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Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan; and Howard Hughes Medical Institute and Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050
Address all correspondence and requests for reprints to: Dr. Yoshihiro Ogawa, Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: ogawa{at}kuhp.kyoto-u.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using the liver-specific human serum amyloid P component (SAP) promoter, we have generated transgenic mice with elevated plasma BNP concentrations (BNP-transgenic mice) (18) comparable to those in patients with cardiovascular disorders (3, 8, 19) and suggested that they serve as a unique experimental model system in which to assess the long-term effects of BNP in vivo. The BNP-transgenic mice show a marked reduction in blood pressure and cardiac weight, accompanied by a significant increase in plasma cGMP concentration (18). These findings indicate that BNP is capable of modifying cardiovascular homeostasis on a long-term basis, thereby suggesting its usefulness for the treatment of cardiovascular disorders such as hypertension and cardiac hypertrophy. Unexpectedly, however, these mice also exhibit marked skeletal overgrowth, accompanied by the activation of endochondral ossification (20). In contrast, transgenic mice overexpressing ANP in the liver (ANP-transgenic mice) have been shown to develop lower blood pressure, but no skeletal phenotypes have been reported to date (21). These findings suggest that ANP and BNP, thought to act through GC-A, may have different signaling pathways. Furthermore, both GC-A-deficient and ANP-deficient mice are hypertensive with cardiac hypertrophy, but show no skeletal defects (22, 23, 24). Recently, it has been reported that C receptor-deficient mice show decreased blood pressure and skeletal abnormalities similar to those in BNP-transgenic mice (25). These findings suggest complex interplay among natriuretic peptides and receptors in vivo.
In this study we use a genetic model to establish that BNP exerts its biological effects solely through a GC-A-mediated pathway in some cases (blood pressure regulation), but in other cases (regulation of skeletal growth) a pathway clearly independent of GC-A mediates the BNP response.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasma BNP and ANP concentration measurements
Plasma BNP and ANP concentrations were determined by RIAs
specific for mouse BNP and ANP, respectively (18).
Blood pressure measurements
Systolic blood pressure was measured by a noninvasive
computerized tail-cuff method (BP-98A, Softron Corp., Tokyo, Japan)
(18) using 5-month-old conscious mice. Mice were
acclimated to the tail-cuff method for 3 days. At least six readings
were taken for each measurement. Blood pressure was always measured by
the same individual, who was not informed of the genotype of each
animal.
Soft x-ray analysis
Five-month-old female mice were killed, skinned, eviscerated,
and subjected to soft x-ray analysis (36 KVp, 4 mA for 2 min; Softron
type SRO-M5, Softron Corp.) (20).
Skeletal preparation and histology
Vertebrae and tibias from 7-day-old mice were fixed in 4%
paraformaldehyde, decalcified in 10% EDTA/0.1 M Tris-HCl,
pH 7.4, for 7 days, and embedded in paraffin. Five-micron-thick
sections were cut from paraffin-embedded specimens and stained with
Alcian blue (pH 2.5) and hematoxylin-eosin (20).
Digoxigenin-labeled antisense and sense riboprobes were obtained from
the RT-PCR product for type II collagen (a gift from Dr. Y. Yamada,
NIH, Bethesda, MD), type X collagen (a gift from Dr. B. R. Olsen,
Harvard Medical School, Boston, MA), and GC-B (nucleotides 762-1394 of
rat GC-B complementary DNA, GenBank M26896) using a DIG RNA labeling
kit (Roche, Mannheim, Germany). In situ
hybridization analysis was performed as previously described
(27).
cGMP concentration measurements
The apical half of cardiac ventricles from 5-month-old male mice
and tail bones from 7-day-old neonates were homogenized in 1 ml 6%
trichloroacetic acid with a Physcotron homogenizer (NITI-ON Medical
Supply, Chiba, Japan). The cGMP concentrations in ventricular and bone
tissues, plasma, and urine were determined by a RIA for cGMP as
previously reported (13).
