AJP - Heart Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 289: H151-H159, 2005. First published February 25, 2005; doi:10.1152/ajpheart.00066.2005
0363-6135/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/1/H151    most recent
00066.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, X.
Right arrow Articles by Dhalla, N. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.
Right arrow Articles by Dhalla, N. S.

Upregulation of {beta}-adrenergic receptors in heart failure due to volume overload

Xi Wang, Emmanuelle Sentex, Harjot K. Saini, Donald Chapman, and Naranjan S. Dhalla

Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre and Department of Physiology, Faculty of Medicine, University of Manitoba, Winnipeg, Canada

Submitted 24 January 2005 ; accepted in final form 23 February 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To examine the mechanisms of changes in {beta}-adrenergic signal transduction in heart failing due to volume overload, we studied the status of {beta}-adrenoceptors ({beta}-ARs), G protein-coupled receptor kinase (GRK), and {beta}-arrestin in heart failure due to aortocaval shunt (AVS). Heart failure in rats was induced by creating AVS for 16 wk, and {beta}-AR binding, GRK activity, as well as their protein content, and mRNA levels were determined in both left and right ventricles. The density and protein content for {beta}1-ARs, unlike those for {beta}2-ARs, were increased in the failing hearts. Furthermore, protein contents for GRK isoforms and {beta}-arrestin-1 were decreased in membranous fractions and increased in cytosolic fractions from the failing hearts. On the other hand, steady-state mRNA levels for {beta}1-ARs and GRK2, as well as protein content for G{beta}{gamma}-subunits, did not change in the failing heart. Basal cardiac function was depressed; however, both in vivo and ex vivo positive inotropic responses of the failing hearts to isoproterenol were augmented. Treatment of AVS animals with imidapril (1 mg·kg–1·day–1) or losartan (20 mg·kg–1·day–1) retarded the progression of heart failure; partially prevented changes in {beta}1-ARs, GRKs, and {beta}-arrestin-1 in the failing myocardium; and attenuated the increase in positive inotropic effect of isoproterenol. These results indicate that upregulation of {beta}1-ARs is associated with subcellular redistribution of GRKs and {beta}-arrestin-1 in the failing heart due to volume overload. Furthermore, attenuation of alterations in {beta}-adrenergic system by imidapril or losartan may be due to blockade of the renin-angiotensin system in the AVS model of heart failure.

{beta}-adrenoceptors; congestive heart failure; G protein-coupled receptor kinase; renin-angiotensin system blockade

{beta}-ADRENOCEPTOR ({beta}-AR)-coupled signal transduction in the heart mediates the positive inotropic and lusitropic effects of catecholamines (5). This system is composed of {beta}-ARs, guanine nucleotide binding proteins (G proteins), and adenylyl cyclase (AC). Binding of catecholamines with {beta}-ARs activates Gs proteins, stimulates AC, increases the intracellular concentration of Ca2+, and thus augments cardiac contractility (12). Furthermore, G protein-coupled receptor kinases (GRKs), a family of serine/threonine kinases, regulate the {beta}-AR signal transduction through phosphorylation of the agonist-occupied receptors (15, 24). Among the seven isoforms of GRKs identified to date, GRK2 or {beta}-AR kinase-1 ({beta}-ARK1), GRK3 ({beta}-ARK2), and GRK5 are expressed abundantly in cardiac tissue (24). On stimulation by catecholamines in cultured adult rat cardiomyocytes, GRK2 translocates from the cytosol to the membrane, phosphorylates {beta}-ARs, and uncouples Gs proteins from {beta}-ARs (17). Overexpression of GRK2 in mice was observed to attenuate isoproterenol-stimulated left ventricular (LV) contractility, decreased cardiac AC activity, and reduced functional coupling of {beta}-ARs (21). On the other hand, overexpression of an inhibitor of GRK2 in mice enhanced the basal and isoproterenol-stimulated cardiac contractility (21). Chronic {beta}-AR stimulation has been shown to result in upregulation of GRK2 and downregulation of {beta}-AR in rat hearts (17), whereas chronic {beta}-AR blockade results in downregulation of GRK and enhanced {beta}-adrenergic signaling in porcine hearts (26).

Data from human end-stage heart failure (3, 5, 34) and several experimental models of heart failure such as spontaneously hypertensive heart failure (1), pressure overload hypertrophy (8), and myocardial infarction (18, 36) have indicated different degrees of changes in {beta}-adrenergic signal transduction, including downregulation of {beta}1-AR and upregulation of GRK2. However, the information regarding the alterations in {beta}-AR signal transduction in heart failure induced by volume overload is conflicting. In patients with volume overload due to left heart valvular disease and mitral regurgitation, a decrease in {beta}-AR density was observed (14, 28). Hammond et al. (16) have also shown that {beta}-AR density, AC activity, and isoproterenol responsiveness were decreased in pigs 5 wk after aortocaval shunt (AVS). Isoproterenol-induced systolic shortening and peak Ca2+ transients were markedly attenuated in cardiomyocytes isolated from rabbit hearts 12–15 wk after the AVS (30). On the other hand, in rats 6 wk after the AVS, the maximal binding (Bmax) and dissociation constant (Kd) characteristics of {beta}-AR remained unaltered, whereas the maximal AC response to stimulation by catecholamines was markedly reduced (31). Furthermore, an increase in {beta}-AR density was observed 1–3 wk, whereas a decrease occurred 8 wk without having any effect on Kd after AVS in rats (6). Such a behavior of {beta}-AR signal transduction in heart failure due to volume overload may be due to a high cardiac output state for AVS animals in contrast to the more common experimental models of heart failure associated with low cardiac output state.

