Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adrenomedullin (AM) is a potent
vasodilating peptide and is involved in cardiovascular and renal
disease. In the present study, we investigated the role of AM in
cardiac and renal function in streptozotocin (STZ)-induced diabetic
rats. A single tail-vein injection of adenoviral vectors harboring the
human AM gene (Ad.CMV-AM) was administered to the rats 1-wk post-STZ
treatment (65 mg/kg iv). Immunoreactive human AM was detected in the
plasma and urine of STZ-diabetic rats treated with Ad.CMV-AM.
Morphological and chemical examination showed that AM gene delivery
significantly reduced glycogen accumulation within the hearts of
STZ-diabetic rats. AM gene delivery improved cardiac function compared
with STZ-diabetic rats injected with control virus, as observed by decreased left ventricular end-diastolic pressure, increased cardiac output, cardiac index, and heart rate. AM gene transfer significantly increased left ventricular long axis (11.69 ± 0.46 vs. 10.31 ± 0.70 mm, n = 10, P < 0.05) and rate of
pressure rise and fall (+6,090.1 ± 597.3 vs. +4,648.5 ± 807.1 mmHg/s), (4,902.6 ± 644.2 vs.
3,915.5 ± 805.8 mmHg/s,
n = 11, P < 0.05). AM also significantly attenuated renal glycogen accumulation and tubular damage in
STZ-diabetic rats as well as increased urinary cAMP and cGMP levels,
along with increased cardiac cAMP and Akt phosphorylation. We also
observed that delivery of the AM gene caused an increase in body weight along with phospho-Akt and membrane-bound GLUT4 levels in skeletal muscle. These results suggest that AM plays a protective role in
hyperglycemia-induced glycogen accumulation and cardiac and renal
dysfunction via Akt signal transduction pathways.
streptozotocin; adrenomedullin; gene delivery; adenovirus
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ADRENOMEDULLIN
(AM) is a potent 52-amino acid vasodilator originally isolated from
tissue extracts of human pheochromocytoma (20). AM has
been detected in a variety of organs, such as the adrenal gland,
kidney, heart, lung, spleen, and brain, and has also been found to be
secreted from endothelial and vascular smooth muscle cells (17,
31). AM is involved in a variety of biological activities,
including vasodilation, diuresis, and inhibition of aldosterone
secretion (6, 20). Plasma AM levels are increased in
patients with cardiac hypertrophy, heart failure, renal dysfunction, and hyperglycemia (10, 19, 26). AM has been reported to act in an autocrine/paracrine fashion to prevent cardiovascular and
renal damage (33). Recently, AM has been shown to have a key role in cardiovascular development. AM knockout (/
) mice were
embryonically lethal by midgestation due to cardiovascular abnormalities (1). These results suggest a possible role
for AM in the regulation of cardiac and renal function
(17).
AM's effect on cardiac function has been suggested to result from the activation of an autonomic baroreflex response, which increases the heart rate to counteract the hypotensive effects of AM (8, 22). However, observations in sheep and rat papillary muscle suggest that AM has direct inotropic effects (18, 28). Even with conflicting reports of AM's effects on cardiac function, there are studies showing direct effects on cardiac cells. AM is not only produced in cardiomyocytes but is capable of binding to and triggering physiological responses of cardiomyocytes (33). AM has been reported to stimulate cAMP and cGMP levels and to reduce extracellular matrix formation along with DNA and protein synthesis in cardiomyocytes (25, 31, 33). Recently, AM has been linked to embryonic cardiovascular development (1).
