Download Atrial Natriuretic Factor Binding to Its Receptor Is Dependent on

Atrial Natriuretic Factor Binding to Its Receptor Is
Dependent on Chloride Concentration
A Possible Feedback-Control Mechanism in Renal Salt Regulation
Kunio S. Misono
Downloaded from http://circres.ahajournals.org/ by guest on August 9, 2017
Abstract—Although considerable evidence indicates a role for atrial natriuretic factor (ANF) in renal salt regulation, other
studies have found a lack of natriuretic response to high-plasma ANF under certain physiological and pathophysiological
conditions. The mechanism for this apparent insensitivity to ANF is unknown. In the present study, it was found that ANF
binding to its receptor requires the presence of chloride and occurs in a chloride concentration– dependent manner. ANF
binding was measured using the purified recombinant hormone-binding domain of the ANF receptor in the presence of 0.1
mol/L NaCl or other selected salt. High specific binding was detected in the presence of NaCl, KCl, or NH4Cl. However,
binding was undetectable when the salt was replaced with NaHCO3, CH3COONa, or CH3COONH4, indicating that binding
requires the presence of chloride. Chloride dependence was also found with the native receptor in bovine adrenocortical
membrane preparations. ANF binding to the recombinant protein was chloride concentration– dependent over a range from
0.05 to 10 mmol/L, and a half-maximum binding was attained at ⬇0.6 mmol/L equivalent chloride concentration.
Competitive-binding assays at several fixed concentrations of NaCl showed that lowering chloride concentration caused a
decrease in maximum binding but did not alter Kd values, suggesting that a loss of chloride turns off ANF binding rather than
reducing affinity for ANF. Saturation-binding studies showed that excess ANF cannot overcome loss of binding caused by
low chloride. Chloride-dependent ANF-receptor binding may function as a feedback-control mechanism regulating the
ANF-receptor action and, hence, renal sodium excretion. (Circ Res. 2000;86:1135-1139.)
Key Words: atrial natriuretic factor 䡲 receptors 䡲 chloride 䡲 sodium 䡲 kidney
A
trial natriuretic factor (ANF) is a hormone involved in
cardiovascular homeostasis through its natriuretic and
vasodilator actions. Although considerable evidence indicates
a direct role of ANF in renal salt regulation, other studies
have found a lack of natriuretic response to high-plasma ANF
under certain physiological and pathophysiological conditions.1,2 For example, infusion of ANF in normovolemic
animals to mimic endogenous ANF increase on a high-salt
diet fails to induce comparable natriuresis.3 In edematous
disease states such as congestive heart failure,4,5 nephrotic
syndrome,5 and liver cirrhosis,6 plasma ANF levels are
elevated, yet sodium is retained. In addition, transgenic
animals overexpressing ANF, with plasma ANF levels elevated 10-fold or more, maintain normal salt excretion.7 The
apparent lack of correlation between plasma ANF levels and
salt excretion led to the suggestion that ANF may not be a
physiological regulator of salt excretion.2 Thus, the physiological role of ANF in renal salt regulation still remains
unclear.
It is evident from these studies that there are certain
experimental and disease situations in which high-plasma
ANF levels do not result in renal salt excretion. The mecha-
nism responsible for this apparent insensitivity to ANF is not
known. In the present study, it was found that binding of ANF
to its receptor requires the presence of chloride and occurs in
a chloride concentration– dependent manner. This finding
suggests that ANF-receptor binding and, hence, physiological
responses to the hormone may be regulated by changes in
chloride concentration at the target sites. In the kidney, this
chloride-mediated control of ANF and ANF-receptor function may play a role in the regulation of renal salt excretion.
The apparent insensitivity to high-plasma ANF observed in
edematous states and in transgenic animals that overexpress
ANF may also be explained by this chloride-mediated control
mechanism.
Materials and Methods
The extracellular hormone– binding domain of the ANF receptor was
expressed in COS-1 cells and purified from the culture medium of
transfected cells by ANF affinity chromatography, as previously
described.8 The purified recombinant hormone-binding domain has
been shown to bind the hormone with an affinity and specificity
consistent with that of the native ANF receptor.8 Bovine adrenocortical plasma membranes were isolated by differential centrifugation
and partially purified by sucrose density-gradient centrifugation, as
Received April 6, 2000; accepted April 27, 2000.