Recently, Wang et al. (38) have characterized a rat model of volume overload-induced cardiac hypertrophy and heart failure due to AVS and have shown hypersensitization to {beta}-AR stimulation at different times (4–16 wk) of AVS induction. In addition, we (39) have reported that increased protein content and activity of AC, rather than any changes in Gs and Gi proteins, may contribute to the enhanced {beta}-AR stimulation in the 16-wk failing hearts due to volume overload. The present study was undertaken to examine the hypothesis that an upregulation of {beta}-ARs is associated with changes in the subcellular distribution of GRK protein contents in heart failure due to volume overload. Thus the density and protein content for {beta}-AR as well as protein content and distribution of GRK isoforms were examined in failing hearts due to the AVS in rats. Given the regulatory role of {beta}-arrestins and G{beta}{gamma} proteins in the {beta}-AR signal transduction (24, 36), alterations in protein contents for {beta}-arrestins and G{beta}{gamma}-subunits were also determined in the failing hearts. The significance of changes in {beta}-AR signal transduction was investigated by studying the inotropic responses of the failing heart to isoproterenol. Because angiotensin-converting enzyme (ACE) inhibitors and angiotensin II type 1 receptor (AT1R) antagonists have been shown to reduce cardiac hypertrophy and improve cardiac function in AVS-induced heart failure (38), we used a long-acting ACE inhibitor imidapril and an AT1R blocker losartan in the AVS rat model to study whether changes in {beta}-AR, {beta}-arrestin, and GRK activities and translocation in the failing hearts are attenuated by blockade of the renin-angiotensin system (RAS).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal model and experimental groups. The protocols for experiments were approved by the University of Manitoba Animal Care and Use Committee in accordance with guidelines by the Canadian Council on Animal Care. The AVS was produced as described earlier (38, 39), whereas sham-operated (Sh) animals served as controls. Briefly, the abdominal aorta was isolated and ligated caudal to the renal artery and cephalic to the aortic bifurcation. The aorta was punctured with a 18-gauge needle into the inferior vena cava to create the shunt. The needle was removed, the aortic puncture site was sealed with a drop of cyanoacrylate glue, and the ligation was removed. The experimental rats were randomly divided into three groups: untreated AVS, AVS treated with losartan (AVS + Los), and AVS treated with imidapril (AVS + Imp). Treatments with losartan (20 mg·kg–1·day–1) and imidapril (1 mg·kg–1·day–1), dissolved in tap water, were started 3 days after surgery by gastric gavage; tap water served as a vehicle for both the AVS and sham groups. In some experiments, sham control animals were treated with imidapril (Sh + Imp) and losartan (Sh + Los). The selection of doses for drug treatments and the experimental protocol were the same as used earlier (39). Physical examination of untreated AVS animals showed sluggishness in their movements and difficulties in breathing in addition to the clinical signs for heart failure, such as edema and pleural effusion.

In vivo and ex vivo hemodynamic assessment. The LV systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP), heart rate, maximal rate of pressure development (+dP/dt), and maximal rate of pressure decay (–dP/dt) were recorded in vivo in the anesthetized animals and ex vivo in the isolated perfused hearts under controlled conditions as described previously (38). In brief, the rats were anesthetized with an intraperitoneal injection of a mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg). The right carotid artery was exposed and cannulated with a microtip pressure transducer (model PR-249, Millar Instruments, Houston, TX), which was inserted through a proximal arteriotomy. The catheter was advanced carefully through the lumen of the carotid artery until the tip of the transducer entered the LV; the catheter was secured with a silk ligature around the artery, and after 15 min, stabilization of heart function was measured. LV developed pressure (LVDP) was calculated as a difference between the LVSP and LVEDP values. In some experiments, isoproterenol was given as a bolus dose (10 µg/kg) through a femoral vein for in vivo effects. To study the ex vivo effects of isoproterenol, isolated rat hearts were perfused with oxygenated Krebs-Henseleit solution at a constant flow of 10 ml/min at 37°C (38). These hearts were paced at 300 beats/min, and the LV function was measured after 30 min of stabilization (38). In some experiments, isoproterenol was infused at 1 µM concentration, whereas in other experiments, the effects of different concentrations (0.25–2 µM) of isoproterenol were studied in the isolated perfused heart preparations.

Preparation of crude membranes and determination of {beta}-AR binding. Crude membranes were isolated as described previously (25) and used for the measurement of {beta}-AR binding. Briefly, the ventricular tissue was minced and then homogenized in 50 mM Tris·HCl, pH 7.4 (15 ml/g tissue) with a PT-20 polytron (Brinkman Instruments, Westbury, NY) twice for 20 s each at a setting of 5. The resulting homogenate was centrifuged at 1,000 g for 10 min, and the pellet was discarded. The supernatant was centrifuged at the same speed in the same buffer. The final pellet was resuspended in 50 mM Tris·HCl, pH 7.4, containing 25 mM sucrose and 10 mM histidine, and stored at –80°C for further experiments. The purification of these membranes was determined by measuring the Na+-K+-ATPase (a sarcolemmal marker enzyme) activities in the presence of 1 mM ouabain and by comparing it with that detected in the heart homogenate as described previously (13). The crude membrane yield was 11.7 ± 0.5 and 12.0 ± 0.4 mg/g tissue for sham and AVS with a membrane purification factor (ratio of Na+-K+-ATPase activity in membrane and homogenate preparations) of 3.2 ± 0.2 and 3.0 ± 0.3, respectively, for these groups. No significant difference in these parameters was observed in sham and AVS-treated groups.

{beta}-AR binding was determined in the crude membranes by using [125I]iodocyanopindolol ([125I]ICYP) as described previously (25). Specific binding to {beta}1-AR was calculated as the difference between [125I]ICYP binding values in the presence and absence of CGP-20712A, a selective {beta}1-AR antagonist, whereas {beta}2-AR specific binding was the difference between [125I]ICYP binding values in the presence and absence of ICI-118,551, a selective {beta}2-AR antagonist. The binding characteristics for {beta}-ARs, Bmax, and Kd were calculated by the Scatchard plot analysis according to the interactive LIGAND program.