Diabetics suffer from vessel damage leading to both vision and circulation problems and are three to eight times more likely to suffer from abnormal cardiac function, termed "diabetic cardiomyopathy." Diabetic cardiomyopathy ranges in severity from mild dysfunction to failure (29). Rats treated with streptozotocin (STZ) not only develop hypoinsulinimia but also cardiomyopathy, polydypsia, hyperglycemia, and glucosuria (7, 34). Previous studies showed that hearts and kidneys from STZ-treated rats contain abnormally high levels of glycogen (3). Glycogen accumulation may be a contributing factor to the development of cardiac and renal dysfunction. Nishimatsu et al. (27) recently reported that AM is capable of directly activating Akt via phosphatidylinositol 3-kinase (PI 3-kinase) in rat aorta. Akt activation inhibits apoptosis and enhances the survival of cardiomyocytes. Phosphorylated Akt can also stimulate cellular glucose usage and stimulate glucose transporter 4 (GLUT4) to the membrane in skeletal muscle (21, 23). In this study, we investigated the potential protective role and mechanism of action of AM in hyperglycemia-induced cardiac and renal damage.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Administration of STZ. STZ (Sigma Chemical) was dissolved in ice-cold 0.05 M citrate buffer (pH 4.5). Two-week-old male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were injected with 65 mg/kg STZ via the tail vein.
Replication-deficient adenoviral vectors and animal preparations. Adenoviral vectors harboring the human AM with the 4F2 enhancer (Ad.CMV-AM) or luciferase cDNA (Ad.CMV-Luc) under the control of the cytomegalovirus enhancer/promoter (CMV) were constructed and prepared as previously described (16). Experimental rats received 1.2 × 1010 plaque formation units, or pfu, of Ad.CMV-AM or Ad.CMV-Luc 1 wk post-STZ treatment. Animal care for all rats in this study conformed to the rules set forth by National Institutes of Health (NIH) guidelines.
Plasma and urine collection and analysis of physiological
parameters.
Rat plasma was collected at various time points throughout the
experiment. Blood glucose levels were measured 7 days post-STZ treatment and at the end of the experiment (OneTouch Profile, Johnson
and Johnson, Milpitas, CA). Twenty-four-hour urine was collected 7 days
after adenoviral vector-mediated gene delivery. Animals were allowed
free access to food and water during the collection period. Urine was
collected and centrifuged at 1,000 g for 10 min to remove
particles, and the volume was recorded. Urine and plasma samples were
stored at 20°C for biochemical analysis.
RIA for human AM, cAMP, insulin, and cGMP. Immunoreactive human AM was determined in plasma by an RIA for human AM by use of rabbit anti-human AM 1-52 antiserum (Peninsula Laboratories, San Carlos, CA), as previously described (2). Plasma insulin levels were measured by a commercially available rat insulin kit according to the manufacturer's instructions (Linco, St. Charles, MO). Urinary and cardiac cAMP and cGMP levels were also determined by RIA (11, 14).
Morphological and histological investigation. Paraffin-embedded tissues were cut at 4 µm and stained with Periodic acid Schiff (PAS). Stained sections were analyzed microscopically and morphometrically. PAS-stained heart sections were examined for glycogen content within the cellular compartment. PAS-stained kidney sections were also evaluated and graded on a scale of 1 to 5, where 5 represented the greatest damage. The detailed scale follows: 1 = normal tissue; 2 = some outer medullary tubule damage; 3 = damage to outer medullary tubules, as well as at least one-third of the cortical distal tubules; 4 = >50% of distal tubule damage; and 5 = majority of distal tubules disrupted as well as proximal brush border damage. All sections were evaluated by researchers under double-blind conditions.
Tissue glycogen assay. Glycogen levels in the heart, kidney, skeletal, and liver extracts were determined as previously described (5). Briefly, 0.1 g of each tissue extract was dissolved in 30% KOH and heated at 100°C for 10 min, followed by a 3-min room temperature incubation. The samples were diluted (1:10) with 30% KOH and vortexed. Anhydrous ethanol was added, and samples were centrifuged at 5,700 rpm for 15 min. The supernatant was carefully removed, and the pellet was resuspended in 0.5 ml of H2O. One milliliter of 0.2% anthrone reagent (0.2 g in 100 ml of 98% H2SO4) was added and mixed, and the mixture was incubated at room temperature for 30 min. The samples were then measured at 620 nm with a spectrophotometer.
Cardiac function.