From the Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio.
Correspondence to Kunio S. Misono, Department of Molecular Cardiology, Lerner Research Institute, NB50, Cleveland Clinic Foundation, 9500 Euclid
Ave, Cleveland, OH 44195. E-mail [email protected]
© 2000 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
1135
1136
Circulation Research
June 9, 2000
Downloaded from http://circres.ahajournals.org/ by guest on August 9, 2017
described.9 BSA was obtained from Boehringer Mannheim Biochemicals and bacitracin from United States Biochemical. All other
reagents were of analytical grade or highest available grades.
ANF-binding assay with the purified hormone-binding domain
was performed by a modification of the method described previously.8 Briefly, binding was measured by incubating the hormonebinding domain (2.5 ng) with 125I-ANF (200 000 to 300 000 cpm per
incubation) in 200 ␮L of the assay medium consisting of 20 mmol/L
sodium phosphate buffer, pH 7.4, 0.05% BSA, and 0.01% bacitracin
in the presence of 0.1 mol/L NaCl or other selected salt. After
incubation at room temperature for 30 minutes, bound 125I-ANF was
separated from unbound 125I-ANF by rapid gel filtration on a
Sephadex G-75 column and counted for 125I-radioactivity. Nonspecific binding was measured by incubation in the presence of
0.1 ␮mol/L unlabeled ANF.
The chloride concentration dependence of ANF binding to the
hormone-binding domain was measured by varying NaCl concentration in the assay medium while maintaining the total salt concentration at 0.1 mol/L by supplementing with sodium acetate. Incubation was carried out with a constant amount of 125I-ANF tracer.
Competitive-binding assays were performed at several fixed concentrations of NaCl with a constant amount of 125I-ANF tracer and
varying concentrations of unlabeled ANF using the purified
hormone-binding domain.
Saturation-binding assays were carried out using 125I-ANF with the
specific radioactivity of 0.02 ␮Ci/pmol, prepared as described.10 The
hormone-binding domain (2.5 ng) was incubated with increasing
concentrations of 125I-ANF from 10 pmol to 0.1 ␮mol/L in 100 ␮L
of the assay medium containing 0.1 mol/L NaCl or sodium acetate.
A parallel set of control incubations was performed in the absence of
the hormone-binding domain to measure background binding. Bound
125
I-radioactivity values obtained in the control incubations were
subtracted from those in corresponding experimental incubations.
ANF-binding assays with the bovine adrenocortical membranes
were carried out according to the method described previously.9
Before the assay, the membranes were freed from chloride by
dialyzing against 20 mmol/L sodium phosphate buffer, pH 7.5,
containing a suspension of an anion exchange resin AG 1X8 in
formate form (Bio-Rad) at 4°C overnight. The membranes (5 ␮g
membrane protein) were incubated in the assay medium containing
0.1 mol/L NaCl or sodium acetate. After incubation at 0°C for 1
hour, membrane-bound 125ANF was separated by filtration and
counted. Nonspecific binding was measured in the presence of
0.1 ␮mol/L unlabeled ANF.
Figure 1. Effect of various salts on binding of ANF to the purified hormone-binding domain of the ANF receptor. Binding
assays were carried out in a medium containing 0.1 mol/L NaCl
or other salt. Binding of 125I-ANF was measured in the absence
(–) and presence (⫹) of 0.1 ␮mol/L unlabeled ANF. Data are
mean⫾SEM; n⫽6.
binding in the presence of 0.1 mol/L sodium acetate ranged
from 70% to 90% of that found in the presence of 0.1 mol/L
NaCl.
Binding of ANF to Its Receptor Occurs in a
Chloride Concentration–Dependent Manner
The chloride concentration dependence of ANF binding to
the hormone-binding domain was examined by increasing the
concentration of NaCl while maintaining the total salt concentration in the assay medium constant at 0.1 mol/L by
supplementing with sodium acetate (Figure 2). ANF binding
was essentially absent at NaCl concentrations below
0.05 mmol/L. ANF binding rose with increasing concentrations of NaCl, reaching a maximum level above 10 mmol/L
NaCl. The half-maximum binding was attained at
⬇0.6 mmol/L NaCl.