Homogenate, cytosolic, and membranous fractions and measurement of GRK activity. Cytosolic and membranous fractions were isolated as described by Benovic et al. (2). An aliquot of tissue homogenate without centrifugation was saved as the homogenate fraction. For GRK activity assay, the fractions were further purified by using 50% (vol/vol) diethylaminoethyl Sephacel column; the eluted protein was concentrated by using microconcentrator Centricon 30 (Amicon) and employed without freezing. The GRK activity assay was based on the light-dependent phosphorylation of rhodopsin as described by Benovic et al. (2). Rhodopsin purified from dark-adapted bovine outer-rod segments was purchased from Calbiochem-Novabiochem (La Jolla, CA). The phosphorylating activity was completely inhibited by 1 µM heparin but not by a protein kinase A inhibitor.

Western blot analysis. Western blot analysis was used to determine {beta}-ARs, GRKs, G{beta}- and G{gamma}-subunits, as well as {beta}-arrestin-1 protein contents. The protein content of each sample was determined by using the Lowry method (13). Equal amounts of proteins (25 µg) from each group were dissolved in SDS-PAGE sample buffer and denatured by boiling for 5 min. The same volume (10 µl in each well) of sample was loaded for each group. The samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Nepeon, Ontario, Canada), followed by immunoblotting using polyclonal antibodies to {beta}1-AR and {beta}-arrestin-1, as well as to GRK2, GRK3, and GRK5 isoforms (Santa Cruz Biotechnology, Santa Cruz, CA), according to protocols provided by the manufacturer. Polyclonal antibodies for G{gamma}- and G{beta}-protein subunits were obtained from Calbiochem-Novabiochem (La Jolla, CA). To ensure uniform protein loading for all groups, gels were stained with Commassie blue after blotting was completed, and blots were stained with Ponceau S solution.

Northern blot analysis. Total RNA from the cardiac tissue was isolated by the method of Chomczynski and Sacchi (9). Twenty micrograms of total RNA were electrophoresed in a 1.2% agarose-formaldehyde gel and transferred to a Nytran Plus membrane (Schleicher and Schuell, Keene, NH). The membrane was hybridized with specific cDNA probes labeled with 32P for {beta}1-AR and GRK2. Autoradiographic results were quantified by densitometry. Glyceraldehyde-3-phosphate dehydrogenase was used to normalize the loading and transfer of RNA.

Statistical analysis. All values were expressed as means ± SE. The difference between the control and experimental groups were evaluated statistically by one-way ANOVA, followed by the Newman-Keuls test. Differences were considered statistically significant at a level of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
General characteristics and hemodynamic changes. As observed earlier (38), the untreated AVS rats showed marked cardiac hypertrophy, congestion of liver and lung, pleural effusion, ascites, and edema of limbs indicating the presence of congestive heart failure. Table 1 shows the general characteristics and hemodynamic data of rats 16 wk after AVS with or without imidapril and losartan treatments. No changes in the body weights of animals were seen in both untreated and treated groups. The increased heart weight and heart-to-body weight ratio in the untreated AVS group were attenuated by both imidapril and losartan. Although the interpretation of an increase in the heart-to-body weight ratio can be problematic under certain conditions, the gain in body weight in experimental animals was not different from the sham control animals. Imidapril decreased the hypertrophic response in LV and right ventricle (RV) by 22% and 32%, and losartan attenuated it by 21% and 22%, respectively (Table 1). These treatments also reduced the increase in lung and liver weights due to the AVS. The results in Table 1 indicate that the basal values for LVDP, +dP/dt and –dP/dt were significantly depressed, whereas LVEDP was significantly increased in the AVS group indicating both systolic and diastolic dysfunction at this stage of heart failure. However, basal heart rate in the AVS group was not different from the sham group. These alterations in the AVS group were partially prevented by treatments with imidapril and losartan. No difference with respect to general characteristics of the animals and different hemodynamic parameters was evident in the sham group with or without drug treatments (Table 1); therefore, no sham treatment group was included in further experiments. All these alterations in the untreated 16-wk AVS animals are similar to those reported earlier at this stage of heart failure due to volume overload in rats (38).


View this table:
[in this window]
[in a new window]
  		Table 1. General characteristics of rats with heart failure induced by AVS for 16 wk with and without Imp or Los treatments

 
Positive inotropic effect of isoproterenol. Previously, we have shown that the positive inotropic effect of isoproterenol is augmented when measured at different times (4–16 wk) of inducing volume overload (38). In this study we have examined the inotropic effect of a bolus dose (10 µg/kg) of isoproterenol in the untreated and drug-treated 16-wk AVS animals under in vivo conditions. The results in Table 2 indicate that the basal values for LVDP, +dP/dt, and –dP/dt were significantly depressed in the AVS group and partially prevented by treatments with imidapril and losartan; however, the increases due to isoproterenol at maximal stimulation time-point over the respective basal values for all parameters (fold stimulation) were greater in the AVS group than those in the sham group. Such increases in the positive inotropic responses to isoproterenol in AVS animals were attenuated by treatments with imidapril and losartan (Table 2). No difference for the basal heart rate or the positive chronotropic effect was evident in the sham and AVS groups with or without drug treatments (Table 2).


View this table:
[in this window]
[in a new window]
  		Table 2. Effect of bolus isoproterenol on in vivo hemodynamics of rats with heart failure induced by the AVS for 16 wk with and without Imp or Los treatments

 
To eliminate the possibility of interference by hemodynamic and neurohormonal factors under in vivo conditions, we evaluated the contractile responses to isoproterenol stimulation ex vivo by using an isolated perfused heart preparation. Whereas the basal LVDP, +dP/dt and –dP/dt were depressed in the AVS group, the fold stimulation (after infusion of 1 µM isoproterenol) over the respective basal value for all these parameters indicated that the positive inotropic effect of isoproterenol was greater in the AVS group compared with that in the sham group; these changes were partially attenuated by both imidapril and losartan treatments (Table 3). In addition, the increased positive inotropic response of the AVS group, as monitored by measuring changes in LVDP in the isolated perfused heart, was apparent at different concentrations of isoproterenol (0.25 -2.00 µM); these responses were attenuated by treatment of AVS animals with imidapril or losartan (Table 4).