Cardiac function was performed as previously described
(30). Briefly, animals were anesthetized with
pentobarbital sodium (50 mg/kg body wt). The femoral and carotid
arteries were cannulated. Heart rate, arterial blood pressure, and left
ventricular end-diastolic pressure (LVEDP) were recorded. Fluorescent
microspheres (FluoSpheres; Molecular Probes, Eugene, OR) were injected
directly into the left ventricle while arterial blood was collected for
a total of 90 s from the femoral artery. At the end of cardiac
function surgery, the collected blood and one kidney were subjected to digestion solution for 2 days to release the microspheres. The microspheres were then quantitated in a spectrofluorometer and excited
at 570 nm, and emission was read at 598 nm. The rats were perfused with
normal saline (0.9% NaCl). The whole heart, left ventricle (including
the intraventricular septum), and left and right kidneys were removed,
blotted, and weighed. Tissue samples were removed, frozen immediately
in liquid nitrogen, and stored at 80°C or fixed in 4% buffered
formaldehyde solution and embedded in paraffin.
Tissue preparation and Western blot. At the end of the experiment, tissue samples (0.1-0.2 g) were harvested for extraction. Briefly, the tissue was minced, placed in RIPA buffer (1× PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and proteinase inhibitor cocktail), and subjected to two 15-s polytron cycles on ice. Skeletal muscle membrane fractions were isolated, as previously described (37). Briefly, skeletal muscle was minced and homogenized, and the supernatant was collected by centrifugation at 9,000 g for 20 min. The resulting supernatant was then centrifuged at 180,000 g for 90 min. The pellet was resuspended and loaded onto a 10-30% (wt/wt) continuous sucrose gradient and centrifuged at 48,000 rpm for 55 min. Protein concentrations were determined by Lowry's method.
Aliquots containing equal amounts of protein (50-100 µg) were subjected to SDS-PAGE and blotted onto nitrocellulose membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). Membranes were incubated with specific antibodies against phosphorylated Akt (pAkt), Akt (Cell Signaling Technology, Beverly, MA), PKC-Statistical analysis. Results are expressed as means ± SE. Comparisons among groups were made by ANOVA followed by Fisher's protected least significant difference test or by an unpaired Student's t-test. Differences were considered significant at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Diabetic state and blood glucose levels.
Blood glucose levels were measured at various time points throughout
the experiment. Five days post-STZ treatment, blood glucose levels of
all treated animals reached 436.2 ± 12.7 mg/dl (n = 10). At the end of the experiment, there were no significant
differences in blood glucose levels in treated STZ-diabetic
rats injected with the AM or luciferase gene (447.0 ± 64.0 vs.
417.3 ± 65.8 mg/dl, n = 10; Table
1). All animals treated with STZ
developed hypoinsulinemia (Table 1).
|
Expression of human AM in STZ-induced diabetic rats. Immunoreactive human AM levels in rats receiving AM gene delivery were measured by a specific RIA for human AM. Human AM levels in the plasma 6 days after gene delivery reached 78.9 ± 81.7 ng/ml, and urine levels 7 days after gene delivery reached 15.9 ± 6.8 ng/ml (n = 7). Linear displacement curves of serial dilutions of urine and plasma from rats injected with the human AM gene displayed parallelism to the human AM standard curve, indicating their immunological identity (data not shown).
Effect of adenoviral-mediated gene delivery of human AM on
body weight of STZ-induced diabetic rats.
AM gene delivery improved the body weight of STZ-diabetic rats.
Normally, rats gain ~5 g · body
wt1 · day
1, but after
STZ treatment the animals neither gained nor lost weight. By the end of
the experiment, the STZ-diabetic rats treated with Ad.CMV-AM developed
a slight but significant increase in body weight compared with the
STZ-diabetic rats treated with Ad.CMV-Luc (Table 1,
n = 10, P < 0.05).
Effects of human AM gene delivery on morphology and tissue glycogen
content in STZ-induced diabetic rats.
