Departments of 1 Pharmacology and 2 Medicine and 3 Biomedical Sciences Ph.D. Program, School of Medicine, University of California at San Diego, La Jolla, California 92093-0636
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ABSTRACT |
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Cardiac fibroblasts (CFs) are
an important cellular component of myocardial responses to
injury and to hypertrophic stimuli. We studied G protein-coupled
receptors to understand how CFs integrate signals that activate
Gq,
Gs, and
Gi. We predicted that the second messenger pathways present in CFs were distinct from those in cardiac
myocytes and that unique signaling interactions existed in the CFs. ANG
II, bradykinin, ATP, and UTP stimulated inositol phosphate (IP)
production 2.2- to 7-fold. Each of these agonists elevated
intracellular Ca2+ concentration
([Ca2+]i)
via release from the intracellular
Ca2+ storage compartment.
Endothelin-1 (ET-1), carbachol, and norepinephrine failed to increase
either IP production or
[Ca2+]i.
Although agonists that activated IP and
Ca2+ transients had no effect on
cAMP production when administered alone, these agents potentiated the
2-adrenergic response two- to
fourfold. Hormones known to inhibit adenylyl cyclase activity in
cardiac myocytes, such as ET-1 and carbachol, failed to lower the
-adrenergic response in fibroblasts. Order of potency and inhibitor
data indicate that the functional receptor subtypes in these cells are
2,
P2Y2, and
AT1 for isoproterenol, ATP, and ANG II, respectively. We conclude that CFs express functional G
protein-linked receptors that couple to
Gq and
Gs, with little or no coupling to
Gi. The expression of receptors
and their coupling to Gq- but not
to Gi-linked responses
distinguishes the signaling in CFs from that in myocytes. Furthermore,
agonists that activate Gq in CFs
potentiate stimulation of Gs, an
example of signaling cross talk not observed in adult myocytes. These
data suggest that G protein-mediated signaling in CFs is unique and may
contribute to the specificity of hormone and drug action on individual
cell types within the heart.
cyclic adenosine 3',5'-cyclic monophosphate; inositol phosphates; intracellular calcium; potentiation; receptor signaling
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INTRODUCTION |
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FIBROBLASTS COMPRISE AS much as two-thirds of cell number in cardiac tissue (9) and play an active role in wound healing, hypertrophy, and fibrosis in the heart. All of these processes are thought to be regulated by hormones and paracrine factors in the adult myocardium. Studies in cardiac fibroblasts (CFs) have focused on gene regulation and secretion of various extracellular matrix (ECM) proteins, most notably collagen, and on the hormones that regulate these ECM proteins (reviewed in Ref. 42). Few reports to date have thoroughly examined G protein-coupled second messenger systems in CFs. Direct ligand binding studies and functional studies indicate the presence of receptors for endothelin (ET-1) and ANG II (16, 23, 42). ANG II promotes proliferation and collagen deposition in cultured CFs (3, 8). Fibroblasts also produce ANG II and other putative growth factors that may act as autocrine and paracrine modulators of cell function and growth within the heart (9, 25, 28). Understanding which hormones and signaling pathways control the proliferative and synthetic capacities of the CFs is necessary to assess their contribution to cardiac remodeling, fibrosis, and hypertrophy.
This laboratory has previously studied cardiac signal transduction
using whole ventricles excised from isolated perfused adult rat hearts
as well as isolated ventricular myocytes (5, 6, 20, 31). Many
differences in transmembrane signaling events have been observed in
whole ventricles vs. isolated ventricular myocytes, indicating that
nonmyocytes play a significant role in the hormonal responsiveness of
intact cardiac tissue. For instance, adult rat ventricular myocytes
have relatively few 2 receptors (
1 receptors predominate),
whereas the intact ventricle has an abundance of
2 receptors (6, 26).
