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Am J Physiol Heart Circ Physiol 278: H633-H642, 2000;
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Vol. 278, Issue 2, H633-H642, February 2000

Ventricular adrenomedullin concentration is a sensitive biochemical marker for volume and pressure overload in rats

Fumiki Yoshihara1, Toshio Nishikimi1, Takeshi Horio2, Chikao Yutani3, Noritoshi Nagaya1, Hisayuki Matsuo1, Tohru Ohe4, and Kenji Kangawa1

1 Research Institute, 2 Division of Hypertension, and 3 Division of Pathology, National Cardiovascular Center, Suita, Osaka 565-8565; and 4 Department of Cardiovascular Medicine, Okayama University Medical School, Okayama 700-8558, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was designed to investigate the pathophysiological significance of adrenomedullin (AM) concentration in volume- and pressure-overloaded cardiac hypertrophy. We measured ventricular AM concentrations and compared them with changes of alpha -actin and myosin heavy chain (MHC) mRNA isoforms after the creation of an aortocaval (AC) shunt as a volume-overload model or the injection of monocrotaline (MCT) as a pressure-overload model, respectively. The left ventricular AM levels after the creation of AC shunt and the right ventricular AM levels after the injection of MCT were significantly increased and correlated with changes of the alpha -actin and MHC mRNA isoforms. However, the ventricular AM mRNA expressions were increased and correlated with ventricular AM concentrations only in the AC shunt model. These results suggest that the ventricular AM levels are upregulated in both the volume- and pressure-overloaded cardiac hypertrophy by differential transcriptional regulation and that the ventricular AM may be a biochemical marker for the volume and pressure overload to the ventricle.

hypertrophy; ribonucleic acid; alpha -actin; myosin heavy chain


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A NOVEL HYPOTENSIVE PEPTIDE adrenomedullin (AM) and its mRNA are known to be highly expressed in the heart as well as in the adrenal gland (18, 19). Previous reports demonstrated that the plasma AM and the ventricular AM levels are increased in patients with heart failure (14, 16) and with cardiac hypertrophy (25). A recent report revealed that AM has a positive inotropic effect in rats (26). These results suggest that ventricular AM may be involved in the pathophysiology of cardiac hypertrophy and function. Indeed, the ventricular AM peptide and mRNA levels are known to be increased in some rat models of pressure-overloaded cardiac hypertrophy, such as the monocrotaline-induced pulmonary hypertension (MCT-PH) (21), Dahl salt-sensitive rats (23), and spontaneously hypertensive rats (22). However, Romppanen et al. (17) recently reported that the ventricular AM levels were significantly higher in hypertensive TGR(mREN-2)27 rats than in normotensive Sprague-Dawley rats without an increase of the ventricular AM mRNA expression. In addition, Kaiser et al. (9) very recently reported that the ventricular AM mRNA levels did not increase during the development of cardiac hypertrophy induced by abdominal aortic banding in rats. In contrast, Nishikima et al. (14) recently found that the ventricular AM peptide and mRNA levels were increased in volume-overloaded cardiac hypertrophy. These results suggest that the mechanisms of AM biosynthesis may be different between the pressure- and volume-overloaded cardiac hypertrophy. Furthermore, the pathophysiological significance and the behavior of ventricular AM in these conditions remain unknown.

In the present study, to investigate the pathophysiological significance and the regulation of AM concentration in the pressure- and volume-overloaded hypertrophy, we created aortocaval (AC) shunt as a volume-overload model and MCT-PH as a pressure-overload model in rats and determined the time course of ventricular AM peptide and mRNA levels, hemodynamics, and the degree of ventricular hypertrophy. We also measured the ventricular expression of alpha -actin mRNA and myosin heavy chain mRNA isoforms as qualitative markers of ventricular hypertrophy (1, 2, 20) and studied the relationship between these markers and the ventricular AM levels.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was performed in accordance with the guidelines of the Animal Care Committee of the National Cardiovascular Center Research Institute.

Preliminary study. To elucidate whether there are significant changes of ventricular AM concentrations and their mRNA expressions in a growth-dependent manner, we evaluated the right and left ventricular AM concentrations and their mRNA expressions in 5-, 6-, 7-, 9-, 10-, and 11-wk-old male Wistar rats (n = 31).