Morphological evaluation of cardiac tissue revealed a beneficial effect
of AM gene delivery. PAS stains sugar moieties bright red, clearly
distinguishing cardiac and renal tissues of STZ-diabetic control rats
from STZ-diabetic rats treated with human AM (Figs. 1, A-C, and
2, A-C). AM gene
delivery resulted in reduction of glycogen accumulation in the heart
and kidney. A chemical glycogen assay was used to quantify tissue
glycogen levels. Cardiac glycogen levels were significantly reduced in
the STZ-diabetic animals receiving AM gene delivery compared with the
control STZ animals injected with the luciferase gene (6.14 ± 1.56 vs. 9.51 ± 3.25 mg/g tissue, n = 5, P < 0.05; Fig. 1D). Similarly, kidney
glycogen levels were also significantly reduced by AM gene delivery
compared with the luciferase control diabetic rats (1.26 ± 0.27 vs. 1.73 ± 0.32 mg/g tissue, n = 4, P < 0.05; Fig. 2D). Scaled kidney damage was also significantly reduced in AM-treated STZ-diabetic rats (3.30 ± 0.20 vs. 4.20 ± 0.37 scale, n = 5, P < 0.05; Fig. 2E). However, glycogen
levels within the skeletal muscle were not affected (0.5 ± 0.1 vs. 0.4 ± 0.1 mg/g tissue, n = 4).
|
|
Effect of human AM gene delivery on cardiac function of
STZ-diabetic rats.
Three weeks post-STZ treatment, which correlates to 2 wk after gene
delivery, animals were subjected to cardiac function evaluation. AM
gene delivery was observed to provide an overall protective effect on
the function of diabetic hearts. Figure 3
(top) depicts typical maximal rate of left ventricular (LV)
pressure rise and fall (±P/
t) in waveforms from each
of the experimental groups. The ±
P/
t was
significantly improved in the STZ-diabetic rats injected with human AM
compared with the STZ-diabetic rats injected with the luciferase gene
(+6,090.1 ± 597.3 vs. +4,581.8 ± 810.9 and
4,903.4 ± 643.4 vs.
3,919.6 ± 837.1 mmHg/s, n = 11, P < 0.05; Fig. 3, bottom). An index of
congestive heart failure, left ventricular end-diastolic pressure, also
showed significant improvement in the STZ-diabetic rats treated with
the human AM gene compared with the STZ-diabetic rats treated with the
luciferase gene (6.15 ± 1.75 vs. 8.82 ± 2.47 mmHg,
n = 11, P < 0.01, Fig.
4). Table 1 shows that AM protects
against cardiac dysfunction of STZ-diabetic rats via improvement in
cardiac output, cardiac index, and left ventricle long axis.
|
|
Effect of human AM gene delivery on cAMP and cGMP levels.
AM gene delivery significantly increased cardiac cAMP levels in
STZ-diabetic rats compared with control rats (6.30 ± 1.34 vs.
4.94 ± 1.51 nmol/µg protein, n = 6 and 4, P < 0.05). In addition, urinary cAMP (55.2 ± 2.9 vs. 39.3 ± 3.3 nmol · 100 g body
wt1 · day
1,
n = 6 and 4, P < 0.05) and cGMP levels
were also increased in the AM-treated STZ-diabetic rats (7.65 ± 0.79 vs. 5.1 ± 0.23 nmol · 100 g body
wt
1 · day
1;
n = 4, P < 0.05, Table 1).
Alteration of cardiac Akt.
Figure 5 shows the effect of AM gene
delivery on phosphorylated Akt (pAkt) and total Akt (tAkt) in the
heart. Densitometric analysis revealed that STZ-diabetic rats
treated with AM had a significantly increased pAkt-to-tAkt ratio
compared with STZ-diabetic rats treated with luciferase
(0.385 ± 0.105 vs. 0.130 ± 0.027 arbitrary units,
n = 3, P < 0.05).
|
Membrane-bound GLUT4 in skeletal muscle.