Additionally, elevation of inositol phosphates (IPs) is negligible in
isolated adult rat ventricular myocytes following exposure to ANG II
(20), whereas the intact ventricle has a prominent response to ANG II
(22). These studies suggest that components of signal-transducing
pathways are not uniformly distributed among the different cell types
in the heart. The cell-cell communication that exists between myocytes and nonmyocytes is physiologically important, since these cells interact via paracrine mechanisms to produce the hormonal responses characteristic of intact cardiac tissue. For example, conditioned medium from nonmyocyte cultures (the majority of which were CFs) induced neonatal cardiac myocyte hypertrophy in vitro (28). In a
similar study, conditioned medium from neonatal CFs treated with ANG II
stimulated hypertrophic growth of neonatal myocytes (25). Mechanical
and other cell-cell interactions may also exist among cells of the
myocardium (fibroblasts, myocytes, endothelial cells, and smooth muscle
cells), adding to the complexity of cellular communication and
signaling in the heart. Identifying signaling components in each
cardiac cell type is essential to understanding the basis of hormonal
effects on cardiac function.
In this study, we examine G protein-mediated second messenger
production in the major nonmyocyte cell, the CF. We hypothesized that
the G protein-coupled second messenger pathways present in CFs were
distinct from those known to exist in cardiac myocytes. The data
support the existence of unique G protein-coupled receptors in CFs:
strong coupling to Gq [ANG
II, bradykinin (BK), and purinergic agonists] and
Gs (isoproterenol and
epinephrine), with little or no functional coupling to
Gi (carbachol, ET-1, and
norepinephrine). We also observed a potentiative interaction between
the Gq- and Gs-linked signaling in which
activation of Gq-phospholipase C (PLC)-IP signaling enhanced the -adrenergic response to isoproterenol.
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MATERIALS AND METHODS |
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Preparation and culture of adult rat CFs. Fibroblasts were prepared by the methods of Eghbali et al. (11) and Villarreal et al. (43). Briefly, the ventricles of three to five hearts from adult male 300- to 350-g Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were minced, pooled, and placed in a collagenase-pancreatin digestion solution. After four digestions, cells were pooled and debris and myocytes were removed by unit gravity sedimentation. Fibroblasts were isolated from enriched fractions on Percoll and suspended in DMEM (GIBCO BRL, Grand Island, NY) supplemented with penicillin, streptomycin, fungizone, and 10% fetal bovine serum (FBS; Gemini Bio-Products, Calabasas, CA). After a 30-min period of attachment to uncoated culture plates, cells that were weakly attached or unattached were rinsed free and discarded. After 48-72 h, confluent cultures were passaged by trypsinization. For signaling assays, only early passage (<5) cells were used. Cells were seeded onto 35-mm plates (0.5-1.0 × 105 cells/plate) or 60-mm plates (1.5-2.0 × 105 cells/plate) and grown to 80-90% confluency (3-4 days). The purity of these cultures was >95% CFs as measured by vimentin and collagen (types I and III) expression as previously described (43, 44).
Phosphoinositide hydrolysis. Cells in 35-mm dishes were washed with DMEM and labeled for 18 h with myo-[3H]inositol (5 µCi/ml; Amersham, Arlington Heights, IL) in DMEM without serum. Free [3H]inositol was removed by rinsing, after which cells were equilibrated with DMEM containing 10 mM LiCl, 10 mg/ml leupeptin, and 1 mg/ml BSA. After 10 min, agonists were added, and incubation continued at 37°C for 15 min. Incubations (performed in duplicate) were terminated by aspiration of medium and addition of cold 5% TCA, from which [3H]IPs were purified and quantified as previously described (20). Total [3H]IPs were counted by liquid scintillation spectrometry, and data were expressed as counts per minute (cpm) per plate or cpm per milligram protein.
Intracellular
Ca2+
measurements.
