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Endocrine-Hypertension Division, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1–5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan
Address all correspondence and requests for reprints to: Dr. Yukio Hirata, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1–5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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120, 90, and 50 kDa) and
transiently increased association of a tyrosine-phosphorylated protein
(120 kDa) and Shc with the
glutathione-S-transferase-Grb2 fusion protein. A MAPK
kinase inhibitor (PD98059) also reduced the AM-induced MAPK activation,
c-fos messenger RNA expression, and cell proliferation.
Although AM has been shown to induce vasodilation through cAMP
production in VSMC, a cAMP antagonist (Rp-cAMP-thionate) and a protein
kinase A inhibitor (KT5720) failed to block AM-induced MAPK activation
and DNA synthesis. Moreover, 8-bromo-cAMP and forskolin did not affect
the MAPK activity. AM had no effect on either the intracellular
Ca2+ concentration or inositol 1,4,5-trisphosphate
formation. In addition, a protein kinase C inhibitor (GF109203X) did
not inhibit the AM-induced MAPK activation. These data suggest that in
addition to its vasodilatory effect through the cAMP-dependent pathway,
AM exerts its mitogenic activity via protein tyrosine kinase-mediated
MAPK activation in quiescent rat VSMC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, it has been shown that AM is an autocrine growth factor for various tumor cell lines (11), and that AM stimulates DNA synthesis and cell proliferation in Swiss 3T3 fibroblasts (12). It has also been reported that CGRP stimulates the proliferation of cultured human EC (13). It has been reported that plasma AM concentrations increase in patients with certain cardiovascular diseases, such as essential hypertension (14, 15), congestive heart failure (16, 17), and septic shock (18). Thus, AM may be involved not only in the regulation of vascular tone but also in the process of vascular lesion formation associated with hypertension and atherosclerosis.
p42/p44 mitogen-activated protein kinase (MAPK) is a member of a family of serine/threonine kinases that may participate in the regulation of cell growth and differentiation (19, 20, 21). MAPK is rapidly activated by activation of growth factor/tyrosine kinase receptors and G protein- coupled receptors (22, 23). Receptor tyrosine kinases and G protein-coupled receptors can recruit a set of adaptor proteins (Shc, Grb2, and Sos), and Sos catalyzes the exchange of GDP to GTP on the membrane-bound p21ras, thereby initiating MAPK cascade. The activation of MAPK results in phosphorylation of a nuclear transcriptional factor, Elk1, which induces expression of the growth-associated nuclear protooncogene c-fos (24, 25).
In the present study, we examined the role of the signal transduction cascade activated by AM in cultured rat VSMC. We found that AM stimulates MAPK activity, c-fos expression, and subsequent proliferation of quiescent rat VSMC via a protein tyrosine kinase (PTK) pathway, but not via a cAMP/protein kinase A (PKA) or Ca2+/protein kinase C (PKC) pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]deoxy-CTP (SA, 111 tetrabecquerels/mmol), and
[
-32P]ATP (SA, 37 megabecquerels/mmol) were obtained
from Amersham International (Aylesbury, UK). Human c-fos
complementary DNA (cDNA) was provided by the Japanese Cancer Research
Bank (Tokyo, Japan).
Cell culture
Rat VSMCs were prepared from the thoracic aorta of 6-week-old
male Wistar rats using the explant method and cultured in DMEM
containing 10% FCS at 37 C in a humidified atmosphere of 95% air-5%
CO2 as previously described (26). Subcultured cells
(10–20th passages) were made quiescent by incubation with serum-free
DMEM for about 2–3 days.
MAPK activity
Quiescent VSMCs (5 x 105 cells) grown on a
24-well plate were stimulated with AM at 37 C in serum-free DMEM for
the indicated times. The reaction was terminated by the replacement of
medium with ice-cold lysis buffer [10 mM Tris-HCl (pH
7.4), 20 mM NaCl, 2 mM EGTA, 2 mM
dithiothreitol, 1 mM Na3VO4, 1
mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and
10 µg/ml aprotinin]. After brief sonication, the sample was
centrifuged at 14,000 x g for 5 min at 4 C, and the
supernatant was assayed for MAPK activity by measurement of the
incorporation of [-32P]ATP into a synthetic peptide
(KRELVEPLTPAGEAPNQALLR) as a specific MAPK substrate using an assay kit
(Amersham). The reaction was carried out with the cell lysate (1
µg protein) in 75 mM HEPES buffer, pH 7.4, containing 1.2
mM MgCl2, 2 mM substrate peptide,
and 1.2 mM ATP/1 µCi [-32P]ATP for 30
min at 30 C. The resultant solution was applied to a phosphocellulose
membrane (Amersham), which was extensively washed in 1% acetic acid
and then in H2O. The radioactivity trapped on the membrane
was measured in a liquid scintillation counter (1900TR, Packard
Instrument Co., Meriden, CT).