Experimental animals. Four-week-old male Wistar rats, weighing 100-120 g, were used for the model of pressure-overloaded cardiac hypertrophy. MCT-PH was produced in rats by a method previously described (12). Experimental animals were given a single subcutaneous injection of 60 mg/kg MCT, whereas control animals were injected subcutaneously with 0.9% saline (n = 46). Approximately 10% of MCT-treated rats died. As a result, 46 MCT-PH rats were studied. On the other hand, 7- to 8-wk-old male Wistar rats weighing 250-300 g were used for the model of volume-overloaded cardiac hypertrophy. The AC shunt was produced in rats by a method described previously (4) and modified in our laboratory (14). Control rats underwent identical operation, but no shunt was established (n = 25). Approximately 30% of AC shunt rats died. As a result, 36 AC shunt rats were studied.

Hemodynamic study. Hemodynamic studies were performed 1, 2, and 3 wk after either the AC shunt operation or the injection of MCT as previously described (14). Animals were anesthetized with pentobarbital sodium (40 mg/kg ip), and a catheter (PE-10 fused to PE-50) filled with heparin-saline solution was inserted into the thoracic aorta of each animal through the right carotid artery to measure the mean arterial pressure and heart rate. In rats with an AC shunt, the catheter was advanced into the left ventricle (LV) to measure the left ventricular end-diastolic pressure. A similar catheter was inserted into the right atrium and ventricle through the right jugular vein to measure the right atrial and ventricular pressure. In rats with MCT-PH, a similar catheter was also inserted into the right ventricle (RV) through the right jugular vein to measure the right ventricular systolic pressure. The rats were then killed, and their hearts were excised. Each heart was divided into the right ventricular free wall, the left ventricular free wall, and the septum, and each portion was weighed separately. We also calculated the ratio of right ventricular free wall weight to body weight (RV/BW) and the ratio of left ventricular free wall and septum weight to body weight (LV+Sep/BW) as indexes of ventricular hypertrophy.

RIA for ventricular AM. Each ventricular free wall to be used for RIA was weighed, diced, and boiled in 10 vol of 1 mol/l acetic acid for 10 min to inactivate intrinsic proteases. After the boiled tissue was cooled, it was homogenized with a Polytron mixer for several minutes. The homogenate was centrifuged at 3,000 g for 30 min, and the supernatant was centrifuged again at 15,000 g for 10 min. The supernatant was evaporated under vacuum until dry. RIA for rat AM was performed as described previously (19).

Oligonucleotide and cDNA probes and radiolabeling of probes. Synthetic oligonucleotide probes 20 bases in length were prepared in an Applied Biosystems DNA Synthesizer (model 392) and purified by electrophoresis. The sequences of the oligonucleotide probes used were as follows: alpha -myosin heavy chain, 5'-TTGTGGGATAGCAACAGCGA-3' (10); beta -myosin heavy chain, 5'-GGTCTCAGGGCTTCACAGGC-3' (5); skeletal alpha -actin, 5'-GCAACCATAGCACGATGGTC-3' (10); and cardiac alpha -actin, 5'-TGCACGTGTGTAAACAAACT-3' (10). The oligonucleotide probes were labeled with [gamma -32P]ATP (Amersham) at the 5' end using T4 polynucleotide kinase, and the labeled probes were purified by column chromatography (NAP column, Pharmacia Biotech, Uppsala, Sweden). The cDNA used as a probe was an EcoR I/Nae I restriction fragment of rat AM cDNA, corresponding to nucleotides -153 to 436 (14), which was radiolabeled by random priming with [alpha -32P]dCTP (Amersham). The labeled probe was purified by column chromatography (NICK column, Pharmacia Biotech).

RNA extraction. Total RNA was extracted from the ventricles by the acid guanidinium thiocyanate-phenol-chloroform method, using the procedure previously described (24). The RNA pellet was finally dissolved in 0.1% diethyl pyrocarbonate-treated water and stored at -80°C until use. The RNA concentration was determined based on absorbance at 260 nm.