CFs were grown on 22-mm glass coverslips, grown in serum-free medium
overnight, and then loaded with 1 µM indo 1-AM (Calbiochem, La Jolla,
CA) in HEPES-buffered saline (HBS; in mM: 130 NaCl, 5 KCl, 10 glucose,
1 MgCl2, 1.0 CaCl2, and 25 HEPES, pH 7.4 at 37°C). Briefly, the cells were washed twice in HBS and incubated in
2 ml HBS containing 1 µM indo 1-AM at 37°C for 30 min. Cells were
washed after the loading period and placed in a 37°C chamber containing HBS, where groups of five to eight cells were viewed using
an inverted Nikon Diaphot microscope. Fluorometric measurements were
collected using the DX-1000 system (Solamere Technology, Salt Lake
City, UT), in which the field was excited at 385 nm and the emission
ratio (R) was collected at 405 and 495 nm, as previously described
(29). Nanomolar Ca2+ values were
calculated using the formula
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Assay of cAMP accumulation. Cells in 60-mm dishes were incubated with DMEM without serum for 2 h and then equilibrated with DMEM containing 0.1 mM IBMX. After 15 min, agonist or diluent was added, and the incubations continued at 37°C for 10 min. Incubations were terminated by aspiration of the medium and the addition of cold 5% TCA to the adherent cells. TCA extracts were collected, purified, and assayed for cAMP as previously described (20). Data, corrected for recovery, are expressed as picomoles of cAMP per dish or per milligram protein.
Assay of cGMP accumulation. Experiments to assess cGMP were similar to those for cAMP. Briefly, fibroblasts were pretreated with 0.5 mM IBMX for 15 min before 6-min incubations with agonists; 5% TCA supernatants were extracted four times with water-saturated ether, and the cGMP content was determined by RIA using the method of Harper and Brooker (17). Data are expressed as picomoles of cGMP per dish.
Estimation of protein content. When necessary, acid-precipitable material was suspended in 0.4 N NaOH and the protein content was estimated by the method of Bradford (4).
Analysis of data. Statistical comparisons (t-tests and single-factor ANOVA) were performed with the program InStat, and curve fitting was performed with the program GraphPad Prism 2.0 (GraphPad Software, San Diego, CA)
Materials. Unless otherwise noted, all chemicals were reagent grade and were purchased from Sigma (St. Louis, MO).
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RESULTS |
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IP production.
We tested a variety of drugs and hormones for their capacity to
stimulate IP production (Fig.
1). Hormones that we expected to enhance IP production, on the basis of previous studies using intact
ventricles, failed to do so: muscarinic cholinergic agonists (carbachol) and -adrenergic agonists (norepinephrine and
phenylephrine) were without effect. These results suggest an absence of
functional (G protein-coupled) muscarinic and
1-adrenergic receptors on the
cells. Of the agents that did stimulate IP production, ATP, UTP, and
ANG II were the most efficacious, producing increases of three- to
sevenfold, and BK doubled IP production. Each of these effective
agonists produced a statistically significant increase over control
[3H]phosphoinositide
hydrolysis (P < 0.05). Surprisingly,
ET-1 elicited a small and statistically insignificant increase
(~30%) in IP production over basal.
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Intracellular
Ca2+
measurements.
To confirm the existence of a hormone-sensitive
Ca2+ release system in adult CFs,
we employed the Ca2+-sensitive dye
indo 1-AM to make fluorometric measurements in multiple fields of five
to eight adherent cells. We first sought to extend the data from the IP
assays to demonstrate a measurable increase in
[Ca2+]i
following administration of those agonists that were effective agonists
of IP accumulation (see Fig. 1). Indo 1 emission ratios (405 nm/495 nm)
were collected in the presence (Fig. 3) and
absence (Fig. 4) of extracellular
Ca2+ to determine whether the
source of the Ca2+ increase was
extracellular or internal stores. The actual
[Ca2+]i
values were calculated and are summarized in Table
1; they range from 81 to 1,260 nM (1 mM
extracellular Ca2+) and from 47 to 1,197 nM (0 extracellular
Ca2+). The agonists that were
most effective in increasing IP production (ANG II, UTP, ATP, and BK)
also elevated
[Ca2+]i.
Hormones that elicited little or no significant stimulation of IP
production, such as carbachol and ET-1, failed to increase [Ca2+]i
(data not shown). We conclude that the agonist-induced
Ca2+ transients were evident only
in response to agents most efficacious in producing IPs. Furthermore,
experiments performed in the absence of extracellular
Ca2+ (by chelation with EGTA; Fig.