Results
Binding of ANF to Its Receptor Requires the
Presence of Chloride
In the presence of 0.1 mol/L NaCl, a significant level of
specific binding of 125I-ANF to the purified hormone-binding
domain was observed (Figure 1). Similar levels of binding
were found when NaCl was replaced with KCl or NH4Cl.
However, when the salt was replaced with sodium bicarbonate, sodium acetate, or ammonium acetate, binding was
undetectable. These results indicate that ANF binding requires the presence of chloride, and that other anions such as
bicarbonate, acetate, and phosphate contained in the assay
medium cannot substitute for the chloride requirement. On
the other hand, the type of cation (Na⫹, K⫹, or NH4⫹) in the
medium had no apparent effect on ANF binding. Chloride
requirement for ANF binding was also observed with the
native ANF receptor in bovine adrenocortical membrane
preparations. The specific binding of ANF obtained in the
presence of 0.1 mol/L sodium acetate in the assay medium
was 16-fold less than that in the presence of 0.1 mol/L NaCl.
When the membranes were used without dialysis, the specific
Figure 2. Chloride concentration dependence of ANF binding to
the ANF-receptor hormone-binding domain. 125I-ANF binding
was measured with increasing concentrations of NaCl. The total
salt concentration in the assay medium was maintained at 0.1
mol/L by supplementing with sodium acetate. Data are
mean⫾SEM; n⫽6.
Misono
Chloride-Mediated Feedback Control of ANF Receptor
Downloaded from http://circres.ahajournals.org/ by guest on August 9, 2017
Figure 3. Effect of chloride concentration on ANF binding to the
ANF-receptor hormone-binding domain examined by
competitive-binding assays performed at several fixed concentrations of NaCl. Data shown are mean of 3 incubations.
Lack of Chloride Completely Abolishes
ANF-Receptor Binding
Competitive-binding assays were performed at several fixed
concentrations of NaCl by incubations with varying concentrations of unlabeled ANF competing against a constant
amount of 125I-ANF tracer (Figure 3). The results showed that
lowering chloride concentration caused a decrease in the
maximum binding but did not alter binding affinity toward
ANF (Kd remained at ⬇1 nmol/L).
Excess ANF Cannot Overcome Inhibition of ANF
Binding by Low Salt
Incubation of the hormone-binding domain in the presence of
0.1 mol/L NaCl with the increasing concentrations of 125Ilabeled ANF showed binding saturation at 0.1 ␮mol/L ligand
concentration (Figure 4). However, in the absence of chloride, binding was undetectable, even when the concentration
of the radioligand was increased to 0.1 ␮mol/L, a concentration 100-fold higher than the Kd value. These results indicate
that loss of ANF binding due to low chloride concentration
cannot be overcome by excess ANF.
Discussion
The data presented in this study show that ANF binding to its
receptor required the presence of chloride. In the absence of
chloride, there was no detectable binding of ANF to the
hormone-binding domain of the ANF receptor. Other anions
such as bicarbonate, acetate, and phosphate could not substitute for the chloride requirement. On the other hand, the type
of cation present showed no effect on ANF binding. Binding
was chloride concentration– dependent over the concentration
range from ⬇0.05 to 10 mmol/L with the half-maximal ANF
binding being attained at ⬇0.6 mmol/L equivalent chloride
concentration. Chloride requirement for ANF binding was
also observed with the partially purified and dialyzed bovine
1137
Figure 4. Saturation binding of ANF to the ANF-receptor
hormone-binding domain performed in the presence of 0.1
mol/L NaCl (F) or sodium acetate (E). Data are mean⫾SEM;
n⫽6.
adrenocortical membranes, although a low level of ANF
binding was still observed when NaCl was replaced with
sodium acetate. The residual ANF-binding activity may have
been due to the presence of chloride ions bound to or trapped
in membrane vesicles in the adrenocortical membrane preparations. The chloride dependence of ANF binding to the
receptor is consistent with our recent finding of a buried,
protein-bound chloride ion in the structure of the ANFreceptor hormone-binding domain as determined by X-ray
crystallography (K.S. Misono, X. Zhang, F. van den Akker,
V.C. Yee, unpublished data, 2000).