Northern blot analysis. Total RNA (20 µg/lane) was denatured with Formalin and formamide and electrophoresed on a 1% agarose gel containing Formalin. The 28S and 18S ribosomal RNAs in gels were stained with ethidium bromide to confirm the integrity of the RNA applied. RNA in the gel was then transferred to a Nylon membrane (Zeta-Probe blotting membrane, Bio-Rad) and fixed by ultraviolet irradiation. For hybridization with oligonucleotide probes, the membranes were prehybridized, hybridized, and washed as previously described (10). For hybridization with cDNA probes, the conditions for prehybridization, hybridization, and membrane washing have been described previously (14). Band intensity was estimated using a radio-image analyzer (BAS 5000, Fuji). To normalize the rat AM signal to the amounts of RNA loaded and the transfer efficiencies, the same membrane was rehybridized with an 18S oligonucleotide probe. Rehybridization was carried out after probes were stripped by boiling in 0.1% SDS for 20 min.

Immunohistochemistry. For the immunohistochemical analysis, ventricles were immediately fixed with 10% Formalin. The tissues were embedded in paraffin, and 4-µm-thick sections were cut and mounted on glass slides treated with silica. The slides were incubated overnight at 60°C and deparaffinized with graded concentrations of xylene and ethanol. Immunohistochemical analysis was performed by using a monoclonal antibody recognizing AM (46-52) (dilution of ascites, 1:200) as previously reported (11). Nonimmune mouse IgG was used as a control. The presence of immunoreactive AM was assessed with light microscopy by trained observers who were unaware of the treatment conditions used.

Statistical analysis. Values are means ± SD. Comparisons of ventricular AM concentrations and its mRNA expressions during normal growth of Wistar rats were performed by one-way ANOVA. Comparisons between two groups were performed by two-way repeated-measures ANOVA with Newman-Keuls post hoc test. Differences were considered statistically significant at a level of P < 0.05. Correlation coefficients were calculated using linear regression analysis.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preliminary study. There were no significant differences in the right and left ventricular AM concentrations and its mRNA expressions during normal growth of Wistar rats between the ages of 5-11 wk old (Table 1).

                              
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  		Table 1.   Right and left ventricular adrenomedullin concentrations and its mRNA expression during normal growth of Wistar rats

Characterization and hemodynamic study. The number of experimental rats, body weight, heart rate, and mean arterial pressure in AC shunt rats and MCT-PH rats are presented in Tables 2 and 3, respectively. Body weight was comparable between the AC shunt rats and the control rats, whereas it was significantly lower in the MCT-PH rats than in the control rats at 1, 2, and 3 wk after the injection of MCT. Heart rate was comparable between the AC shunt rats and the control rats at 1, 2, and 3 wk after the operation. It was also comparable between the MCT-PH rats and the control rats at 1, 2, and 3 wk after the injection of MCT. Mean arterial pressure was significantly decreased in the AC shunt rats compared with the control rats at 1, 2, and 3 wk after the operation. It was significantly decreased in the MCT-PH rats compared with the control rats at 2 and 3 wk after the injection of MCT. Left ventricular end-diastolic pressure, mean right atrial pressure (Fig. 1), and right ventricular systolic pressure (1 wk, sham vs. AC shunt 40.9 ± 3.6 vs. 55.3 ± 4.5 mmHg, P < 0.05; 2 wk, 36.7 ± 3.7 vs. 55.8 ± 8.2 mmHg, P < 0.05; 3 wk, 40.0 ± 3.5 vs. 62.2 ± 6.2 mmHg, P < 0.05) were significantly increased in the AC shunt rats compared with the control rats at 1, 2, and 3 wk after the operation. Right ventricular systolic pressure was significantly increased in the MCT-PH rats compared with the control rats at 2 and 3 wk after the injection of MCT (Fig. 2).

                              
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  		Table 2.   Number, body weight, heart rate, and mean arterial pressure in AC shunt and control rats


                              
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  		Table 3.   Number, body weight, heart rate and mean arterial pressure in MCT-PH and control rats



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  		Fig. 1.   Effects of volume overload induced by aortocaval (AC) shunt on left ventricular (LV) end-diastolic pressure (A), mean right atrial pressure (B), ratio of LV free wall and septum (LV+Sep) weight to body weight (C), and ratio of right ventricular (RV) free wall weight to body weight (D) at 1, 2, and 3 wk after operation, respectively. See Table 1 for no. of rats. Values are means ± SD. * P < 0.05, ** P < 0.01 vs. sham.