Figure 6 shows the effect of AM gene
delivery on skeletal muscle tissue preparations. Figure 6A
shows an increase in the pAkt-to-tAkt ratio in the skeletal muscle of
AM-treated STZ-diabetic rats (0.675 ± 0.027 vs. 0.132 ± 0.013 arbitrary units, n = 2, P < 0.05). Membrane fractions of skeletal muscle were probed with a
specific antibody for GLUT4 (Fig. 6B). Densitometric
analysis showed a significant increase in GLUT4 present in the membrane
of skeletal muscle tissues from STZ-diabetic rats treated with the AM
gene compared with STZ-diabetic rats treated with the luciferase gene
(390.8 ± 113.2 vs. 226.9 ± 54.7 arbitrary units,
n = 3, P < 0.05). However,
membrane-bound PKC-2 identified by Western blotting
revealed no differences between AM-treated and control STZ-diabetic
rats (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The STZ-diabetic model has proved useful in a variety of research
studies for the characterization of treatments associated with diabetes
in humans. Since 1971, STZ has been used to create experimental
diabetic animal models. STZ causes hyperglycemia by specifically
inducing DNA strand breaks in pancreatic islet -cells and stimulates
nuclear poly(ADP-ribose) synthetase, thus depleting intracellular
NAD+ and NADP+ levels. Reduction of
intracellular NAD+ and NADP+ inhibits
proinsulin synthesis, leading to a diabetic state (38). It
has been suggested (32) that the production of activated oxygen species (superoxide, hydrogen peroxide, hydroxyl radical, and
singlet oxygen) has a major role in the development of STZ-induced diabetes.
In this study, we have shown that AM gene delivery is capable of improving the cardiac function of STZ-diabetic rats. Immunoreactive human AM levels were observed in both urine and plasma of STZ-diabetic rats receiving an AM-harboring adenovirus, indicating that human AM was being produced and secreted from both the liver and the kidney. In previous studies we observed AM mRNA expression in the liver, heart, and kidney, along with a reduction in blood pressure in hypertensive rats after intravenous injection of Ad.CMV-AM (36). The STZ-treated rats did not develop hypertension, and AM gene delivery did not produce a significant reduction in blood pressure. The use of metabolic cages failed to allow detection of a diuretic effect in the STZ-diabetic rats receiving the human AM transgene. This may indicate either that the expression level of recombinant human AM achieved in this study was not high enough to produce an effect, or more likely that AM's diuretic effect was masked due to the massive urinary excretion from STZ-diabetic rats (>100 ml/24 h).
AM has also been suggested to have a major role in cardiac and renal
function in addition to blood pressure regulation (26). Morphological evaluation and chemical assays demonstrated that somatic
human AM gene delivery is capable of preventing cardiac glycogen
accumulation in STZ-diabetic rats, suggesting increased glucose
utilization. Extensive cardiac glycogen levels could be attributed to
cardiovascular dysfunction and the development of cardiomyopathy.
Although an increase in PKC-2 activity has been linked
to diabetic cardiomyopathy (35), we found that AM has no
effect on membrane-bound PKC-
2 in STZ-diabetic rats
(data not shown). Reduction of cardiac damage was observed as improved ±
P/
t, LVEDP, LV weight, heart weight, and LV long
axis of the STZ-diabetic rats treated with human AM. These observations
are consistent with the notion that AM provides cardiovascular protection.
AM's biological effects on vasodilation and natriuresis have been shown to be mediated by both cAMP and nitric oxide-cGMP signaling pathways (15, 32). We observed an increase in both cardiac and renal (or urinary) cAMP levels, along with increased urinary cGMP levels, in the STZ-diabetic rats receiving AM gene delivery. In addition, AM increases phosphorylation of cardiac Akt. These results suggest that AM's protective effects are mediated, at least partially, through the PI 3-kinase-Akt pathway, along with cAMP and cGMP second messenger cascades (13, 15, 27). Akt is known to inhibit apoptosis and glycogen synthase kinase (GSK)3 while stimulating GLUT4 translocation and phosphofructokinase 2 (PFK-2) (9). These known Akt activities could help explain the observed protective effects of AM gene delivery in STZ-induced diabetic rats. Interaction with GSK3, PFK-2, and GLUT4 could lead to increased utilization of glucose, leading to a reduction in glycogen accumulation and thus improvement in body weight, cardiac function, renal damage, and overall health of the animal. The cAMP second messenger cascade could also be involved in the reduction of glycogen accumulation. Increased cAMP levels can activate protein kinases, leading to inactivation of glycogen synthase and thus inhibition of glycogen production (12). These same pathways could also be active in skeletal muscle, leading to the observed increased membrane-bound GLUT4 via the Akt pathway, and thus improved glucose utilization and body weights of AM-treated STZ-diabetic rats.