Competitive ANF-binding assays performed at several
fixed concentrations of NaCl showed that lowering chloride
concentration caused a decrease in maximum binding but did
not alter Kd values. This indicates that a loss of chloride
completely abolishes ANF binding rather than reducing
binding affinity. Furthermore, saturation-binding data
showed that inhibition of ANF binding by low salt could not
be overcome by excess ANF. Physiologically, these results
suggest that the chloride-dependent ANF binding provides an
on-off mechanism for the receptor, and that loss of ANF
binding on chloride deficit cannot be overcome by excess
levels of ANF.
The chloride dependence of ANF binding demonstrated in
this study suggests a possibility that changes in chloride
concentration at the target sites for this hormone may modulate hormone-receptor binding and regulate physiological
responses. In the kidney, this chloride-mediated regulation of
ANF-receptor binding may function as a feedback-control
mechanism in ANF-induced natriuresis to maintain normal
salt and fluid balance. In the loop of Henle and the distal
tubule, sodium reabsorption is mediated by Na⫹ and Cl⫺
cotransport systems and, hence, is accompanied by chloride
reabsorption. Thus, the chloride concentrations at these tubular sites are tightly coupled with the state of electrolyte and
1138
Circulation Research
June 9, 2000
Downloaded from http://circres.ahajournals.org/ by guest on August 9, 2017
water transport. In the medullary collecting ducts immediately downstream (the main tubular site of ANF action11), the
tubular chloride concentration may reach millimolar ranges
under certain physiological and pathophysiological conditions. For example, aldosterone treatment lowers the tubular
fluid chloride concentration to ⱕ4 mmol/L,12,13 which falls
within the range where ANF-receptor binding is chloride
concentration– dependent. Additionally, the ANF receptor is
located in both apical and basal membranes of the medullary
collecting ducts regulating sodium reabsorption and
vasopressin-stimulated water reabsorption.14 –17 ANF natriuresis and diuresis may then be regulated through control of
ANF-receptor binding by the chloride concentrations in the
tubule and peritubular microcirculation. It is probable that
such a control mechanism plays an important role in renal
regulation of electrolyte and fluid homeostasis.
In edematous disease states such as congestive heart
failure,4,5 nephrotic syndrome,5 and liver cirrhosis,6 the
plasma levels of ANF are markedly elevated, yet sodium is
retained. Congestive heart failure is characterized by activation of the renin-angiotensin-aldosterone system, increased
vasopressin secretion, and heightened sympathetic nervous
activity. ANF, elevated because of volume expansion, is
thought to be one of the principal factors opposing these
activities. Although studies using the ANF-receptor antagonist HS-142-1 have demonstrated the suppressive effects of
ANF on the sympathetic and renin-angiotensin-aldosterone
systems as well as on sodium reabsorption,18 –20 the high
plasma levels of ANF fail to produce correspondingly enhanced natriuresis in congestive heart failure.4,5 In such an
edematous state, the proposed chloride-mediated feedback
mechanism for the ANF receptor may function to prevent
excessive salt loss. The chronically elevated plasma ANF in
this state would be expected to activate the ANF receptor and
induce persistent ANF natriuresis, which would consequently
reduce the tubular chloride concentration. ANF natriuresis
might then be turned off by inhibition of ANF binding by
insufficient chloride when the tubular chloride concentration
falls below a threshold level. This chloride-mediated
feedback-control mechanism may be responsible, aside from
the counterregulatory action of the renin-angiotensin system,
for the attenuated response to high-plasma ANF observed in
the edematous states. Such a salt-conserving feedback mechanism may likewise operate in ANF-overexpressing transgenic animals, in which salt is also retained in spite of high
plasma levels of ANF.
The lack of natriuretic response to high-plasma ANF
observed in these states and animals is consistent with our
finding in vitro that excess ANF cannot overcome the loss of
ANF binding caused by low chloride. Additionally, it has
been found that KCl infusion21 or acute volume expansion by
isotonic saline infusion7 in ANF-overexpressing transgenic
animals causes exaggerated natriuretic responses. This exaggerated natriuresis may also be explained by the chloridemediated feedback mechanism operating in the kidney. With
KCl infusion or acute volume expansion, the resultant increase in the filtered chloride concentration may unblock this
chloride-mediated inhibition of ANF binding to the receptor,
allowing maximal natriuresis to occur in response to high
plasma levels of ANF. It has also been reported that highdose ANF infusion in normovolemic animals induces only
modest natriuresis.3 The suppressed natriuresis in response to
high-dose ANF infusions may similarly have resulted from
the chloride-mediated feedback mechanism, in which a decreased tubular chloride concentration after acute ANF natriuresis may have prevented further natriuresis by inhibiting
binding of ANF to its receptor.