Western blotting
Quiescent VSMCs (2 x 106 cells) grown on a
six-well plate were stimulated with AM at 37 C in serum-free medium for
the indicated times. The reaction was terminated by the replacement of
medium with 100 µl SDS-PAGE buffer, pH 6.8, containing 62.5
mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM
dithiothreitol, and 0.1% bromophenol blue. After brief sonication,
samples were boiled for 5 min at 95 C and centrifuged (14,000 x
g, 5 min) at 4 C, and aliquots of the supernatants were
subjected to 10% SDS-PAGE. Proteins in the gel were transferred to a
nitrocellulose membrane (Hybond-ECL, Amersham) by electroblotting. The
membrane was treated with polyclonal phospho-specific MAPK antibody
(1:1000) that recognizes p42/p44 MAPK only when catalytically activated
by phosphorylation at Tyr204, with recombinant
antiphosphotyrosine antibody (1:1000), or with polyclonal rabbit
anti-ERK2 antibody (1:1000). After incubation with the appropriate
secondary antibodies, immunoreactive proteins were detected using the
ECL Western blotting detection kit (Amersham).
For immunoblot analysis of Grb2 associatable proteins, quiescent VSMCs
(5 x 106 cells) were stimulated for the indicated
times and lysed in 0.8 ml lysis buffer (pH 7.4, containing 20
mM Tris-HCl, 150 mM NaCl, 2.5 mM
EDTA, 1.0% Triton-X, 0.1% SDS, 10% glycerol, 50 mM NaF,
10 mM Na3P2O7, 1.0%
deoxycholic acid, 1 mM Na3VO4, 1
mM phenylmethylsulfonylfluoride, and 10 µg/ml aprotinin).
Lysates were sonicated for 5 sec and then centrifuged at 14,000 x
g for 5 min at 4 C, and the supernatant was rocked with
agarose-conjugated GST-Grb2 fusion protein (6 µg) for 16 h at 4
C. The beads were washed three times with lysis buffer, solubilized in
Laemmli sample buffer, and subjected to immunoblotting. After the
membrane was initially treated with mouse monoclonal
antiphosphotyrosine antibody (1:1,000) or polyclonal anti-Shc antibody
(1:3,000) and then with secondary antibodies, immunoreactive proteins
were detected by the ECL detection kit.
Northern blot analysis
Quiescent VSMCs (5 x 106 cells) were
incubated with AM in fresh serum-free DMEM for the indicated times.
Total RNAs from VSMCs were extracted using the acid guanidinium
thiocyanate-phenol-chloroform method (27). Northern blot analysis was
carried out as previously described (28). Briefly, cellular RNAs (20
µg) were separated by formaldehyde-1.1% agarose gel electrophoresis
and transferred to a MagnaGraph nylon membrane (Micron Separations,
Westborough, MA). After UV wave cross-linking, RNA immobilized on the
membrane was hybridized with human c-fos cDNA as a probe in
the presence of 50% formamide at 42 C. The probe was labeled with
[-32P]deoxy-CTP triphosphate by the random primed
labeling method. The membrane was washed finally in 0.1 x SSPE
(15 mM NaCl, 1 mM
NaH2PO4, and 0.1 mM EDTA)-0.5% SDS
at 50 C and autoradiographed with intensifying screens for 24
h.
Mitogenic assays
DNA synthesis was assessed by incorporation of
[3H]thymidine into cells as previously described (29). In
brief, after preincubation in serum-free DMEM for 48 h, the
quiescent VSMC (5 x 105 cells) were incubated with
AM for 20 h, after which 1 µCi [3H]thymidine was
added, and the cells were further incubated for 4 h. After
completion, trichloroacetic acid-insoluble radioactivity was measured
with a liquid scintillation counter (1900TR, Packard).