Adenoviral gene delivery is capable of producing high levels of the transgene; however, due to the lack of viral genome integration, transgene expression is only temporary. Adenoviral vectors can also stimulate host immune responses, leading to inflammation, loss of infected host cells, and thus transgene expression (4). To minimize the host immune and inflammatory responses, and the possibility of readministration without the production of notable host immune response, it is essential to develop improved viral vectors for prolonged transgene expression. Improved delivery vectors like adeno-associated viral vectors would make a prolonged experimental time line possible, which could potentially improve the elucidation of AM effects.
In this study, we have demonstrated that adenoviral-mediated gene delivery of human AM not only provided protection from cardiac dysfunction but also prevented body weight loss and kidney tubule damage in STZ-diabetic rats. The observed beneficial effects may result from AM's ability to counteract deleterious effects due to a diabetic state and suggest a potential therapeutic use for AM and/or its signal transduction pathway in cardiovascular and renal diseases, especially cardiomyopathy.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Heart, Lung, and Blood Institute Grant HL-29397.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: L. Chao, Dept. of Biochemistry and Molecular Biology, Medical Univ. of South Carolina, Charleston, SC 29425-2211 (E-mail: chaol{at}musc.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/ajpendo.00147.2002
Received 8 April 2002; accepted in final form 23 July 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Caron, KM,
and
Smithies O.
Extreme hydrops fetalis and cardiovascular abnormalities in mice lacking a functional adrenomedullin gene.
Proc Natl Acad Sci USA
98:
615-619,
2000
2.
Chao, J,
Jin L,
Lin KF,
and
Chao L.
Adrenomedullin gene delivery reduces blood pressure in spontaneously hypertensive rats.
Hypertens Res
20:
269-277,
1997[Medline].
3.
Chen, V,
Ianuzzo CD,
Fong BC,
and
Spitzer JJ.
The effects of acute and chronic diabetes on myocardial metabolism in rats.
Diabetes
33:
1078-1084,
1984[Abstract].
4.
Christ, M,
Lusky M,
Stoeckel F,
Dreyer D,
Dieterle A,
Michou AI,
Pavirani A,
and
Mehtali M.
Gene therapy with recombinant adenovirus vectors: evaluation of the host immune response.
Immunol Lett
57:
19-25,
1997[ISI][Medline].
5.
Chung, Y,
and
Dao YZ.
Glycogen assay for diagnosis of female genital chlamydia trachomatis infection.
J Clin Microbiol
36:
1081-1082,
1998
6.
Ebara, T,
Miura K,
Okumura M,
Matsuura T,
Kim S,
Yukimura T,
and
Iwao H.
Effect of adrenomedullin on renal hemodynamics and functions in dogs.
Eur J Pharmacol
263:
69-73,
1994[ISI][Medline].
7.
Fein, FS,
and
Sonnenblick EH.
Diabetic cardiomyopathy.
Prog Cardiovasc Dis
27:
255-270,
1985[ISI][Medline].
8.
Fukuhara, M,
Tsuchihashi T,
Abe I,
and
Fujishima M.
Cardiovascular and neurohormonal effects of intravenous adrenomedullin in conscious rabbits.
Am J Physiol Regul Integr Comp Physiol
269:
R1289-R1293,
1995
9.
Galetic, I,
Andjelkovic M,
Meier R,
Brodbeck D,
Park J,
and
Hemmings BA.
Mechanism of protein kinase B activation by insulin/insulin-like growth factor-1 revealed by specific inhibitors of phosphoinositide 3-kinasesignificance for diabetes and cancer.
Pharmacol Ther
82:
409-425,
1999[ISI][Medline].
10.
Garcia-Unzueta, MT,
Montalban C,
Pesquera C,
Berrazueta JR,
and
Amado JA.
Plasma adrenomedullin levels in type 1 diabetes. Relationship with clinical parameters.
Diabetes Care
21:
999-1003,
1998[Abstract].
11.
Gettys, TW,
Okonogi K,
Tarry WC,
Johnston J,
Horton C,
and
Taylor IL.
Examination of relative rates of cAMP synthesis and degradation in crude membranes of adipocytes treated with hormones.