Statistical analysis
Data were expressed as the mean ± SE. The
statistical significance of differences in mean value was assessed by
two-way ANOVA or Fisher’s test. Differences among mean values were
considered significant as values of P < 0.05 and
P < 0.01.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Plasma BNP
concentrations in GC-A-/-
mice were 39.2 ± 7.0 fmol/ml and were significantly higher than
those in GC-A+/+ mice (P < 0.05). Plasma
BNP concentrations in BNP-Tg and
BNP-Tg/GC-A-/- mice
(2734 ± 480 and 1941 ± 295 fmol/ml, respectively) were
markedly elevated (
50- to 100-fold increases) relative to those in
GC-A+/+ and
GC-A-/- mice. No
significant difference in plasma BNP concentration was noted between
BNP-Tg and BNP-Tg/GC-A-/-
mice. Plasma ANP concentrations in GC-A+/+,
BNP-Tg, GC-A-/-, and
BNP-Tg/GC-A-/- mice were
52.9 ± 6.1, 91.1 ± 33.1, 82.3 ± 11.9, and 100.1
± 28.0 fmol/ml, respectively (n = 6; Fig. 1B). No significant
differences in plasma ANP concentration were noted among genotypes.
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). The blood pressure of
GC-A-/- mice (145 ±
3 mm Hg) was approximately 30 mm Hg higher than that of
GC-A+/+ mice. These data are consistent with
previous reports (18, 22, 23). On the other hand, no
significant differences in blood pressure were observed between
GC-A-/- and
BNP-Tg/GC-A-/- mice
(144 ± 4 mm Hg).
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). GC-A-/- mice
had a significant increase in heart to body weight ratio (8.1 ±
0.5 mg/g BW) relative to GC-A+/+ mice (170%;
P < 0.01). The
BNP-Tg/GC-A-/- mice
developed cardiac hypertrophy (7.9 ± 0.4 mg/g BW), and no
significant difference was noted between
GC-A-/- and
BNP-Tg/GC-A-/- mice.
Skeletal phenotypes
Figure 3A shows a gross appearance
of 7-month-old female GC-A+/+, BNP-Tg,
GC-A-/-, and
BNP-Tg/GC-A-/- mice. The
BNP-Tg/GC-A-/- mice
developed skeletal deformities, characterized by hunch back and
elongated limbs, paws, and tails, which are similar to those in BNP-Tg
mice as previously reported (20). Soft x-ray analysis
revealed that at 7 months of age, BNP-Tg and
BNP-Tg/GC-A-/- mice had
longer vertebral bodies and tibias compared with
GC-A+/+ and
GC-A-/- mice,
respectively (Fig. 3B). At 10 and 20 weeks of age, the naso-anal length
of BNP-Tg mice was significantly greater than that of
GC-A+/+ mice (n = 4 each; P
< 0.05 at 10 weeks of age; P < 0.01 at 20 weeks of
age; Fig. 3C). No significant difference in naso-anal length was noted
between GC-A+/+ and
GC-A-/- mice. The
naso-anal length of
BNP-Tg/GC-A-/- mice was
increased significantly compared with that of
GC-A-/- mice
(P < 0.05 at 10 weeks of age; P <
0.01 at 20 weeks of age). No significant difference was noted between
BNP-Tg/GC-A-/- and BNP-Tg
mice. The longitudinal lengths of fifth lumbar vertebrae and tibias
were also significantly greater in
BNP-Tg/GC-A-/- and BNP-Tg
than those in GC-A-/- and
GC-A+/+ mice, respectively (n = 4;
P < 0.01; Fig. 3D).
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, A–D). The heights of hypertrophic
chondrocyte zone in BNP-Tg and
BNP-Tg/GC-A-/- mice were
280 ± 12 and 290 ± 5 µm, respectively, which were
significantly larger than those in GC-A+/+ and
GC-A-/- mice (200 ±
9 and 220 ± 5 µm, respectively; n = 6; P
< 0.01). No significant differences in the heights of nonhypertrophic
chondrocyte zone were noted among GC-A+/+,
BNP-Tg, GC-A-/-, and
BNP-Tg/GC-A-/- mice
(870 ± 16, 870 ± 8, 890 ± 10, and 910 ± 20
µm, respectively; n = 6).