In animal models of congestive heart failure and nephrotic
syndrome, ANF infusion lowers the arterial mean pressure
and increases the glomerular filtration rate to levels similar to
those in the respective control animals, whereas ANF natriuresis is markedly blunted. 5 In addition, in ANFoverexpressing transgenic animals, high-plasma ANF is accompanied by systemic hypotension but normal salt excretion
is maintained.7 These findings suggest that the chloridemediated feedback control operates at renal tubular sites but
not at the renal or systemic vascular sites.
Attenuated natriuretic response to ANF has also been
observed in animals on a low-salt diet.22 The NaCl deficit in
low-salt animals is associated with marked elevation in
sodium reabsorption in the medullary collecting ducts. ANF
infusion in these animals does not reduce this sodium
reabsorption. On the other hand, the systemic hypotensive
effect of ANF is still present. On the basis of these findings,
it has been suggested that a salt-conserving counterregulatory
mechanism operates in the kidney and overcomes ANF
natriuresis under salt-deprived conditions. It was also found
that the lack of responsiveness to ANF in these salt-deficient
animals is reversed within 1 hour of salt repletion, suggesting
that this counterregulatory mechanism is not mediated by
downregulation of the ANF receptor.22 Additional studies
showed that this salt-conserving mechanism operates primarily in the medullary collecting duct23 and functions independently of the sympathoadrenergic and renin-angiotensin-aldosterone systems.24 The suppressed natriuretic response to
ANF could not be explained on the basis of reduced renal
perfusion pressure or glomerular filtration rate.24 These findings are consistent with direct inhibition of the ANF receptor
by chloride deficit as demonstrated in this study and are in
agreement with the present hypothesis that chloride-mediated
feedback control of the ANF receptor occurs at the renal
tubular sites, regulating renal salt excretion.
In summary, binding of ANF to the ANF-receptor
hormone-binding domain was found to require the presence
of chloride and occur in a chloride concentration– dependent
manner. It is proposed that chloride-mediated feedback control of the ANF receptor occurs in the kidney and plays a role
in the regulation of ANF-mediated natriuresis and, hence,
renal salt excretion. In light of this possible feedback-control
mechanism in ANF natriuresis mediated by chloride, the
physiological and pathophysiological roles of ANF in salt and
volume homeostasis warrant additional investigation.
Acknowledgments
This work was supported by grants from the American Heart
Association National Center (95012310) and the National Institutes
of Health (HL54329). I thank S. Misono for valuable discussions and
constructive review of the manuscript. I am grateful to Xiaolun
Zhang for able technical assistance.
Misono
Chloride-Mediated Feedback Control of ANF Receptor
References
Downloaded from http://circres.ahajournals.org/ by guest on August 9, 2017
1. de Zeeuw D, Janssen WM, de Jong PE. Atrial natriuretic factor: its (patho)physiological significance in humans. Kidney Int. 1992;41:1115–1133.
2. Goetz KL. Evidence that atriopeptin is not a physiological regulator of
sodium excretion. Hypertension. 1990;15:9 –19.
3. Bie P, Wang BC, Leadley RJ Jr, Goetz KL. Hemodynamic and renal
effects of low-dose infusions of atrial peptide in awake dogs. Am J
Physiol. 1988;254:R161–R169.
4. Burnett JC Jr, Kao PC, Hu DC, Heser DW, Heublein D, Granger JP,
Opgenorth TJ, Reeder GS. Atrial natriuretic peptide elevation in congestive heart failure in the human. Science. 1986;231:1145–1147.
5. Koepke JP, DiBona GF. Blunted natriuresis to atrial natriuretic peptide in
chronic sodium-retaining disorders. Am J Physiol. 1987;252:F865–F871.
6. Warner L, Skorecki K, Blendis LM, Epstein M. Atrial natriuretic factor
and liver disease. Hepatology. 1993;17:500 –513.
7. Field LJ, Veress AT, Steinhelper ME, Cochrane K, Sonnenberg H.
Kidney function in ANF-transgenic mice: effect of blood volume
expansion. Am J Physiol. 1991;260:R1–R5.