Second Messengers Phosphoproteins
13:
37-49,
1990[ISI][Medline].
12.
Grekinis, D,
Reimann EM,
and
Schlender KK.
Phosphorylation and inactivation of rat heart glycogen synthase by cAMP-dependent and cAMP-independent protein kinases.
Intl J Biochem Cell Biol
27:
565-573,
1995[Medline].
13.
Griffith, TM,
and
Taylor HJ.
Cyclic AMP mediates EDHF-type relaxations of rabbit jugular vein.
Biochem Biophys Res Commun
263:
52-57,
1999[ISI][Medline].
14.
Harper, JF,
and
Brooker G.
Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'O acetylation by acetic anhydride in aqueous solution.
J Cyclic Nucleotide Res
1:
207-218,
1975[ISI][Medline].
15.
Hayakawa, H,
Hirata Y,
Kakoki M,
Suzuki Y,
Nishimatsu H,
Nagata D,
Suzuki E,
Kikuchi K,
Nagano T,
Kangawa K,
Matsuo H,
Sugimoto T,
and
Omata M.
Role of nitric oxide-cGMP pathway in adrenomedullin-induced vasodilation in the rat.
Hypertension
33:
689-693,
1999
16.
He, TC,
Zhou S,
Costa LT,
Yu J,
Kinzler KW,
and
Vogelstein B.
A simplified system for generating recombinant adenovirus.
Proc Natl Acad Sci USA
95:
2509-2514,
1998
17.
Ichiki, Y,
Kitamura K,
Kangawa K,
Kawamoto M,
Matsuo H,
and
Eto T.
Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma.
FEBS Lett
338:
6-10,
1994[ISI][Medline].
18.
Ihara, T,
Ikeda U,
Tate Y,
Ishibashi S,
and
Shimada K.
Positive inotropic effects of adrenomedullin on rat papillary muscle.
Eur J Pharmacol
390:
167-172,
2000[ISI][Medline].
19.
Ishimitsu, T,
Nishikimi T,
Saito Y,
Kitamura K,
Eto T,
Kangawa K,
Matsuo H,
Omae T,
and
Matsuoka H.
Plasma levels of adrenomedullin, a newly identified hypotensive peptide, in patients with hypertension and renal failure.
J Clin Invest
94:
2158-2161,
1994[ISI][Medline].
20.
Kitamura, K,
Kangawa K,
Kawamoto M,
Ichiki Y,
Nakamura S,
Matsuo H,
and
Eto T.
Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma.
Biochem Biophys Res Commun
192:
553-560,
1993[ISI][Medline].
21.
Kohn, AD,
Summers SA,
Birnbaum MJ,
and
Roth RA.
Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation.
J Biol Chem
271:
31372-31378,
1996
22.
Lainchbury, JG,
Troughton RW,
Lewis LK,
Yandle TG,
Richards AM,
and
Nicholls MG.
Hemodynamic, hormonal, and renal effects of short-term adrenomedullin infusion in healthy volunteers.
J Clin Endocrinol Metab
85:
1016-1020,
2000
23.
Matsui, T,
Li L,
del Monte F,
Fukui Y,
Franke TF,
Hajjar RJ,
and
Rosenzweig A.
Adenoviral gene transfer of activated phosphatidylinositol 3'-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro.
Circulation
100:
2373-2379,
1999
24.
Nagaya, N,
Nishikimi T,
Horio T,
Yoshihara F,
Kanazawa A,
Matsuo H,
and
Kangawa K.
Cardiovascular and renal effects of adrenomedullin in rats with heart failure.
Am J Physiol Regul Integr Comp Physiol
276:
R213-R218,
1999
25.
Nishikimi, T,
Horio T,
Yoshihara F,
Nagaya N,
Matsuo H,
and
Kangawa K.
Effect of adrenomedullin on cAMP and cGMP levels in rat cardiac myocytes and nonmyocytes.
Eur J Pharmacol
353:
337-344,
1998[ISI][Medline].
26.
Nishikimi, T,
Saito Y,
Kitamura K,
Ishimitsu T,
Eto T,
Kangawa K,
Matsuo H,
Omae T,
and
Matsuoka H.
