rats from
both strains that were maintained on either a regular (n=12) or a
Japanese+1% NaCl (n=12) diet. To detect brain ANP and BNP mRNAs and
atria ANP mRNA, we used an RNase protection assay;
[
-32P]UTP antisense RNA fragments were
prepared with the use of a Maxiscript kit (Ambion) from ANP, BNP, and
ß-actin cDNAs, respectively. The RNase protection assay was performed
as reported previously.26 Plasma ANP levels were also
measured.
Statistical Analysis
Values for ANP mRNA, plasma ANP levels, cGMP levels, and CAT
fold induction are reported as mean±SEM. Comparisons between groups
were performed with one-way ANOVA followed by nonparametric
post hoc tests. A value of P<0.05 was considered
statistically significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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Sequence Analysis
Sequence analysis of the ANP cDNA revealed a G
A
substitution in the SHRSP strain at position 1125, within exon 2. As a
result, a glycine at position 100 of SHR prepro-ANP is replaced by a
serine in the SHRSP. Because this single–base pair substitution
abolishes the recognition site for the BstUI enzyme, we were
able to confirm it with a PCR-RFLP assay, followed by agarose gel
electrophoresis (Figure 1
).
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The analysis of the 5' flanking region of ANP
revealed a CA transposition localized at position -463 from the CAP
site, changing a putative PEA-2, polyoma enhancer, binding
site27 from 3'-GCCCGCA-5' in SHR to 3'-GCACGCA-5' in
SHRSP. This mutation was clearly confirmed by SSCP (Figure 2). No other sequence difference was
found between the two strains.
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Comparative sequence analysis of the coding region of BNP failed to show any difference between the two strains.
Protein Analysis
A typical result of the Western blot analysis of the
cellular extracts from COS-7 and AtT-20 cells, transfected with either
the SHR- or the SHRSP-derived ANP cDNA, is shown in Figure 3A. These experiments provided evidence
of a differential processing of the two translational products,
depending on their strain of origin. In particular, although the SHR
construct yielded predominantly the unprocessed 
17-kDa protein, with
some fully processed ANP, the SHRSP-derived construct resulted in the
production of a partially processed peptide of 6.5 kDa and
of some fully processed ANP. Consistently, the analysis
of the ex vivo processing of atrial pro-ANP, which confirmed the
production of a 6.5-kDa peptide in SHRSP only, provided further
evidence of interstrain differences (Figure 3B). Of note, both
the in vitro and ex vivo differential processing of the SHRSP pro-ANP
peptide occurred despite an identical length of the ANP mRNA in
transfected cells and in tissues, as shown by Northern blotting and
reverse transcription–PCR (Figure 4),
and despite an identical length of the atrial pro-ANP peptide (Figure 3B).
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Due to the very limited amount of the whole brain ANP, we chose
to consider the ANP peptide forms present in the hypothalamus,
where the peptide is relatively more abundant than elsewhere, and we
found evidence for differential profiles of peptides between the two
strains (Figure 5). Remarkably, the
observed processing patterns resembled those seen in the in vitro and
ex vivo experiments, although the sites of cleavage used for processing
are known to differ in brain and atrial tissues.28
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Functional Studies of Coding Mutation
Transfection of AtT-20 cells with the rat GC-A receptor cDNA and
their subsequent stimulation with the processed pro-ANP forms, produced
by cells transfected in parallel with either the SHR or the SHRSP ANP
cDNA construct, resulted in a statistically significantly higher cGMP
production after stimulation with the supernatant obtained from
SHRSP ANP-transfected cells. In particular, after
normalization for the ANP concentration of the supernatants, the
cGMP-to-ANP ratio (average of 6 independent experiments) was 3.8±0.4
and 5.1±0.4 with SHR and SHRSP ANP stimulation, respectively
(P<0.05 versus SHR).
Analysis of 5' End Mutation
The activity of the ANP promoter, as determined with
the CAT assay of 4 independent experiments, was significantly lower in
endothelial cells transfected with the construct
containing the SHRSP variant at position -463 compared with the SHR
construct (-50%, P<0.05 versus SHR; Figure 6). Preliminary observations in primary
rat cardiomyocyte cells are consistent with the
findings in endothelial cells.
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In Vivo Regulation of ANP and
BNP Genes
The expression of ANP was significantly reduced
in the cerebral tissue of SHRSP compared with SHR (Figure 7) for both regular and stroke-promoting
diets (3-fold lower, P<0.0001). In contrast, atrial
expression of ANP was comparable in the two strains (Figure 8). Consistently, because cardiac
atria largely represent the major source of circulating ANP, we
observed comparable plasma ANP levels in SHR and SHRSP for both dietary
regimens: 419±45 versus 346±73 pg/mL (regular diet) and 326±53
versus 287±48 pg/mL (Japanese diet) in SHR and SHRSP, respectively
(P=NS).
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Of note, measured plasma ANP levels are not indicative of differential processing of the propeptide in the two strains due to the fact that the radioimmunoassay antibody (raised against the final 28–amino acid peptide) is not predicted to differentiate between processed and unprocessed forms.
No differences were found between the two strains with regard to BNP expression.
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Discussion |
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In a previous study in the SHRSP/SHR F2 intercross, we localized a stroke-relevant gene, STR2, within an area of rat chromosome 5 that also carries the genes encoding ANP and BNP.12 Both genes colocalize with the peak of linkage of this quantitative trait locus. The known biological cardiovascular properties of these two peptides,12 13 14 as well as their localization in the brain,15 16 made them legitimate candidate genes. In addition, the availability of both rat sequence data and rat-specific antibodies facilitated our task of interrogating the possible contributory role of these genes into the higher susceptibility to stroke in the model studied. Although the present findings do not permit us to conclude that the ANP gene is causally linked to stroke in the SHRSP, the observation of altered structure, regulation, and function of ANP is certainly consistent with the hypothesis that this peptide may modulate susceptibility to acute cerebrovascular accidents in our animal model.