8. Misono KS, Sivasubramanian N, Berkner K, Zhang X. Expression and
purification of the extracellular ligand-binding domain of the atrial natriuretic peptide (ANP) receptor: monovalent binding with ANP induces 2:2
complexes. Biochemistry. 1999;38:516 –523.
9. Misono KS, Grammer RT, Rigby JW, Inagami T. Photoaffinity labeling
of atrial natriuretic factor receptor in bovine and rat adrenal cortical
membranes. Biochem Biophys Res Commun. 1985;130:994 –1001.
10. He X, Nishio K, Misono KS. High-yield affinity alkylation of the atrial
natriuretic factor receptor binding site. Bioconjug Chem. 1995;6:541–548.
11. Schulz-Knappe P, Honrath U, Forssmann WG, Sonnenberg H. Endogenous natriuretic peptides: effect on collecting duct function in rat kidney.
Am J Physiol. 1990;259:F415–F418.
12. Hanley MJ, Kokko JP. Study of chloride transport across the rabbit
cortical collecting tubule. J Clin Invest. 1978;62:39 – 44.
13. Moe OW, Berry CA, Rector FC. Renal transport of glucose, amino acid,
sodium, chloride, and water. In: Brenner BM, ed. The Kidney. 6th ed.
Philadelphia, Pa: WB Saunders Co; 2000:375– 415.
1139
14. Sonnenberg H, Honrath U, Wilson DR. In vivo microperfusion of inner
medullary collecting duct in rats: effect of amiloride and ANF. Am J
Physiol. 1990;259:F222–F226.
15. Nonoguchi H, Sands JM, Knepper MA. Atrial natriuretic factor inhibits
vasopressin-stimulated osmotic water permeability in rat inner medullary
collecting duct. J Clin Invest. 1988;82:1383–1390.
16. Ritter D, Dean AD, Guan ZH, Greenwald JE. Polarized distribution of
renal natriuretic peptide receptors in normal physiology and ischemia.
Am J Physiol. 1995;269:F918 –F925.
17. Ritter D, Dean AD, Gluck SL, Greenwald JE. Natriuretic peptide
receptors A and B have different cellular distributions in rat kidney.
Kidney Int. 1995;48:5758 –5766.
18. Wada A, Tsutamoto T, Matsuda Y, Kinoshita M. Cardiorenal and neurohumoral effects of endogenous atrial natriuretic peptide in dogs with
severe congestive heart failure using a specific antagonist for guanylate
cyclase– coupled receptors. Circulation. 1994;89:2232–2240.
19. Nishikimi T, Miura K, Minamino N, Takeuchi K, Takeda T. Role of
endogenous atrial natriuretic peptide on systemic and renal hemodynamics in heart failure rats. Am J Physiol. 1994;267:H182–H186.
20. Zhang PL, Mackenzie HS, Totsune K, Troy JL, Brenner BM. Renal
effects of high-dose natriuretic peptide receptor blockade in rats with
congestive heart failure. Circ Res. 1995;77:1240 –1245.
21. Veress AT, Field LJ, Steinhelper ML, Sonnenberg H. Effect of potassium
infusion on renal function in ANF-transgenic mice. Clin Invest Med.
1992;15:483– 488.
22. Honrath U, Chong CK, Wilson DR, Sonnenberg H. Dietary salt extremes
and renal function in rats: effect of atrial natriuretic factor. Clin Sci
(Colch). 1994;87:525–531.
23. Veress AT, Chong CK, Field LJ, Sonnenberg H. Blood pressure and
fluid-electrolyte balance in ANF-transgenic mice on high- and low-salt
diets. Am J Physiol. 1995;269:R186 –R192.
24. Veress AT, Honrath U, Chong CK, Sonnenberg H. Renal resistance to
ANF in salt-depleted rats is independent of sympathetic or ANG-aldosterone systems. Am J Physiol. 1997;272:F545–F550.
Atrial Natriuretic Factor Binding to Its Receptor Is Dependent on Chloride
Concentration: A Possible Feedback-Control Mechanism in Renal Salt Regulation
Kunio S. Misono
Downloaded from http://circres.ahajournals.org/ by guest on August 9, 2017
Circ Res. 2000;86:1135-1139
doi: 10.1161/01.RES.86.11.1135
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2000 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/86/11/1135
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/