View this table:
[in this window]
[in a new window]
  		Table 3. Effect of isoproterenol on ex vivo cardiac performance of rats with heart failure induced by the AVS for 16 wk with and without Imp or Los treatments

 

View this table:
[in this window]
[in a new window]
  		Table 4. Effect of different concentrations of isoproterenol on ex vivo LVDP of rats with heart failure induced by the AVS for 16 wk with or without Imp or Los treatments

 
Changes in {beta}-ARs. The data in Table 5 reveal a significant increase in Bmax of {beta}1-AR in the untreated AVS group in both the LV and RV, indicating an increase in {beta}1-AR density. Treatment with imidapril or losartan partially depressed this increment. The affinity (1/Kd) of {beta}1-AR was not altered in the experimental animals for both the LV and RV. No changes in {beta}2-AR were observed for Kd and Bmax in both the LV and the RV from AVS animals with or without drug treatments. Western blot analysis for {beta}1-ARs and {beta}2-ARs showed an increase in {beta}1-AR protein content without any change in the {beta}2-AR protein content in the failing heart (Fig. 1), except that the extent of increase in protein content is much higher than that in the receptor density for {beta}1-ARs (148% and 129% vs. 57% and 54% in the LV and RV, respectively). This may be due to a homogenous increase of {beta}1-AR, including functionally active and inactive receptors, or due to differences in the sensitivity of these two methods. The increase in {beta}1-AR content in AVS animals was significantly attenuated by treatments with imidapril or losartan (Fig. 1). To gain some information regarding the molecular basis of changes in {beta}1-AR protein contents in the failing heart, we examined {beta}1-AR mRNA levels in the myocardium by using Northern blot analysis. No changes in {beta}1-AR mRNA levels resulted in both the LV and RV from untreated or treated groups (data not shown). However, the immunoblot data on {beta}1-AR is not an artifact because protein content for {beta}2-AR in the present study as well as Gi- and Gs-protein content (39) in the same experimental preparation as used in this study were not altered.


View this table:
[in this window]
[in a new window]
  		Table 5. Cardiac {beta}-AR-binding characteristics in rats with heart failure induced by the AVS for 16 wk with and without Imp or Los treatments

 


View larger version (36K):
[in this window]
[in a new window]
  		Fig. 1. {beta}1-Adrenoceptor ({beta}1-AR, A) and {beta}2-adrenoceptor ({beta}2-AR, B) protein contents in left ventricle (LV) and right ventricle (RV) of rats 16 wk after aortocaval shunt (AVS) with and without treatment of imidapril (Imp) and losartan (Los). Typical Western blots are given for each group in both panels. Values are means ± SE of 4 separate hearts in each group. *P < 0.05 vs. sham group (Sh); {dagger}P < 0.05 vs. AVS group.

 
Alterations in GRKs. Neither the GRK activities (250 ± 15.6 and 182 ± 7.8 fmol·mg–1·min–1 in the LV and RV, respectively) nor the protein contents of GRK2, GRK3, and GRK5 (as determined by Western blots) were altered in the LV and RV homogenate from untreated or drug-treated AVS animals (data not shown). On the other hand, Fig. 2 shows a significant reduction of GRK activity in the membranous fraction and a significant increase in the cytosolic fraction isolated from both the LV and RV 16 wk after the AVS. The redistribution of GRK activity was partially attenuated by treatment with imidapril or losartan. To examine whether changes in GRK activity in the failing heart were associated with alterations of GRK isoforms in the membranous and cytosolic fractions, protein contents for different GRK isoforms were determined. A decrease in GRK2, GRK3, and GRK5 protein expression in the membranous fraction and an increase in the cytosolic fraction were observed in the failing LV and RV (Figs. 3 and 4). These changes in protein contents for GRK isoforms in both membranous and cytosolic fractions were partially prevented when treating AVS animals with imidapril or losartan. Because myocardial GRK2 expression is closely related with the {beta}-AR function (15), mRNA levels of GRK2 were detected. No difference of GRK2 mRNA levels in untreated or drug-treated groups was observed in the present study (data not shown).



View larger version (35K):
[in this window]
[in a new window]
  		Fig. 2. G protein-coupled receptor kinase (GRK) activities in membranous (A) and cytosolic (B) fractions from LV and RV of rats 16 wk after AVS with and without treatment of Imp and Los. Values are means ± SE of 4 separate hearts in each group. *P < 0.05 vs. Sh; {dagger}P < 0.05 vs. AVS group.

 


View larger version (42K):
[in this window]
[in a new window]
  		Fig. 3. Protein content for GRK2 in membranous (A) and cytosolic (B) fractions of LV and RV from rats 16 wk after AVS with or without treatment of Los and Imp. Typical Western blots are given for each group in both panels. Values are means ± SE from the same 4 hearts in each group as used in Fig. 2. *P < 0.05 vs. Sh; {dagger}P < 0.05 vs. AVS group.

 


View larger version (62K):
[in this window]
[in a new window]
  		Fig. 4. Membranous (A and C) and cytosolic (B and D) GRK3 and GRK5 protein content in LV and RV of rats 16 wk after AVS with and without treatment of Imp and Los. Typical Western blots are given for each group in both panels. Values are means ± SE from the same 4 hearts in each group as used in Fig. 2. *P < 0.05 vs. Sh; {dagger}P < 0.05 vs. AVS group.