In the present experiments, we demonstrate sequence differences between SHR and SHRSP in both the regulatory and the coding regions of ANP. Furthermore, we provide evidence that these differences are associated with both differential regulation and differential processing of ANP, with the latter likely related to predicted structural differences. Furthermore, the lower levels of expression found in SHRSP brains are consistent with the results of promoter studies with the CAT system, in which the SHRSP-derived construct showed decreased activity. Likewise, the results of a number of different approaches, aimed at the study of the resultant peptides, consistently demonstrated differential, typical patterns of processing that are characterized by an accumulation of an intermediate-size peptide from SHRSP transcripts and of larger peptides from SHR transcripts in both transfection experiments and ex vivo observations. No indication for differential length or stability of the ANP message was observed. Last, our cGMP data indicate that the differentially processed SHRSP ANP is also associated with altered biological activity. The latter finding could be explained by the additional intermediate-size peptide or by a different receptor-binding process of ANP products in SHRSP.
Failure to detect the 28–amino acid mature peptide in AtT-20 cellular extracts and in atrial extracts, which may represent a study limitation, could be due to the lower protein production of the AtT-20 cell model and to a major degree of peptide degradation in tissue extracts. On the other hand, the consistently observed differential pattern of processing supports the validity of all of the experimental models that were used.
A large body of experimental work has shown the presence of ANP in the central nervous system,18 19 20 particularly in the hypothalamus and the anteroventral region of the third ventricle—areas that are critical for body fluid regulation and circulatory control. Brain ANP levels have been previously investigated in the SHR strain and compared with its corresponding normotensive control, the Wistar-Kyoto (WKY) rat.29 30 31 32 In those studies, a role was postulated for ANP in the development of hypertension. Our data demonstrate substantially lower brain ANP expression in the SHRSP compared with the SHR in the absence of any difference in blood pressure.12 Given the facts that the SHRSP (low expression) variant of ANP has been previously linked to a protective effect toward stroke and that recent experiments have demonstrated a relation between stroke and expression of brain adrenomedullin, another potent vasorelaxant peptide, in a model of surgically induced cerebral infarction,33 which was worsened by the administration of this peptide, it is intriguing to speculate about a stroke-promoting effect of central nervous system vasodilatation.
The high degree of evolutionary conservation that the glycine at position 100 of ANP shows suggests that amino acid substitution at this position may well affect the translational product. The differential processing, as well as the differential cGMP activity observed for the two transcripts, is consistent with the putative biological relevance of an amino acid that is very well conserved among species.
A recent study34 that examines the possible role of ANP in stroke in the rat and fails to find evidence for such a contribution is actually quite consistent with our observations: in this study, SHRSP and WKY rats subjected to middle cerebral artery occlusion showed no differences in brain ANP expression. Of note, there was no difference between the ANP genes of the two strains used with regard to sequence, structure, and functional parameters, as demonstrated previously.34 An earlier, erroneous interpretation that stroke in an intercross of SHRSP and WKY rats was linked to the ANP locus35 was indeed corrected by additional data on chromosomal localization of ANP in this recent report.34
Although the present data support the notion that ANP may indeed be a candidate gene for contribution to stroke given its localization within the stroke-linked locus STR2, final validation of this result must be based on congenic and positional cloning experiments, which are in progress. It is of interest, however, that we were recently able to show that an allelic variant of ANP is associated with a 2-fold risk for stroke in humans.36 Thus, data from studies in both animals and humans favor further exploration of what seems to be a significant contributory or modulatory role for genetic variants of ANP in the pathogenesis of stroke.
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Acknowledgments |
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This work was supported by Research Career Development Award (KOA-HL03138-01) and Grant R01-HL5641101 from the National Heart, Lung, and Blood Institute (Dr Lindpaintner), a Special Program Grant from the Italian Ministry of Health (IRCCS), and a grant from the Italian Telethon, Project D.45 (Dr Volpe).
Received June 22, 1999; accepted September 8, 1999.
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C. F. Deschepper, I. Boutin-Ganache, A. Zahabi, and Z. Jiang In Search of Cardiovascular Candidate Genes: Interactions Between Phenotypes and Genotypes Hypertension, February 1, 2002; 39(2): 332 - 336. [Abstract] [Full Text] [PDF]
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 A. Hassan, N. Ali, Y. Dong, N. D. Carter, and H. S. Markus Atrial natriuretic peptide gene G664A polymorphism and the risk of ischemic cerebrovascular disease Neurology, November 13, 2001; 57(9): 1726 - 1728. [Abstract] [Full Text] [PDF]
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 M. van Eickels, C. Grohe, J. P.M. Cleutjens, B. J. Janssen, H. J.J. Wellens, and P. A. Doevendans 17{beta}-Estradiol Attenuates the Development of Pressure-Overload Hypertrophy Circulation, September 18, 2001; 104(12): 1419 - 1423. [Abstract] [Full Text] [PDF]
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 D. L Vesely Atrial natriuretic peptides in pathophysiological diseases Cardiovasc Res, September 1, 2001; 51(4): 647 - 658. [Full Text] [PDF]
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L. Sironi, E. Tremoli, I. Miller, U. Guerrini, A. M. Calvio, I. Eberini, M. Gemeiner, M. Asdente, R. Paoletti, and E. Gianazza Acute-Phase Proteins Before Cerebral Ischemia in Stroke-Prone Rats : Identification by Proteomics Stroke, March 1, 2001; 32(3): 753 - 760. [Abstract] [Full Text] [PDF]
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