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  		Fig. 2.   Effects of pressure overload induced by monocrotaline-induced pulmonary hypertension (MCT-PH) on RV systolic pressure (A), ratio of RV weight to body weight (B), and ratio of LV weight to body weight (C) at 1, 2, and 3 wk after injection. Values are means ± SD. * P < 0.05, ** P < 0.01 vs. sham.

Quantitative changes in ventricles. LV+Sep/BW and RV/BW were significantly increased in the AC shunt rats compared with the control rats at 1, 2 and 3 wk after the operation (Fig. 1). Whereas LV+Sep/BW was unchanged in the MCT-PH rats compared with controls throughout the experimental period, RV/BW was significantly increased in the MCT-PH rats compared with the control rats at 2 and 3 wk after the injection of MCT (Fig. 2).

Ventricular AM concentrations. Both the left and right ventricular AM concentrations were significantly increased in the AC shunt rats compared with the controls at 1, 2, and 3 wk after the operation. The right ventricular AM concentration was also significantly increased in the MCT-PH rats compared with the controls at 3 wk after the injection of MCT, whereas the left ventricular AM concentration was unchanged throughout the experimental period (Fig. 3).


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  		Fig. 3.   Effects of volume overload induced by AC shunt and pressure overload induced by MCT injection on ventricular adrenomedullin (AM) concentration at 1, 2, and 3 wk after operation or injection. Values are means ± SD. * P < 0.05, ** P < 0.01 vs. sham.

Qualitative changes in ventricles and ventricular AM gene expressions. The ratio of skeletal to cardiac alpha -actin mRNA and the ratio of beta - to alpha -myosin heavy chain mRNA were increased in the LV in the AC shunt rats compared with the control rats at 2 and 3 wk after the operation (Figs. 4 and 6), and they already tended to be increased in RV in the MCT-PH rats compared with the control rats at 1 wk after the injection of MCT (Figs. 5 and 6).


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  		Fig. 4.   Representative Northern blots showing AM in LV and RV free wall, alpha -actin isoforms, myosin heavy chain (MHC) isoforms, and 18S in LV free wall in rats with AC shunt. Skel-Act, skeletal actin; Card-Act, cardiac actin.



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  		Fig. 5.   Representative Northern blots showing AM in LV and RV free wall, alpha -actin isoforms, myosin heavy chain isoforms, and 18S in RV free wall in rats with MCT-PH.



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  		Fig. 6.   Effects of volume overload induced by AC shunt and pressure overload induced by MCT injection on ratio of skeletal to cardiac alpha -actin mRNA (A and B) and ratio of beta - to alpha -myosin heavy chain mRNA (C and D) at 1, 2, and 3 wk after operation or injection, respectively. Sham-operated control rats (n = 6 for 1 wk, n = 7 for 2 wk, and n = 6 for 3 wk); AC shunt rats (n = 8 for 1 wk, n = 8 for 2 wk, and n = 9 for 3 wk); saline-injected control rats (n = 7 for 1 wk, n = 7 for 2 wk, and n = 11 for 3 wk); and MCT-PH rats (n = 4 for 1 wk, n = 9 for 2 wk, n = 9 for 3 wk). Values are means ± SD. * P < 0.05, ** P < 0.01 vs. sham.

The ventricular AM gene expression was significantly increased in both the LV and RV in the AC shunt rats compared with the control rats at 1, 2, and 3 wk after the operation (Figs. 4 and 7). In contrast, the ventricular AM gene expression was unchanged both the LV and RV in the MCT-PH rats compared with the control rats throughout the experimental period (Figs. 5 and 7).


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  		Fig. 7.   Summary of data of Northern blot analysis of ventricular AM mRNA in AC shunt rats and MCT-PH rats at 1, 2, and 3 wk after operation or injection, respectively. Density abundance was defined as ratio between AM mRNA and 18S mRNA. Values are means ± SD. * P < 0.05, ** P < 0.01 vs. sham.