 
{beta}-Arrestin and G{beta}{gamma}-protein contents. In view of the important role of {beta}-arrestins in regulating {beta}-AR signal transduction (36), we determined {beta}-arrestin-1 protein content in the heart. Fig. 5 reveals that {beta}-arrestin-1 protein content in both the LV and RV was decreased in the membranous fraction and increased in the cytosolic fraction similar to that observed for GRK2 content; however, the extent of changes in {beta}-arrestin-1 was not as dramatic as that seen for GRK2 (Fig. 3). These alterations in {beta}-arrestin-1 contents in AVS animals were attenuated by imidapril or losartan treatments. Because G{beta}{gamma}-protein subunits help in translocation of the GRK2 and GRK3 to the membrane and facilitate the phosphorylation of receptors, we measured the cardiac G{beta}2 and G{gamma}7 proteins. Results reveal no change in either of the G{beta}2- and G{gamma}7-protein contents in the LV and RV after 16 wk of inducing the AVS (Fig. 6). Treatments of animals with imidapril or losartan also did not affect the G{beta}{gamma}-protein contents.



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 5. {beta}-Arrestin-1 protein contents in membranous and cytosolic fractions in LV (A) and RV (B) from failing hearts induced by AVS with and without treatment of Imp and Los. Typical Western blots for each group are given in both panels. Values are means ± SE of 4 separate hearts in each group. *P < 0.05 vs. Sh; {dagger}P < 0.05 vs. AVS group.

 


View larger version (45K):
[in this window]
[in a new window]
  		Fig. 6. Protein contents of G{beta} (A) and G{gamma} (B) subunits in LV and RV of failing heart induced by AVS with and without treatment of Imp and Los. Typical Western blots for each group are given in each panel. Values are means ± SE of 4 separate animals for each group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Upregulation of {beta}-AR signal transduction in heart failure due to volume overload. We have shown that the stimulation of ventricle function of the failing heart by isoproterenol was augmented in vivo. Increased inotropic responses of the failing hearts due to volume overload were also evident at different concentrations of isoproterenol in isolated perfused heart preparations under controlled conditions. This contrasts with most other types of heart failure in which depressed positive inotropic response to {beta}-AR stimulation is usually evident (3, 12, 37). Such a difference in inotropic responses to catecholamines between the high and low cardiac output states may be due to differences in the etiological basis of heart failure. It can be argued that the augmented positive inotropic response to isoproterenol in heart failure because of volume overload may be due to depressed contractile function. However, this may not be the case because depressed contractile function in failing hearts due to myocardial infarction was not associated with augmented inotropic effect of isoproterenol (29). On the other hand, it is likely that the increased positive inotropic effect of isoproterenol in the AVS model of heart failure may be due to increased AC activity (39) as well as an increased density of {beta}-ARs in the failing hearts. In this regard, we have detected an increase in {beta}1-AR density, without any change in {beta}2-AR density, in the failing hearts due to volume overload. The results from Western blot analysis also confirmed an increase in {beta}1-AR protein content, unlike {beta}2-AR protein content, in the failing heart 16 wk after the AVS. Such a unique change in {beta}1-AR mechanisms in the volume-overloaded failing heart may be related to the lack of myocardial fibrosis in this model of heart failure (27) compared with other types of congestive heart failure, such as cardiomyopathy, ventricular pacing, myocardial infarction, and pressure overload (37).

Previous studies from different laboratories have reported a wide variety of alterations in {beta}-AR-mediated signal transduction in failing hearts due to volume overload. Communal et al. (10) demonstrated an increased {beta}-AR density in epicardium and decreased density in endocardium but no alteration in receptor density or {beta}1-to-{beta}2 ratio in the total LV myocardium 4 wk after the induction of AVS in rats. Cartagena et al. (6) found a significant increase of total {beta}-AR density in the heart from 7 to 21 days, followed by a decrease on day 56 without any change in the {beta}-AR affinity in hypertrophied hearts due to volume overload. Earlier studies (38) from our laboratory have revealed that the positive inotropic effect of isoproterenol was increased at 4, 8, and 16 wk volume overload was induced in rats. Such observations as well as the results in the present study indicate upregulation of {beta}-AR mechanisms at a time (16 wk after AVS induction) when the clinical signs of heart failure were evident.

Because previous studies (23) have revealed that chronic stimulation by {beta}-agonist leads to downregulation, whereas chronic inhibition of {beta}-adrenergic signaling results in upregulation of {beta}-ARs (19, 26), it is possible that the upregulation of {beta}1-ARs observed in the AVS model is associated with a decrease in the catecholamine level. However, Willenbrock et al. (40) have shown a significant increase in plasma norepinephrine levels without any alteration in ventricular norepinephrine concentration in rats 4 wk after the AVS. Although in the present study we did not measure the norepinephrine concentration, it is expected that upregulation of {beta}1-AR, without a change in {beta}2-AR, may be due to a decrease in the neuronally released norepinephrine, which has a selective affinity to {beta}1-AR. It should be mentioned that a depletion of norepinephrine stores by reserpine or denervation is known to produce supersensitivity to {beta}-AR stimulation (33), but other investigators (7) have observed reduced norepinephrine responses of the failing hearts in which catecholamine stores were depleted. Thus a clear relationship between the status of cardiac norepinephrine stores and sympathetic stimulation remains to be elucidated. Therefore, extensive studies regarding the release, uptake, and turnover of norepinephrine in the volume-overloaded model will be undertaken to understand the relationship of sympathetic activation and the observed changes in {beta}-AR-mediated signal transduction.