For determination of cell number, subconfluent VSMCs (2 x
104 cells) were preincubated in DMEM containing 0.1% FCS
for 48 h. After medium was renewed by the same fresh medium, cells
were incubated with AM for 3 days. After trypsinization, cell number
was determined in a cell counter (CDA-500, Toua Medical Electronics
Co., Kobe, Japan).
Determination of intracellular calcium concentration
([Ca2+]i)
Measurement of [Ca2+]i was determined
by the Ca2+-fura-2 fluorescence method as described
previously (30). After incubation in serum-free DMEM for 48 h,
cells were trypsinized and incubated with 4 µM fura-2
acetoxymethyl ester (Dojindo Chemical Laboratory, Kumamoto, Japan) at
37 C for 20 min in buffered physiological salt solution. The
Ca2+-fura-2 fluorescence of the suspended cells was
measured by a spectrofluorometer (CA-200DP, Japan Spectroscopic Co.,
Tokyo, Japan) using excitation at 340 and 380 nm and emission at 500
nm. [Ca2+]i values were determined according
to the method of Grynkiewicz et al. (31).
Measurement of inositol 1,4,5-trisphosphate (IP3)
For measurement of IP3, quiescent VSMCs (106
cells/well) were incubated with AM or ET-1 in 2 ml Hanks’ Balanced
Salt Solution, pH 7.4, at 37 C for 30 sec as previously described (32).
Incubation was terminated by the addition of 15% trichloroacetic acid,
and the extracts were used for measurement of IP3 using a protein
binding assay kit (Amersham).
Statistical analysis
All results are expressed as the mean ± SE of
three to five samples (n). Student’s t test was used for
the statistical analysis; P < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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); the approximate half-maximal doses
(ED50) of AM and CGRP were 2.5 x 10-9
and 5.6 x 10-8 M, respectively. To
confirm the mitogenic activity of AM on VSMC, cell number was measured
after treatment with AM for 3 days. AM caused a dose-dependent increase
in cell number; the approximate ED50 was 2.5 x
10-9 M (Fig. 1B).
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, AM
(10-7 M)- and CGRP (10-7
M)-stimulated DNA syntheses were equally and dose
dependently (10-8–10-5 M)
inhibited by a CGRP antagonist, CGRP-(8–37). The AM (5 x
10-8 M)-induced increase in cell number was
also inhibited by CGRP-(8–37) (10-6 M; Fig. 2B).
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, AM (10-7
M) caused a transient MAPK activation, peaking at 5 min,
which returned to the basal level by 30 min, whereas neither
8-bromo-cAMP (10-3 M) nor forskolin
(10-5 M) affected MAPK activity in VSMC. AM
stimulated MAPK activity in a dose-dependent manner
(10-9–10-6 M) with the
approximate ED50 of 2.5 x 10-8
M (Fig. 3B). As p42/p44 MAPK activation is closely
correlated with its phosphorylation at Tyr204, the effect
of AM on phosphorylation of p42/p44 MAPK was analyzed by immunoblotting
with polyclonal antibody, which selectively recognizes
Tyr204-phosphorylated MAPKs. AM (10-7
M) induced tyrosine phosphorylation of both p42 and p44
MAPKs within 5 min (Fig. 4B).
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) and its phosphorylation (Fig. 4B). These data
suggest that the AM-induced MAPK activation and subsequent cell growth
are mediated via the same AM/CGRP receptor.
Role of MAPK pathway in c-fos expression and cell proliferation by
AM
To determine the role of MAPK cascade in the growth-promoting
effect of AM, we examined the effect of a MAPK kinase (MEK-1)
inhibitor, PD98059, on AM-induced MAPK activation, c-fos
expression, and cell growth in VSMC. PD98059 (5 x
10-5 M) decreased the AM-induced MAPK
activation (Fig. 5A) and its
phosphorylation (Fig. 5B). A transient (0.5-h) expression of
c-fos messenger RNA (mRNA) by AM (10-7
M) was markedly (60%) inhibited by PD98059 (5 x
10-5 M; Fig. 6A), whereas PD98059 (5 x
10-5 M) decreased the AM-stimulated increase
in cell number by about 70% (Fig. 6B). These results suggest the
critical role of the MAPK pathway in the mechanism of AM-induced
c-fos expression and cell proliferation.