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, E–H). By contrast, type X collagen-positive hypertrophic
chondrocyte zones were increased in height in BNP-Tg and
BNP-Tg/GC-A-/- mice
relative to those in GC-A+/+ and
GC-A-/- mice (Fig. 4, I–L). In this study GC-B mRNA expression was detected in the
proliferative and prehypertrophic zones of the growth plate in
GC-A+/+ mice (Fig. 4M). This is consistent with a
recent report that GC-B mRNA is expressed predominantly in
proliferating chondrocytes of the fetal mouse tibia
(28).
Urinary, plasma, ventricular, and bone cGMP concentrations
Urinary, plasma, and ventricular cGMP concentrations were
increased significantly in 5-month-old BNP-Tg mice (20.3 ± 5.9
pmol/min, 50.6 ± 11.1 pmol/ml, and 13.7 ± 0.8 pmol/g
tissue, respectively) relative to those in
GC-A+/+ mice (5.2 ± 1.7 pmol/min, 12.8
± 3.5 pmol/ml, and 11.4 ± 0.4 pmol/g tissue, respectively;
n = 6; P < 0.01; Fig. 5, A–C). By contrast,
GC-A-/- mice (1.4 ±
0.3 pmol/min, 2.4 ± 0.5 pmol/ml, and 2.1 ± 0.2 pmol/g
tissue, respectively) had significantly lower urinary, plasma, and
ventricular cGMP concentrations than GC-A+/+ mice
(P < 0.01). No significant difference was noted
between GC-A-/- and
BNP-Tg/GC-A-/- mice
(2.6 ± 0.6 pmol/min, 3.7 ± 0.6 pmol/ml, and 2.1 ± 0.2
pmol/g tissue, respectively).
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). Bone cGMP concentrations were increased
significantly in BNP-Tg and
BNP-Tg/GC-A-/- mice
(52.8 ± 3.9 and 46.3 ± 12.4 pmol/g tissue, respectively)
compared with those in GC-A+/+ and
GC-A-/- mice (n = 4;
P < 0.01). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We observed no significant differences in blood pressure and heart to body weight ratio between GC-A-/- and BNP-Tg/GC-A-/- mice. These results indicate that BNP does not lower blood pressure and inhibit cardiac hypertrophy in the absence of GC-A. Plasma, urinary, and ventricular cGMP concentrations were elevated in BNP-Tg mice but decreased in GC-A-/- mice relative to GC-A+/+ mice. Furthermore, no significant differences were noted between GC-A-/- and BNP-Tg/GC-A-/- mice. Thus, the hypotensive and antihypertrophic effects of BNP are mediated by increased production of cGMP through the activation of GC-A. This is consistent with the previous observation that ANP and BNP fail to relax precontracted aortic rings prepared from GC-A-/- mice (29). A significant reduction of cardiac weight in BNP-Tg mice might be at least partly due to the hypotensive effect of BNP. However, previous studies reported that ANP can reduce the cell volume of cultured rabbit cardiocytes through a cGMP-mediated pathway (30, 31). Taken together, our results suggest that BNP exerts its antihypertrophic effect on the heart through GC-A expressed in both blood vessels and heart.
This study demonstrates that BNP-Tg/GC-A-/- mice continue to exhibit marked longitudinal growth of vertebrae and long bones comparable to that in BNP-Tg mice. Histologically, BNP-Tg mice are indistinguishable from BNP-Tg/GC-A-/- mice, in that both genetic models show increases in the height of the hypertrophic zone of growth plate cartilage as revealed by increased expression of type X collagen mRNA in both genotypes. These findings indicate that BNP can activate endochondral ossification through another pathway in vivo, although GC-A mRNA is expressed in mouse long bones (32). In this study, bone cGMP concentrations in BNP-Tg and BNP-Tg/GC-A-/- mice are increased significantly relative to those in GC-A+/+ and GC-A-/- mice, respectively. These observations suggest that GC-A expressed in the bone is not coupled to the increase the cGMP production. We reported that BNP can increase the longitudinal growth of cultured mouse long bones through a cGMP-mediated pathway (20). Furthermore, mice lacking type II cGMP-dependent protein kinase develop dwarfism as a result of impaired endochondral ossification (33), suggesting the importance of cGMP signaling in endochondral ossification. It is, therefore, likely that BNP can activate GCcoupled receptor(s) other than GC-A in vivo, thereby affecting endochondral ossification.