Regulation of {beta}-AR activity by GRK. Because the status of {beta}-adrenergic activity is determined by the GRK activity in the myocardium (15), the data presented in this study reveal a redistribution of GRK activity between the membranous fraction and the cytosolic fraction in the volume-overloaded heart. This redistribution was related to the changes of GRK isoforms because the protein contents for GRK2, GRK3, and GRK5 showed similar pattern of changes as the GRK activity in AVS-induced heart failure. The altered distribution of GRKs in the membranous and cytosolic fractions in the failing heart does not appear to be due to changes in G{beta}{gamma}-subunit protein content because no changes in G{beta}- or G{gamma}-protein subunits were seen. Furthermore, significant changes in both Gs and Gi proteins were observed in hearts failing due to volume overload (39). However, we detected a decrease of {beta}-arrestin-1 protein content in the membranous fraction and an increase in the cytosolic fraction suggesting its synergistical action in regulating {beta}-adrenergic signaling with GRKs. Ungerer et al. (35) found no alterations in mRNA and protein expression of {beta}-arrestin-1 in the end-stage human heart failure. The discrepancy between their results and ours may be mainly due to the differences in the type and stage of heart failure. GRK2 and {beta}-arrestin-1 have been shown to be associated with the regulation of endothelial function, whereas GRK3 and GRK5 are linked with cardiomyocyte function (36), and thus some caution should be exercised while interpreting these results. Whereas some investigators have shown upregulation of GRK2 without any changes in GRK5 in hypertensive rats, others (42) have reported redistribution of GRK2 to intercalated disc area and GRK5 to nucleus in the same experimental model. Accordingly, the results described in the present study as well as from other laboratories appear to support the concept that alterations in GRK activity and GRK isoforms may be dependent on the type and stage of heart failure, and the redistribution of GRK isoforms in the diseased heart may be associated with the regulation of {beta}-AR activity.

Blockade of RAS and modification of changes in {beta}-AR- linked mechanisms. Our observations regarding attenuation of the progression of AVS-induced cardiac hypertrophy and heart failure upon treatment of animals with an ACE inhibitor (imidapril) and an AT1R antagonist (losartan) are consistent with the role of RAS activation in heart hypertrophy and dysfunction due to volume overload (27, 41, 42). Although we did not measure the levels of plasma angiotensin II in this study, other investigators (32) have shown a significant increase in the levels of this hormone in AVS animals. Nonetheless, this study has also shown that blockade of the RAS by imidapril and losartan partially prevented the augmented inotropic responses of the volume-overloaded failing hearts to isoproterenol, as well as upregulation of {beta}1-ARs and redistribution of GRK activity and isoform contents. In view of the similarities between the effects of ACE inhibitors and AT1R antagonists on changes in {beta}-AR mechanisms in heart failure due to volume overload, our results indicate that the beneficial effects of these drug treatments may be due to blockade of the RAS. Because the stimulation of the RAS has been reported to affect the sympathetic activity in different experimental models (4, 22, 29), it is possible that the blockade of the RAS by ACE inhibitors and AT1R blockers may depress the sympathetic system and produce the effects observed in this study. In addition, ACE inhibitors and AT1R antagonists are known to decrease the oxidative stress in human vascular endothelial cells (43), which is associated with the development of heart failure (11). Imidapril has also been shown to inhibit the production of angiotensin II and decrease the protein content as well as gene expression of inducible nitric oxide synthase, which is associated with the improvement of endothelial function in rats undergoing hypertensive heart failure (20). Because treatments of AVS animals with both imidapril and losartan did not attenuate the increased AC activities or protein content (39), it appears that attenuation of {beta}1-AR density and protein content may be related to some specific effects of these drugs on cardiac remodeling. Thus further investigations are needed to sort out the exact mechanisms of {beta}-AR upregulation in heart failure due to volume overload.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This paper was supported by grants from the Heart and Stroke Foundation of Manitoba and the Canadian Institutes of Health Research. X. Wang and E. Sentex were supported by a Studentship and a Postdoctoral Fellowship from the Heart and Stroke Foundation of Canada, respectively.