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, AM rapidly (within 1
min) induced tyrosine phosphorylation of at least three distinct
proteins (50, 90, and 120 kDa) as revealed by Western blot
analysis using antiphosphotyrosine antibody; these bands were markedly
reduced by pretreatment with genistein (10-4
M), a PTK inhibitor.
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, AM transiently (within 1 min) increased association
of the tyrosine-phosphorylated protein (120 kDa) with Grb2 as
detected by immunoprecipitation with GST-Grb2 and immunoblotting with
an antiphosphotyrosine antibody. The rapid association of all Shc
isoforms (p46, p52, and p66) with Grb2 was also observed upon AM
treatment (Fig. 7B). Pretreatment with genistein
(10-4 M) completely blocked MAPK
activation (Fig. 8A) and the increase in
cell number stimulated by AM (10-7 M; Fig. 8B). ST638 (10-4 M), a highly selective PTK
inhibitor, also blocked the AM-induced MAPK activation (data not
shown). These data suggest that AM induces tyrosine phosphorylation of
p120, a putative PTK, to recruit adaptor proteins (Shc and Grb2),
thereby promoting MAPK activation and subsequent cell growth in
VSMC.
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) or [Ca2+]i
(Fig. 9B). GF109203X (2 x 10-6 M), a
highly specific PKC inhibitor that completely blocked PMA-induced MAPK
activation (33), had no effect on the AM-induced MAPK activation or its
phosphorylation (data not shown).
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) or its phosphorylation (data not
shown); Rp-cAMP-thionate (10-3 M) also did not
affect AM-stimulated DNA synthesis (Fig. 10B). These data suggest that
neither the Ca2+/PKC pathway nor the cAMP/PKA pathway
is involved in the AM-induced mitogenic signal in rat VSMC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported that AM- and CGRP-induced cAMP responses in rat VSMC were inhibited by a CGRP receptor antagonist, CGRP-(8–37), suggesting that AM and CGRP may interact with the same and/or very similar receptors in rat VSMC (7). In the present study, both AM and CGRP stimulated DNA synthesis and proliferation of quiescent rat VSMC in culture, the effects of which were similarly antagonized by a CGRP antagonist, CGRP-(8–37). Recently, a cDNA clone for the putative 395-residue AM receptor containing seven transmembrane domains was identified in rat lung (35). However, CGRP does not interact with the putative AM receptor to increase cAMP generation (35). Northern blot analysis of total RNA from our rat VSMC in culture with PCR-cloned cDNA for the putative AM receptor failed to detect its expression (Iwasaki, H., T. Imai, and Y. Hirata, unpublished observation). Therefore, there exist receptor subtypes for AM/CGRP other than the putative AM receptor in rat VSMC.
Activation of MAPK by the tyrosine kinase receptors, such as platelet-derived growth factor and epidermal growth factor, appears to be a common requirement for cell growth and differentiation (36). Recently, the G protein-coupled receptors for certain vasoconstrictor peptides, such as angiotensin II and ET-1, have been shown to induce MAPK activation in rat VSMC (37, 38). In the present study, we have clearly demonstrated that stimulation with AM resulted in a marked activation of MAPK associated with its tyrosine phosphorylation in rat VSMC. There appears to be some discrepancy between the maximal responses of MAPK activity in response to AM in the present study. This may be due to the different passages of VSMCs used in each experiment and/or very rapid (within 5 min) and transient activation. Thus, Western blotting using phospho-specific MAPK antibody was performed, confirming the data from these kinase assays. Likewise, the apparent difference between the ED50 for DNA synthesis and MAPK activation by AM, although not considerably significant, may be accounted for by the same reasons. Alternatively, other signal transduction(s) in addition to MAPK activation may be involved in the maximal growth response by AM.