Immunohistochemistry. The immunohistochemical analysis revealed that AM immunoreactivity was more intense in pressure- and volume-overloaded ventricular myocytes than in normal ventricular myocytes (Fig. 8).


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  		Fig. 8.   AM immunoreactivity using a monoclonal antibody that recognizes the COOH-terminal structure of AM in ventricle. AM immunoreactivity is more intense in pressure- and volume-overloaded cardiac myocytes. Panels are identified as follows: AC shunting LV (A), sham-operated LV (B), control LV (nonimmune mouse IgG was used; C), MCT-PH RV (D), saline-injected control RV (E), and a control RV (nonimmune mouse IgG was used, F).

Correlation between ventricular AM concentration and other ventricular parameters. We evaluated correlations between ventricular AM concentration and other ventricular parameters using all the points including 1, 2, and 3 wk after the operation or MCT injection. The ventricular AM level was significantly correlated with the ventricular weight, with the ratio of skeletal to cardiac alpha -actin mRNA, and with the ratio of beta - to alpha -myosin heavy chain mRNA in LV in the AC shunt rats and in RV in the MCT-PH rats and their respective control rats (Table 4). The ventricular AM level was significantly correlated with the ventricular expression of AM mRNA only in LV in the AC shunt rats and their control rats (Table 4).

                              
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  		Table 4.   Correlations between ventricular AM concentration and other ventricular parameters in AC shunt and MCT-PH rats and their control rats


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we demonstrated for the first time that the left ventricular AM levels and the left ventricular expression of AM mRNA significantly increased with an increase of the left ventricular end-diastolic pressure and the left ventricular weight at 1, 2, and 3 wk after the creation of an AC shunt compared with the sham-operated controls and that the ventricular AM levels were significantly correlated with the ventricular quantitative and qualitative changes as well as with the expression of AM mRNA. We also showed that the right ventricular AM levels increased with an increase of the right ventricular systolic pressure and the right ventricular weight at 2 and 3 wk after the injection of MCT compared with the saline-injected controls and that the ventricular AM levels were also significantly correlated with the ventricular quantitative and qualitative changes. However, the ventricular expression of AM mRNA was unchanged throughout the experimental period. These results suggest that ventricular AM levels are upregulated in both volume- and pressure-overloaded cardiac hypertrophy by the differential transcriptional regulation and that the ventricular AM may be a sensitive biochemical marker for both volume and pressure overload to the ventricle.

In the volume-overloaded cardiac hypertrophy, the left ventricular AM levels were significantly upregulated, along with an increase of the left ventricular end-diastolic pressure and the left ventricular weight at 1 wk after the creation of the AC shunt. The ratio of skeletal to cardiac alpha -actin mRNA and the ratio of beta - to alpha -myosin heavy chain mRNA, the qualitative markers of cardiac hypertrophy, significantly increased at 2 wk after the creation of the AC shunt. These results suggest that an increased level of ventricular AM may be a sensitive marker reflecting volume overload to the ventricle. In the pressure-overloaded cardiac hypertrophy, the ratio of skeletal to cardiac alpha -actin mRNA and the ratio of beta - to alpha -myosin heavy chain mRNA already tended to be increased at 1 wk after the MCT injection. The right ventricular AM levels significantly increased at 3 wk after the MCT injection, and the ventricular AM levels closely correlated with the ratio of skeletal to cardiac alpha -actin mRNA and with the ratio of beta - to alpha -myosin heavy chain mRNA. Furthermore, the left ventricular AM levels were unchanged throughout the experimental period, suggesting that the ventricular AM levels are upregulated in a pressure overload-dependent manner in MCT-PH rats. AM is also known to be highly expressed in the heart in the fetus (13). These results suggest that the ventricular AM level may be a qualitative marker for pressure-overloaded cardiac hypertrophy.