   ACKNOWLEDGMENTS
 
Imidapril and losartan were gifts from Tanabe Seiyaku, Osaka, Japan and Merck Frosst, Canada, respectively.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. S. Dhalla, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave., Winnipeg, MB R2H 2A6, Canada (E-mail: nsdhalla{at}sbrc.ca)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Anderson KM, Eckhart AD, Willette RN, and Koch WJ. The myocardial beta-adrenergic system in spontaneously hypertensive heart failure (SHHF) rats. Hypertension 33: 402–407, 1999.[Abstract/Free Full Text]
  2. Benovic JL, Mayor F, Staniszewski C, Lefkowitz RJ, and Caron MG. Purification and characterization of the beta-adrenergic receptor kinase. J Biol Chem 262: 9026–9032, 1987.[Abstract/Free Full Text]
  3. Bohm M. Alterations of {beta}-adrenoceptor-G protein regulated adenylyl cyclase in heart failure. Mol Cell Biochem 147: 147–160, 1995.[CrossRef][Web of Science][Medline]
  4. Bohm M, Zolk O, Flesch M, Schiffer F, Schnabel P, Stasch JP, and Knorr A. Effects of angiotensin II type 1 receptor blockade and angiotensin-converting enzyme inhibition on cardiac {beta}-adrenergic signal transduction. Hypertension 31: 747–754, 1998.[Abstract/Free Full Text]
  5. Bristow MR, Hershberger RE, Port JD, Gilbert EM, Sandoval A, Rasmussen R, Cates AE, and Feldman AM. Beta-adrenergic pathways in nonfailing and failing human ventricular myocardium. Circulation 82, Suppl 2: I12–I25, 1990.
  6. Cartagena G, Sapag HM, Jalil J, Tapia V, Guarda E, Foncea R, Corbalan R, Ebensperger R, and Lavandero S. Changes in beta-adrenergic receptors of rat heart and adipocytes during volume-overload induced cardiac hypertrophy. Int J Clin Pharmacol Ther Toxicol 31: 198–203, 1993.[Medline]
  7. Chidsey CA and Braunwald E. Sympathetic activity and neurotransmitter depletion in congestive heart failure. Pharmacol Rev 18: 685–700, 1966.[Abstract/Free Full Text]
  8. Choi DJ, Koch WJ, Hunter JJ, and Rockman HA. Mechanism of beta-adrenergic receptor desensitization in cardiac hypertrophy is increased beta-adrenergic receptor kinase. J Biol Chem 272: 17223–17229, 1997.[Abstract/Free Full Text]
  9. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[Web of Science][Medline]
  10. Communal C, Ribuot C, Durand A, and Demenge P. Myocardial beta-adrenergic reactivity in volume overload-induced cardiac hypertrophy in the rat. Fundam Clin Pharmacol 12: 411–419, 1998.[Web of Science][Medline]
  11. Dhalla NS, Temsah RM, and Netticadan T. Role of oxidative stress in cardiovascular diseases. J Hypertens 18: 655–673, 2000.[CrossRef][Web of Science][Medline]
  12. Dhalla NS, Wang X, Sethi R, Das PK, and Beamish RE. {beta}-Adrenergic linked signal transduction mechanisms in failing hearts. Heart Fail Rev 2: 55–65, 1997.
  13. Dixon IM, Hata T, and Dhalla NS. Sarcolemmal calcium transport in congestive heart failure due to myocardial infarction in rats. Am J Physiol Heart Circ Physiol 262: H1387–H1394, 1992.[Abstract/Free Full Text]
  14. Dzimiri N and Moorji A. Relationship between alterations in lymphocyte and myocardial beta-adrenoceptor density in patients with left heart valvular disease. Clin Exp Pharmacol Physiol 23: 498–502, 1996.[Web of Science][Medline]
  15. Dzimiri N, Muiya P, Andres E, and Al-Halees Z. Differential functional expression of human myocardial G protein receptor kinases in left ventricular cardiac diseases. Eur J Pharmacol 489: 167–177, 2004.[CrossRef][Web of Science][Medline]
  16. Hammond HK, Roth DA, Insel PA, Ford CE, White FC, Maisel AS, Ziegler MG, and Bloor CM. Myocardial beta-adrenergic receptor expression and signal transduction after chronic volume-overload hypertrophy and circulatory congestion. Circulation 85: 269–280, 1992.[Abstract/Free Full Text]
  17. Iaccarino G, Dolber PC, Lefkowitz RJ, and Koch WJ. Beta-adrenergic receptor kinase-1 levels in catecholamine-induced myocardial hypertrophy: regulation by beta- but not apha1-adrenergic stimulation. Hypertension 33: 396–401, 1999.[Abstract/Free Full Text]
  18. Ishigai Y, Mori T, Moriyama S, and Shibano T. Induction of cardiac beta-adrenergic receptor kinase 1 in rat heart failure caused by coronary ligation. J Mol Cell Cardiol 31: 1261–1268, 1999.[CrossRef][Web of Science][Medline]
  19. Karliner JS. Effects of beta-blockade on beta-adrenergic receptors and signal transduction. J Cardiovasc Pharmacol 14, Suppl 5: S6–S12, 1989.
  20. Kobayashi N, Higashi T, Hara K, Shirataki H, and Matsuoka H. Effects of imidapril on NOS expression and myocardial remodeling in failing heart of Dahl salt-sensitive hypertensive rats. Cardiovasc Res 44: 518–526, 1999.[Abstract/Free Full Text]
  21. Koch WJ, Rockman HA, Samama P, Hamilton RA, Bond RA, Milano CA, and Lefkowitz RJ. Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a beta ARK inhibitor. Science 268: 1350–1353, 1995.[Abstract/Free Full Text]
  22. Laflamme AK, Oster L, Cardinal R, and de Champlain J. Effects of renin-angiotensin blockade on sympathetic reactivity and {beta}-adrenergic pathway in the spontaneously hypertensive rats. Hypertension 30: 278–287, 1997.[Abstract/Free Full Text]
  23. Nanoff C, Freissmuth M, Tuisl E, and Schutz W. A different desensitization pattern of cardiac beta-adrenoceptor subtypes by prolonged in vivo infusion of isoprenaline. J Cardiovasc Pharmacol 13: 198–203, 1989.[Web of Science][Medline]
  24. Penn RB, Pronin AN, and Benovic JL. Regulation of G protein-coupled receptor kinases. Trends Cardiovasc Med 10: 81–89, 2000.[CrossRef][Web of Science][Medline]
  25. Persad S, Takeda S, Panagia V, and Dhalla NS. Beta-adrenoceptor-linked signal transduction in ischemic-reperfused heart and scavenging of oxyradicals. J Mol Cell Cardiol 29: 545–558, 1997.[CrossRef][Web of Science][Medline]
  26. Ping P, Gelzer BR, Roth DA, Kiel D, Insel PA, and Hammond HK. Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart. J Clin Invest 95: 1271–1280, 1995.[Web of Science][Medline]
  27. Ruzicka M, Keeley FW, and Leenen FH. The renin-angiotensin system and volume overload-induced changes in cardiac collagen and elastin. Circulation 90: 1989–1996, 1994.[Abstract/Free Full Text]