Increased plasma levels of adrenomedullin in patients with heart failure.
J Am Coll Cardiol
26:
1424-1431,
1995[ISI][Medline].
27.
Nishimatsu, H,
Suzuki E,
Nagata D,
Moriyama N,
Satonaka H,
Walsh K,
Sata M,
Kangawa K,
Matsuo H,
Goto A,
Kitamura T,
and
Hirata Y.
Adrenomedullin induces endothelium-dependent vasorelaxation via the phosphatidylinositol 3-kinase/Akt-dependent pathway in rat aorta.
Circ Res
89:
63-70,
2001
28.
Parkes, DG.
Cardiovascular actions of adrenomedullin in conscious sheep.
Am J Physiol Heart Circ Physiol
268:
H2574-H2578,
1995
29.
Regan, TJ,
Lyons MM,
Ahmed SS,
Levinson GE,
Oldewurtel HA,
Ahmad MR,
and
Haider B.
Evidence for cardiomyopathy in familial diabetes mellitus.
J Clin Invest
60:
884-899,
1977[Medline].
30.
Richer, C,
Fornes P,
Cazaubon C,
Domergue V,
Nisato D,
and
Giudicelli JF.
Effects of long-term angiotensin II AT1 receptor blockade on survival, hemodynamics and cardiac remodeling in chronic heart failure in rats.
Cardiovasc Res
41:
100-108,
1999[ISI][Medline].
31.
Sakata, J,
Shimokubo T,
Kitamura K,
Nishizono M,
Iehiki Y,
Kangawa K,
Matsuo H,
and
Eto T.
Distribution and characterization of immunoreactive rat adrenomedullin in tissue and plasma.
FEBS Lett
352:
105-108,
1994[ISI][Medline].
32.
Sato, Y,
Hotta N,
Sakamoto N,
Matsuoka S,
Ohishi N,
and
Yagi K.
Lipid peroxide level in plasma of diabetic patients.
Biochem Med
21:
104-107,
1979[ISI][Medline].
33.
Shimekake, Y,
Nagata K,
Ohta S,
Kambayashi Y,
Teraoka H,
Kitamura K,
Eto T,
Kangawa K,
and
Matsuo H.
Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells.
J Biol Chem
270:
4412-4417,
1995
34.
Tsuruda, T,
Kato J,
Kitamura K,
Kuwasako K,
Imamura T,
Koiwaya Y,
Tsuji T,
Kangawa K,
and
Eto T.
Adrenomedullin: a possible autocrine or paracrine inhibitor of hypertrophy of cardiomyocytes.
Hypertension
31:
505-510,
1998
35.
Vadlamudi, RV,
Rodgers RL,
and
McNeill JH.
The effect of chronic alloxan- and streptozotocin-induced diabetes on isolated rat heart performance.
Can J Physiol Pharmacol
60:
902-911,
1982[ISI][Medline].
36.
Wakasaki, H,
Koya D,
Schoen FJ,
Jirousek MR,
Ways DK,
Hoit BD,
Walsh RA,
and
King GL.
Targeted overexpression of protein kinase C beta2 isoform in myocardium causes cardiomyopathy.
Proc Natl Acad Sci USA
94:
9320-9325,
1997
37.
Wang, C,
Dobrzynski E,
Chao J,
and
Chao L.
Adrenomedullin gene delivery attenuates renal damage and cardiac hypertrophy in Goldblatt hypertensive rats.
Am J Physiol Renal Physiol
280:
F964-F971,
2001
38.
Wilson, GL,
Hartig PC,
Patton NJ,
and
LeDoux SP.
Mechanisms of nitrosourea-induced b-cell damage. Activation of poly (ADP-ribose) synthetase and cellular distribution.
Diabetes
37:
213-216,
1988[Abstract].
39.
Zhou, M,
Sevilla L,
Vallega G,
Chen P,
Palacin M,
Zorzano A,
Pilch PF,
and
Kandror KV.
Insulin-dependent protein trafficking in skeletal muscle cells.
Am J Physiol Endocrinol Metab
275:
E187-E196,
1998