Recently, it has been reported that AM induces the immediate-early gene, c-fos, in cultured rat VSMC and cardiomyocytes (39). MAPK phosphorylates the transcription factor Elk1, thereby leading to c-fos gene expression via a serum-responsive element in its promoter region (24). Thus, it is suggested that AM stimulates c-fos expression through MAPK activation. In fact, the present study has demonstrated that PD98059, a MEK-1 inhibitor, inhibited AM- induced MAPK activation, c-fos mRNA expression, and mitogenesis. However, the partial inhibition of c-fos induction and cell growth by PD98059 suggest that MAPK activation is necessary, but not sufficient, for the maximal growth response by AM in rat VSMC.
Recently, it has been reported that angiotensin II induced MAPK
activation through a PTK-dependent mechanism in rat VSMC (33). Thus, we
asked whether PTK activation is required for the AM-induced mitogenesis
in rat VSMC. In the present study, AM rapidly induced tyrosine
phosphorylation of at least three distinct proteins with different
molecular sizes (50, 90, and 120 kDa), whose characteristics
are currently unidentified. As tyrosine phosphorylation of these
molecules was very rapid (within 1 min), they may represent targets for
PTK after AM receptor activation. It should be noted that tyrosine
phosphorylation and activation of MAPK and cell proliferation induced
by AM were equally blocked by PTK inhibitors (genistein and ST638).
Taken together, it is suggested that activation of as yet
uncharacterized PTK by AM is required for MAPK activation and
subsequent cell proliferation.
An adaptor protein, Shc, after tyrosine phosphorylation upon activation of both receptor and nonreceptor PTKs (40, 41), can link tyrosine-autophosphorylated PTKs with the Grb2/Sos complex, thereby leading to p21ras activation (42). Recently, Shc has also been implicated in p21ras and MAPK activation by several G protein-coupled receptors (40, 41, 42, 43, 44, 45). In the present study, we have demonstrated that AM rapidly increased the amounts of all three Shc isoforms (p46, p52, and p66) coprecipitated with a GST-Grb2 fusion protein in rat VSMC. Moreover, AM induced association of a 120-kDa tyrosine-phosphorylated protein with GST-Grb2 fusion protein with a time course similar to that of Shc. These data further suggest that AM activates a 120-kDa PTK, as yet uncharacterized, to recruit Shc, thereby initiating the p21ras/MAPK cascade in VSMC. Recently, Pyk2, a nonreceptor tyrosine kinase with an apparent molecular mass of 120 kDa, has been implicated in MAPK activation with several stimuli, such as activation of certain G protein-coupled receptors, PKC activation, UV irradiation, and increases in [Ca2+]i and extracellular osmolarity (46, 47). Whether Pyk2 plays a central role in AM-induced MAPK activation is currently under investigation in our laboratory.
It has been reported that activation of AM receptor coupled to Gq protein in bovine EC resulted in an increase in [Ca2+]i and PKC activation through IP3 and diacylglycerol production, respectively (9). However, it is unclear whether Gq activation is involved in MAPK activation by AM in VSMC. In our experiment, a PKC inhibitor (GF109203X) that completely blocked the PMA-induced MAPK activation (33) had no effect on AM-induced MAPK activation, and AM itself affected neither IP3 formation nor [Ca2+]i in rat VSMC. These data indicate that the AM-induced MAPK activation and subsequent cell growth in rat VSMC are independent of Gq-coupled second messengers.
As AM/CGRP stimulates cAMP formation in rat VSMC (7), whose potencies were almost comparable to stimulate DNA synthesis in the present study, cAMP may be an alternative intracellular messenger for mitogenesis. Recently, it has been reported that PKA stimulated phosphorylation and activation of MAPK via the serine/threonine kinase B-Raf in PC12 cells (48). In fact, AM stimulated DNA synthesis and cell proliferation of Swiss 3T3 cells via a cAMP-dependent mechanism (12). However, the role of a cAMP/PKA pathway in the regulation of VSMC growth by AM is unlikely, due to the following observations. First, both a PKA inhibitor (KT5720) and a cAMP antagonist (Rp-cAMP-thionate) failed to inhibit MAPK activity and [3H]thymidine uptake by AM. Second, forskolin and 8-bromo-cAMP failed to stimulate MAPK activity in VSMC. Third, compounds that increase cAMP formation and activate PKA conversely inhibited platelet-derived growth factor-BB- and thrombin-induced activation of MAPK kinase and DNA synthesis in rat VSMC (49, 50). Collectively, these findings suggest that AM-induced cAMP generation and subsequent PKA activation are not involved in its mitogenic effect by AM in rat VSMC.