The present study also demonstrated that the ventricular AM levels are increased in parallel with the ventricular AM mRNA levels after the creation of an AC shunt, in agreement with a previous report (14). In contrast, the expression of the AM gene was unchanged throughout the experimental period despite the increase of the ventricular AM level in the pressure-overloaded cardiac hypertrophy. The result is also consistent with the recent report of Romppanen et al. (17), who found increased left ventricular AM levels in hypertensive TGR(mREN-2)27 rats without an increase of the left ventricular AM mRNA expression. Kaiser et al. (9) also reported that the ventricular AM mRNA levels did not increase in rats developing pressure-overloaded cardiac hypertrophy because of aortic banding. The mechanisms responsible for the absence of an increase in the expression of the ventricular AM gene despite the persistent upregulation in the ventricular AM levels in the pressure-overloaded cardiac hypertrophy remain uncertain. Romppanen et al. (17) previously reported that arginine-vasopressin infusion for 2 h induced a 1.6-fold increase in the AM mRNA level and a 1.7-fold increase in the AM level in the LV in normotensive Sprague-Dawley rats. Together with our results, these findings suggest that the expression of the AM gene might increase only during a very early period in pressure-overloaded cardiac hypertrophy. Wada et al. (28) reported that phosphorylation of the translational initiation factor eIF-4E significantly increased in pressure-overloaded cardiac hypertrophy, whereas volume overload had no effect on the eIF-4E phosphorylation. The eIF-4 factor is involved in the binding of mRNA to the 43S initiation complex to form the 48S initiation complex, suggesting that the increased eIF-4E phosphorylation may contribute to an accelerated rate of protein synthesis in the pressure-overloaded cardiac hypertrophy. Thus one possible mechanism for the upregulation of the ventricular AM level without an increase of AM gene expression in pressure-overloaded cardiac hypertrophy may be translational upregulation of ventricular AM biosynthesis caused by increased eIF-4E phosphorylation. However, we could not exclude the possibility that the increase in AM mRNA degradation rates or the decrease in the release rates of AM from cardiac myocytes might account for the discrepancy between ventricular AM levels and their gene expression in the pressure-overloaded cardiac hypertrophy. The exact cellular mechanism leading to the difference of AM mRNA levels between the two types of cardiac hypertrophy requires further study.

The immunohistochemical analysis revealed that AM immunoreactivity was more intense in pressure- and volume-overloaded ventricular myocytes than in normal ventricular myocytes. Jougasaki et al. (8) previously reported that AM immunoreactivity in the myocyte is highly expressed in the failing heart than in normal heart and that there was no evidence of AM immunoreactivity in the connective tissues. These results suggest that ventricular myocyte, not nonmyocyte, may be major source of increased ventricular AM levels in cardiac hypertrophy.

The role of increased ventricular AM levels in cardiac hypertrophy remains uncertain at present. There have been many reports (3, 6, 7) that AM induced elevated cAMP levels in various tissues. Nishikimi et al. (15) recently reported that AM induces elevation of the cAMP level of cardiomyocytes via an AM- and CGRP-common receptor and in nonmyocytes via a CGRP-like receptor. Yamazaki et al. (29) reported that the protein kinase A stimulated by cAMP activates Raf-1 and mitogen-activated protein kinase, followed by an increase in protein synthesis in neonatal rat cardiomyocytes. These findings suggest that AM may be involved in producing cardiac hypertrophy. In contrast, Tsuruda et al. (27) recently suggested the possibility that cultured neonatal rat cardiomyocytes produce and secrete AM and that the secreted AM inhibits the protein synthesis of these cells. Further research is necessary to reveal the exact role of increased tissue levels of AM in cardiac hypertrophy.

In summary, the ventricular AM levels were increased in both the volume- and pressure-overloaded cardiac hypertrophy, and these increases were mediated by the differential transcriptional regulation. Furthermore, our results suggest that the ventricular AM may be a biochemical marker for the volume and pressure overload to the ventricle.


    ACKNOWLEDGEMENTS

We thank Yoko Saito for technical assistance and Nobuo Shirahashi for helpful advice of statistical analysis.


    FOOTNOTES

This work was supported in part by Special Coordination Funds for Promoting Science and Technology (Encouragement System of COE) from the Science and Technology Agency of Japan, the Ministry of Health and Welfare, and the Human Science Foundation of Japan.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. Nishikimi, National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565-8565, Japan (E-mail: nishikim{at}dokkyomed.ac.jp).

Received 20 January 1999; accepted in final form 28 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 278(2):H633-H642
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