4) indicated that the Ca2+
transients were mainly due to release from internal stores, since they
were similar in magnitude to those performed in 1 mM extracellular Ca2+. Taken together, the IP and
Ca2+ data strongly suggest that
the IP3 receptor plays a major
role in Ca2+ mobilization and
signaling in CFs.
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cAMP production.
We tested the agonists used in the IP studies to assess their capacity
to influence cAMP metabolism. These experiments were conducted in the
presence of a phosphodiesterase (PDE) inhibitor, 0.1 mM IBMX, so that
changes in cellular cAMP content could be ascribed to enhanced
activation of Gs-adenylyl
cyclase and not to altered PDE activity. Under these conditions, the
basal content of cAMP in control cells was 20 pmol/mg protein. The
agonist isoproterenol stimulated a large increase in cAMP accumulation, to 500 pmol/mg protein. This robust
-adrenergic response indicates that CFs possess a complete and functional
receptor-Gs-adenylyl cyclase
pathway. Order of potency data (epinephrine
norepinephrine; Fig.
5) indicate that the
receptor in these
cells is of the
2-subtype.
PGE2 modestly stimulated cAMP
accumulation (fourfold over basal) indicating the presence of a PG
receptor linked to Gs. No agents
that stimulated IP production increased cAMP levels when added alone.
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Synergistic interaction of Gq-linked IP
pathway with Gs-cyclase pathway.
Frequently, agents that stimulate IP production via
Gq-linked pathways also act at
receptors linked to Gi to inhibit
adenylyl cyclase. Thus we tested the effects of agonists that act
through the Gq signaling pathway
on isoproterenol-induced cAMP production. None of the agonists produced
the anticipated reduction of the -adrenergic response. Rather,
hormones that were strong stimulators of IP production acted
synergistically with isoproterenol to potentiate cAMP production in CFs
(Fig. 6). These agonists included ANG II, BK, UTP, and ATP, none of
which increased cAMP when added alone (see above). The synergism was
observed both in the absence and presence of IBMX, suggesting that
alterations in PDE activity are not involved in the cross talk (data
not shown). ET, which had a statistically insignificant effect on IP
production, did not potentiate the
-adrenergic response.
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Production of cGMP.
In the presence of 0.5 mM IBMX, basal cGMP content of cultured CFs is
0.7-1.0 pmol/60-mm dish. Nitroprusside and atrial natriuretic factor increased cGMP content 3.8- to 6-fold, indicating that the cells
contain both the soluble and membrane-bound forms of guanylyl cyclase.
The presence of a nitroprusside-sensitive guanylyl cyclase suggested
that hormones that stimulate IP production and elevate
[Ca2+]i
(Figs. 1-4) would elevate cellular cGMP via
Ca2+/calmodulin stimulation of
soluble NO synthase, with the resultant NO stimulating the soluble
guanylyl cyclase. Surprisingly, no agonists that stimulated IP
production or elevated
[Ca2+]i
altered cellular cGMP content (Fig. 8). The
addition of the Ca2+ ionophore
A-23187 (10 µM) likewise caused no accumulation of cGMP. Thus it
appears that soluble NO synthase is absent from CFs or is inactive; as
a result, the hormones that elevate
[Ca2+]i
do not increase cGMP levels in CFs.
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DISCUSSION |
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In this report, we show evidence that a number of functional receptors
in the CFs, including -adrenergic, ANG II, BK, and purinergic
P2Y2 receptors, couple to G
protein-linked signaling pathways in a pattern that is distinct from
that of cardiac myocytes. All of these receptors, except the
2 receptor, couple to stimulate phosphoinositide hydrolysis via Gq
and PLC; sufficient IP accumulation causes mobilization of
intracellular Ca2+. A number of
these putative Gq-linked receptors
can be subtyped on the basis of our data. The functional ANG II
receptor of the rat CFs is the AT1
receptor, in agreement with the binding data of Villarreal et al. (43)
and other investigators (9, 12). The identification of this receptor is
based on the effectiveness and appropriate affinity of the
AT1 antagonist losartan.