Several ligands that bind to G protein-coupled receptors use pertussis
toxin (PTX)-sensitive Gi/Go proteins
for their mitogenic responses (51, 52). Recently, it has been shown
that a PTX-sensitive MAPK cascade involving
p21ras was mediated through a PTK pathway
activated by ß-subunits of Gi/Go (24, 53).
However, our preliminary data do not support a role for PTX-sensitive G
proteins in the AM-induced MAPK activation, because pretreatment with
PTX failed to affect AM-induced MAPK activation (unpublished
observation). Thus, the mitogenic signaling by AM occurs independent of
PTX-sensitive G proteins. Identification and characterization of the
putative transducer(s) to activate PTK by AM are currently under
investigation.
Recently, it has been reported that AM inhibited serum-stimulated proliferation and platelet-derived growth factor-stimulated migration of rat VSMC via a cAMP-dependent process (54, 55). We also confirmed that AM only partially inhibited serum-stimulated DNA synthesis in our rat VSMC in culture (Iwasaki, H., S. Eguchi, and Y. Hirata, unpublished observation). Although the reason for the apparent discrepancy is unknown, AM may play dual roles in VSMC growth: one as a growth promoter under a quiescent state, and another as a growth inhibitor under a proliferative state, as is the case with transforming growth factor-ß (56, 57).
A considerable amount of AM is synthesized by and secreted from
cardiovascular tissues in vitro, including VSMCs, ECs, and
cardiomyocytes (3, 4, 6), which may contribute to the circulating AM
levels in vivo. Cytokines, such as interleukin-1ß and
tumor necrosis factor-, and glucocorticoids have been shown to
induce AM gene expression in cultured VSMC, EC, and cardiomyocytes (5, 6). As the local concentrations of AM at the site of vasculature should
be far higher than those in plasma, our in vitro study
suggests that AM may function as an autocrine and/or paracrine growth
factor for VSMC. However, further study is required to clarify the
pathophysiological role of AM in the process of vascular remodeling
associated with hypertension, atherosclerosis, and endothelial
dysfunction.
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Footnotes |
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Received January 28, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 H. Iwasaki, S. Eguchi, H. Ueno, F. Marumo, and Y. Hirata Mechanical stretch stimulates growth of vascular smooth muscle cells via epidermal growth factor receptor Am J Physiol Heart Circ Physiol, February 1, 2000; 278(2): H521 - H529. [Abstract] [Full Text] [PDF]
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K. Shimizu, H. Tanaka, M. Sunamori, F. Marumo, and M. Shichiri Adrenomedullin Receptor Antagonism by Calcitonin Gene-Related Peptide(8-37) Inhibits Carotid Artery Neointimal Hyperplasia After Balloon Injury Circ. Res., December 3, 1999; 85(12): 1199 - 1205. [Abstract] [Full Text] [PDF]
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H. Iwasaki, S. Eguchi, H. Ueno, F. Marumo, and Y. Hirata Endothelin-Mediated Vascular Growth Requires p42/p44 Mitogen-Activated Protein Kinase and p70 S6 Kinase Cascades via Transactivation of Epidermal Growth Factor Receptor Endocrinology, October 1, 1999; 140(10): 4659 - 4668. [Abstract] [Full Text]
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 M. Shichiri, H. Kato, M. Doi, F. Marumo, and Y. Hirata Induction of Max by Adrenomedullin and Calcitonin Gene-Related Peptide Antagonizes Endothelial Apoptosis Mol. Endocrinol., August 1, 1999; 13(8): 1353 - 1363. [Abstract] [Full Text]
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H. Nishimatsu, E. Suzuki, D. Nagata, N. Moriyama, H. Satonaka, K. Walsh, M. Sata, K. Kangawa, H. Matsuo, A. Goto, et al. Adrenomedullin Induces Endothelium-Dependent Vasorelaxation via the Phosphatidylinositol 3-Kinase/Akt-Dependent Pathway in Rat Aorta Circ. Res., July 6, 2001; 89(1): 63 - 70. [Abstract] [Full Text] [PDF]
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