GC-B is the another GC-coupled receptor that BNP is known to activate (4, 6). In this study we have shown that GC-B mRNA is expressed in the proliferative and prehypertrophic zones of the growth plate. We have previously suggested that CNP is an endogenous natriuretic peptide in the bone and may activate endochondral ossification via GC-B in cultured fetal mouse tibias (32). Furthermore, we have recently created transgenic mice expressing CNP under the control of the cartilage-specific type II collagen promoter. They exhibit marked skeletal overgrowth accompanied by increased endochondral ossification (A. Yasoda, Y. Ogawa, N. Tamura, M. Suda, H. Chusho, T. Miyazawa, K. Tanaka, and K. Nakao unpublished data), which is very similar to those found in BNP-Tg and BNP-Tg/GC-A-/- mice. It suggests that CNP can locally affect endochondral ossification. Taken together, the data of this study suggest that in BNP-Tg and BNP-Tg/GC-A-/- mice, BNP is secreted from the liver into the circulation at a high rate and cross-reacts with GC-B in vertebrae and long bones, thereby activating endochondral ossification. In this context, Matsukawa et al. have recently reported that C receptor-deficient mice show hunched backs, elongated long bones, and vertebral bodies (25), which are very similar to those of BNP-Tg and BNP-Tg/GC-A-/- mice. With no significant increases in plasma ANP and BNP concentrations in C receptor-deficient mice, disruption of C receptor should result in the increased availability of an endogenous ligand (i.e. CNP) to GC-B, thereby activating endochondral ossification (25).
On the other hand, no skeletal overgrowth has been reported in ANP-transgenic mice (21), although ANP and BNP can activate both GC-A and GC-B equipotently (12, 13). However, we have observed that ANP and BNP can similarly activate endochondral ossification in cultured fetal mouse tibias (20, 32). Moreover, increased body length was observed in proportion to the copy number of the BNP transgene (20), and no gross skeletal abnormalities were found in BNP-transgenic mice with the lower copy number of the transgene. Therefore, it is likely that high plasma ANP or BNP concentrations are needed to promote endochondral ossification in vivo. Considering that plasma ANP concentrations in ANP-transgenic mice (21) are about 1 order of magnitude lower than plasma BNP concentrations in BNP-transgenic mice (20), ANP-transgenic mice may have few or mild skeletal abnormalities. Detailed histological examinations of the skeleton of ANP-transgenic mice are needed to address this issue.
It has been recognized that the phenotypes of experimental animals sometimes vary, depending on their genetic backgrounds (34). Originally, GC-A-deficient mice were produced on a C57BL/6–129/SvJ hybrid background (22), whereas BNP-transgenic mice have been maintained on a C57BL/6 background (18). Thus, the four genotypes used in this study (GC-A+/+, BNP-Tg, GC-A-/-, and BNP-Tg/GC-A-/- mice) may differ genetically in addition to the GC-A allele and BNP transgene, which might affect their phenotypes. However, the cardiovascular and skeletal phenotypes of BNP-Tg and GC-A-/- mice examined in this study are identical to those of BNP-transgenic and GC-A-deficient mice reported previously (18, 20, 22). It is, therefore, unlikely that the skeletal phenotypes of BNP-Tg/GC-A-/- mice may result from genetic variations among the animals examined.
Nevertheless, it is also possible that there is an as yet unidentified GC- or non-GC-coupled receptor with high affinity to BNP (35, 36), thereby mediating its cardiovascular and skeletal effects. Recently, at least 29 guanylyl cyclase sequences have been reported in Caenorhabditis elegans (37). A recent study suggested the existence of a large number of non-GC-coupled natriuretic peptide receptor other than C receptor in rat FRTL-5 thyroid cells (38). Further studies are required for the identification of an unknown biologically active receptor(s) specific for BNP.
In conclusion, this study provides genetic evidence that BNP exerts its cardiovascular effects through a GC-A-mediated pathway, whereas it can affect endochondral ossification through a GC-coupled receptor(s) other than GC-A, possibly GC-B.
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Acknowledgments |
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Footnotes |
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2 Investigator with the Howard Hughes Medical Institute.
Received January 28, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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