Villarreal et al. (43) found that stimulation of this receptor
increases expression of collagen and fibronectin mRNAs and increases
collagen production. Activation of AT receptors appears to influence or cause proliferative changes associated with cardiac hypertrophy and
failure in the rat model (9, 14, 27, 36). Stimulation of AT receptors
in cardiac myocytes causes hypertrophy (27), increased
mitogen-activated protein (MAP) kinase activity (14, 36), p21 Ras
activation (33), and activation of signal transducers and activators of
transcription (2). The transmembrane signaling that we describe for AT
in the CF distinguishes the AT receptor of the fibroblasts from that of
the cardiac myocyte, since ANG II does not activate significant
phosphoinositide hydrolysis in freshly isolated adult rat ventricular
myocytes (20). Other cardiac cell types express AT receptors as well
(32, 39) but do not comprise a major fraction of the cardiac mass. From
our data and those of the studies discussed above, we conclude that CFs
likely represent a major site of ANG II action in the heart, an action
mediated at least in part by the
Gq-PLC-IP pathway.
We also tested two other peptides that have been associated with
proliferative changes in the myocardium, ET-1 and BK. ET-1 causes
increased CF proliferation and stimulates production of ECM proteins
(15, 16), whereas BK appears to inhibit ECM production (24). Both of
these peptide hormones normally couple to
Gq-PLC-linked signaling pathways.
BK modestly stimulated IP production in the current study; the effect
of ET-1 did not reach statistical significance. This small effect of
ET-1 did not result in the mobilization of Ca2+ as measured by indo 1 fluorescence. This may be due to the low level of IP production in
response to ET-1; even in the presence of 10 mM LiCl we observed only a
30% increase over basal. One other study demonstrated that ET-1
modestly increased
[Ca2+]i
in CFs isolated from Wistar-Kyoto rats (41), a discrepancy that we
attribute to differences in rat strains and/or differences in cell
isolation techniques. In our case, ET-1-induced IP production in CFs
appears insufficient to elicit a measurable
Ca2+ transient. Consistent with
the lack of Ca2+ response is the
failure of ET to potentiate the -adrenergic response (see below).
ET receptor coupling in the fibroblasts differs from that in the myocyte. Our data indicate negligible coupling of fibroblast ET receptors to Gq-PLC and no coupling to Gi-adenylyl cyclase. One binding study found roughly equal expression of ETA and ETB subtypes in adult rat CFs (13); another found predominantly ETB receptors (23). It is surprising that cells with significant numbers of ETA receptors (~10,000 per cell, from Ref. 13) do not show the expected coupling of the ETA receptor to Gi-linked inhibition of adenylyl cyclase. This distinguishes the fibroblast ET receptor and its coupling from that of the adult cardiac myocyte, in which we find a single population of ETA receptors that couples strongly to both Gq and Gi (19, 20).
CFs express functional receptors for the major transmitters released
from adrenergic storage vesicles, catecholamines and ATP. The
catecholamine receptors of CFs couple to stimulate cAMP predominantly
through the 2 receptor subtype,
on the basis of the order of potency data. The predominance of
2 receptors in CFs is
consistent with earlier observations that these receptors exist in
membranes prepared from whole heart and ventricles but appear to be
only a small proportion (~15%) of
receptors in purified cardiac
myocyte preparations (6, 26). Additionally, it has been observed that
2 receptors may couple to
Gi (in addition to
Gs) in isolated cardiac
myocytes, which may suggest that these receptors have differential
effects on cAMP production (45). Judging by the lack of detectable
coupling through Gi to inhibit adenylyl cyclase and through Gq to
stimulate PLC and IP production, we conclude that CFs lack functional
1- and
2-adrenergic receptors. The
purinergic receptors on CFs couple to IP production with an order of
potency indicative of a P2Y2
receptor. Molecular evidence for the existence of this receptor subtype
along with the P2Y1 subtype has
been shown in neonatal rat CFs using Northern analysis (46).