  28. Sakagoshi N, Nakano S, Taniguchi K, Hirata N, and Matsuda H. Relation between myocardial beta-adrenergic receptor and left ventricular function in patients with left ventricular volume overload due to chronic mitral regurgitation with or without aortic regurgitation. Am J Cardiol 68: 81–84, 1991.[CrossRef][Web of Science][Medline]
  29. Sethi R, Shao Q, Ren B, Saini HK, Takeda N, and Dhalla NS. Changes in {beta}-adrenoceptors in heart failure due to myocardial infarction are attenuated by blockade of renin-angiotensin system. Mol Cell Biochem 263: 11–20, 2004.[CrossRef][Web of Science][Medline]
  30. Sugawara H, Sakurai K, Atsumi H, Nakada S, Tomoike H, and Endoh M. Differential alteration of cardiotonic effects of EMD 57033 and beta-adrenoceptor agonists in volume-overload rabbit ventricular myocytes. J Card Fail 6: 338–349, 2000.[CrossRef][Web of Science][Medline]
  31. Sullebarger JT, D'Ambra PM, Clark LC, Thanikarry L, and Fontanet HL. Effect of digoxin on ventricular remodeling and responsiveness of beta-adrenoceptors in chronic volume overload. J Cardiovasc Pharmacol Ther 3: 281–290, 1998.[Medline]
  32. Suzuki H, Maehara K, Yaoita H, and Maruyama Y. Altered effects of angiotensin II type 1 and type 2 receptor blockers on cardiac norepinephrine release and inotropic responses during cardiac sympathetic nerve stimulation in aorto-caval shunt rats. Circ J 68: 683–690, 2004.[CrossRef][Web of Science][Medline]
  33. Trenelenburg U. Supersensitivity and subsensitivity to sympathomimetic amines. Pharmacol Rev 15: 225–276, 1963.[Abstract/Free Full Text]
  34. Ungerer M, Bohm M, Elce JS, Erdmann E, and Lohse MJ. Altered expression of beta-adrenergic receptor kinase and beta 1-adrenergic receptors in the failing human heart. Circulation 87: 454–463, 1993.[Abstract/Free Full Text]
  35. Ungerer M, Parruti G, Bohm M, Puzicha M, De Blasi A, Erdman E, and Lobse MJ. Expression of {beta}-arrestin and {beta}-adrenergic receptor kinases in the failing heart. Circ Res 74: 206–213, 1994.[Abstract/Free Full Text]
  36. Vinge LE, Oie E, Andersson Y, Grogaard HK, Andersen G, and Attramadal H. Myocardial distribution and regulation of GRK and {beta}-arrestin isoforms in congestive heart failure in rats. Am J Physiol Heart Circ Physiol 281: H2490–H2499, 2001.[Abstract/Free Full Text]
  37. Wang X and Dhalla NS. Modification of {beta}-adrenoceptor signal transduction pathway by genetic manipulation and heart failure. Mol Cell Biochem 214: 131–155, 2000.[CrossRef][Web of Science][Medline]
  38. Wang X, Ren B, Liu S, Sentex E, Tappia PS, and Dhalla NS. Characterization of cardiac hypertrophy and heart failure due to volume overload in rat. J Appl Physiol 94: 752–763, 2003.[Abstract/Free Full Text]
  39. Wang X, Sentex E, Chapman D, and Dhalla NS. Alterations of adenylyl cyclase and G proteins in aortocaval shunt induced heart failure. Am J Physiol Heart Circ Physiol 287: H118–H125, 2004.[Abstract/Free Full Text]
  40. Willenbrock R, Stauss H, Scheuermann M, Osterziel KJ, Unger T, and Dietz R. Effect of chronic volume overload on baroreflex control of heart rate and sympathetic nerve activity. Am J Physiol Heart Circ Physiol 273: H2580–H2585, 1997.[Abstract/Free Full Text]
  41. Yamakawa H, Imamura T, Matsuo T, Onitsuka H, Tsumori Y, Kato J, Kitamura K, Koiwaya Y, and Eto T. Diastolic wall stress and ANG II in cardiac hypertrophy and gene expression induced by volume overload. Am J Physiol Heart Circ Physiol 279: H2939–H2946, 2000.[Abstract/Free Full Text]
  42. Yi XP, Gerdes AM, and Li F. Myocyte redistribution of GRK2 and GRK5 in hypertensive, heart-failure-prone rats. Hypertension 39: 1058–1063, 2002.[Abstract/Free Full Text]
  43. Zhang H, Schmeisser A, Garlichs CD, Plotze K, Damme U, Mugge A, and Daniel WG. Angiotensin II-induced superoxide anion generation in human vascular endothelial cells: role of membrane-bound NADH-/NADPH-oxidases. Cardiovasc Res 44: 215–222, 1999.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Y. Takewa, E. R. Chemaly, M. Takaki, L. F. Liang, H. Jin, I. Karakikes, C. Morel, Y. Taenaka, E. Tatsumi, and R. J. Hajjar
Mechanical work and energetic analysis of eccentric cardiac remodeling in a volume overload heart failure in rats
Am J Physiol Heart Circ Physiol, April 1, 2009; 296(4): H1117 - H1124.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
N. S. Dhalla, H. K. Saini-Chohan, D. Rodriguez-Leyva, V. Elimban, M. R. Dent, and P. S. Tappia
Subcellular remodelling may induce cardiac dysfunction in congestive heart failure
Cardiovasc Res, February 15, 2009; 81(3): 429 - 438.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
X. Xu, W. Wan, L. Ji, S. Lao, A. S. Powers, W. Zhao, J. M. Erikson, and J. Q. Zhang
Exercise training combined with angiotensin II receptor blockade limits post-infarct ventricular remodelling in rats
Cardiovasc Res, June 1, 2008; 78(3): 523 - 532.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
R. Sethi, H. K. Saini, X. Guo, X. Wang, V. Elimban, and N. S. Dhalla
Dependence of changes in beta-adrenoceptor signal transduction on type and stage of cardiac hypertrophy
J Appl Physiol, March 1, 2007; 102(3): 978 - 984.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
K. J. Milne, D. B. Thorp, C. W. J. Melling, and E. G. Noble
Castration inhibits exercise-induced accumulation of Hsp70 in male rodent hearts
Am J Physiol Heart Circ Physiol, April 1, 2006; 290(4): H1610 - H1616.
[Abstract] [Full Text] [PDF]

Home page
 J CARDIOVASC PHARMACOL THERHome page
N. S. Dhalla, M. R. Dent, P. S. Tappia, R. Sethi, J. Barta, and R. K. Goyal
Subcellular Remodeling as a Viable Target for the Treatment of Congestive Heart Failure
Journal of Cardiovascular Pharmacology and Therapeutics, March 1, 2006; 11(1): 31 - 45.
[Abstract] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
H. K. Saini, O. N. Tripathi, S. Zhang, V. Elimban, and N. S. Dhalla
Involvement of Na+/Ca2+ exchanger in catecholamine-induced increase in intracellular calcium in cardiomyocytes
Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H373 - H380.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/1/H151    most recent
00066.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, X.
Right arrow Articles by Dhalla, N. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.
Right arrow Articles by Dhalla, N. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.