When we stimulated the 2 and
P2Y2 receptors of CFs
simultaneously, the result was a potentiation of cAMP production over that of
2 stimulation alone,
suggesting a positive interaction between the IP signaling pathway and
the
2-Gs-adenylyl
cyclase pathway. This cross talk appears to be unidirectional, since IP production by Gq-coupled receptors
was not enhanced by treatment with
agonists. The potentiation of
cAMP accumulation by activated phosphoinositide hydrolysis was not
restricted to the interaction of purinergic and
2 agonists. ANG II and BK also
caused a synergistic accumulation of cAMP; the interaction extends to
other stimulators of adenylyl cyclase
(PGE2 and forskolin) as well. A
number of mechanisms are possible. Bell et al. (1) first noted that a stimulation of protein kinase C (PKC) by phorbol esters led to an
enhancement of adenylyl cyclase activity in S49 lymphoma cells, a
finding that may reflect the capacity of types II and V adenylyl cyclase to function as a substrate for PKC (38). Houslay (21) reviewed
the possible targets for regulation of cAMP metabolism by PKC,
including reduction of inhibitory tone by
Gi
2 and the modulation of
activities of PDE isoforms. Houslay also stressed the role of PKC in
the interaction of signal transduction pathways through phospholipases
and eicosanoid production, on the activity of the tyrosine kinase
activities of growth factor receptors, and on the regulation of ion
channels. Mobilization of Ca2+
could stimulate
Ca2+/calmodulin-sensitive adenylyl
cyclase (40). Recent work indicates that cross talk between
Gq and
Gs signaling pathways may also involve
-subunits released when
Gq is activated, with either direct action on adenylyl cyclase or actions mediated by other effectors such as growth factors, receptor tyrosine kinases, and MAP
kinases (37). The precise mechanisms by which cross talk occurs between
Gq and
Gs in CFs are not known and
require further study, as do the long-term or
"downstream" consequences of
Gq/Gs cross talk. We have preliminary evidence that the interaction of the
Gq-PLC with
Gs-cAMP production produces
significant effects on immediate early gene activation: treatment of
CFs with ANG II in combination with isoproterenol reduces c-Jun mRNA
expression compared with that of ANG II alone (Endo-Mochizuki and
Brunton, unpublished observations). These responses to ANG II may
involve not only the Gq pathway
but also a number of
Ca2+-dependent steps, including
activation of PKC, tyrosine kinases, MAP kinases, and S6 kinases (34).
The
-adrenergic responses may involve multiple effects of protein
kinase A and interactions of stimulatory and inhibitory transcriptional
control factors with the cAMP response elements of target genes.
In summary, this study demonstrates that adult rat CFs contain G
protein-coupled signaling systems that are distinct from those of adult
rat cardiac myocytes. A comparison of functional G protein-coupled
signaling in adult rat CFs and myocytes is shown in Table
2. CFs have receptors for the peptide
growth factors, ANG II and BK, that link to
Gq-PLC. These cells also respond
to the two major components of the adrenergic storage vesicle,
norepinephrine and ATP, with 2
receptors linking to adenylyl cyclase via
Gs and
P2Y2 receptors linking to IP
production and Ca2+ mobilization.
Surprisingly, we found no evidence of muscarinic receptor coupling to
Gi or
Gq, linkages that are prominent in cardiac myocytes. ET does not appear to couple to
Gq or
Gi in fibroblasts, also in
contrast to the ETA receptor
coupling observed in myocytes. Last, we describe an intriguing
Gq/Gs
cross talk phenomenon not yet described in myocardial cells, which may
be important in specific signaling events controlling the function of
CFs within the myocardium.
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ACKNOWLEDGEMENTS |
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J. G. Meszaros and A. M. Gonzalez contributed equally to this report.
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FOOTNOTES |
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We thank Dr. Paul A. Insel and Joan R. Kanter for critical review of the manuscript and helpful discussions.
This research was supported by National Institutes of Health Grants HL-41307, HL-09887, HL-07444, and GM-07752 and American Heart Association Grant 91015560.
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: J. G. Meszaros, Dept. of Pharmacology 0636, UCSD School of Medicine, La Jolla, CA 92093-0636 (E-mail: jmeszaros{at}ucsd.edu).
Received 24 May 1999; accepted in final form 27 August 1999.
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