Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208
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ABSTRACT |
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In vascular smooth muscle (VSM) and many other cells, G protein receptor-coupled activation of mitogen-activated protein kinases has been linked, in part, to increases in free intracellular Ca2+. Previously, we demonstrated that ionomycin-, angiotensin II-, and thrombin-induced activation of extracellular signal-regulated kinase (ERK)1/2 in VSM cells was attenuated by pretreatment with KN-93, a selective inhibitor of the multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase II). In the present study, we show that the Ca2+-dependent pathway leading to activation of ERK1/2 is preceded by nonreceptor proline-rich tyrosine kinase (PYK2) activation and epidermal growth factor (EGF) receptor tyrosine phosphorylation and is attenuated by inhibitors of src family kinases or the EGF receptor tyrosine kinase. Furthermore, we demonstrate that pretreatment with KN-93 or a CaM kinase II inhibitor peptide inhibits Ca2+-dependent PYK2 activation and EGF receptor tyrosine phosphorylation in response to ionomycin, ATP, and platelet-derived growth factor but has no effect on phorbol 12,13-dibutyrate- or EGF-induced responses. The results implicate CaM kinase II as an intermediate in the Ca2+/calmodulin-dependent activation of PYK2.
calcium; calcium/calmodulin-dependent protein kinase; proline-rich tyrosine kinase; extracellular signal-regulated kinase 1/2; epidermal growth factor receptor transactivation; src; vascular smooth muscle cells; KN-93; autoinhibitory peptide
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INTRODUCTION |
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MITOGEN-ACTIVATED PROTEIN (MAP) kinases such as extracellular signal-regulated kinase (ERK)1/2 are involved in the regulation of essential cellular processes, including gene expression and cell proliferation (5, 23). Signaling pathways involving ERK1/2 are triggered by diverse cellular stimuli, including receptor tyrosine kinase-coupled growth factors (25), G protein-coupled receptor (GPCR) agonists (8), mechanical stimuli (19), and integrin-dependent cell/matrix interactions (7). Although the signaling cascade initiated by receptor tyrosine kinase-coupled growth factors such as epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) is understood in considerable molecular detail, the signaling pathways by which GPCR ligands activate ERK1/2 are not fully understood.
A number of proximal signaling pathways, including G protein
-subunits (26), protein kinase C, and/or
Ca2+ (4), may converge on the ERK signaling
cascade. The nonreceptor tyrosine kinases, proline-rich tyrosine kinase
(PYK2) and src, have been proposed to be points of
convergence for Ca2+- and protein kinase C-dependent
pathways leading to ERK1/2 activation (6). PYK2 and
src may act via a pathway involving tyrosine phosphorylation
and activation of the EGF receptor, scaffolding of adaptor proteins and
guanine nucleotide exchange factors (SHC/GRB2/sos), and
consequent ras-dependent activation of the well-described protein kinase cascade, culminating in ERK1/2 activation (10, 12). Alternatively, PYK2 and src may lead to
activation of a protease that produces heparin/EGF from extracellular
matrix that then acts as a ligand for the EGF receptor
(20). Also, src has been proposed to directly
activate the SHC/GRB2/sos complex (12). In all
of these models, the proximal events that couple increases in free
intracellular Ca2+ or protein kinase C to
PYK2/src activation are not known.
In vascular smooth muscle (VSM), ERK1/2 activation in response to angiotensin II (ANG II), thrombin, or ATP appears to involve both Ca2+ and protein kinase C-dependent mechanisms (2, 4, 6). We previously reported that activation of ERK1/2 in cultured rat aortic VSM in response to receptor and nonreceptor Ca2+-mobilizing stimuli (ANG II, thrombin, and ionomycin) is temporally preceded by activation of the multifunctional serine/threonine kinase Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) (2). Inhibition of CaM kinase II by pretreatment with a calmodulin antagonist (calmidazolium) or a CaM kinase II inhibitor (KN-93) attenuated ERK1/2 activation in response to these stimuli. This pharmacological evidence, along with limited molecular studies, suggests that CaM kinase II, like protein kinase C, is an intermediate in GPCR-mediated activation of ERK1/2 in VSM cells. However, these studies provided no insight into the mechanisms by which CaM kinase II could activate the ERK signaling pathway.
In the present study, we used the Ca2+ ionophore ionomycin
to selectively stimulate Ca2+-dependent signaling pathways
in VSM cells independent of other GPCR pathways involving either
activated G protein -subunits or protein kinase C and determined
the effects of inhibitors of tyrosine kinases and CaM kinase II on the
activation of PYK2, the EGF receptor, and ERK1/2. The results indicate
that Ca2+-dependent activation of ERK1/2 in VSM
cells is completely dependent on PYK2/src activation and
transactivation of EGF receptor. Pretreatment with the CaM kinase II
inhibitors, KN-93 or autoinhibitory peptide (AIP), attenuates PYK2 and
EGF receptor activation in response to Ca2+-mobilizing
stimuli in VSM, supporting a role for CaM kinase II in the activation
of the nonreceptor tyrosine kinases PYK2 and src.
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EXPERIMENTAL PROCEDURES |
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Cell culture. VSM cells were dispersed from the medial layer of thoracic aortas of Sprague-Dawley rats weighing 200-300 g as described earlier (14), cultured in DMEM/F-12 medium containing 10% fetal bovine serum (Hyclone) at 37°C and 5% CO2, and split twice a week. Cells from passages 3-10 were used in the experiments. Confluent cultures were growth arrested for 16-24 h in DMEM/F-12 containing 0.4% serum. One hour before the experiments, the 0.4% serum-containing medium was removed from the cells and replaced with Hanks' balanced salt solution (HBSS) supplemented with Ca2+/Mg2+ and 10 mM HEPES, pH 7.4.
Cell lysate preparation.
VSM cells were pretreated with inhibitors for 30 min before being
exposed to stimulants for the indicated times. Reactions were stopped
by transferring the dishes to ice, removing the HBSS, and adding
ice-cold lysis buffer [in mM: 25 MOPS, pH 8.6, 1.5 EGTA, 125 NaCl, 50 NaF, 50 sodium pyrophosphate, 1 dithiothreitol, and 0.1 phenylmethylsulfonyl fluoride, with 0.5% NP-40 and 0.2 U/ml aprotinin
(1 ml/100 mm dish, 0.3 ml/60 mm dish)]. The cells were then scraped
and collected into 1.5-ml microcentrifuge tubes and stored at 20°C.
Permeabilization of smooth muscle cells. AIP, a CaM kinase II-specific inhibitor peptide (16), was introduced into the VSM cells by reversible permeabilization, as described previously (17). Briefly, cells were growth arrested for 24 h in serum-free media followed by a series of 2-min incubations in PBS to reduce their temperature to 4°C. The cells were then incubated in a permeabilization buffer (20 mmol/l HEPES, pH 7.4, 10 mmol/l EGTA, 140 mmol/l KCl, 50 µg/ml saponin, 5 mmol/l NaN3, and 5 mmol/l oxalic acid dipotassium salt) containing 10 µM CaM kinase II inhibitor peptide (myristoylated-AIP, autocamtide-2-related inhibitory peptide; Biomol Research Labs, Plymouth Meeting, PA) for 10 min on ice. The cells were then washed with PBS (4°C) three times and allowed to recover in PBS for an additional 20 min at 4°C. After incubation at room temperature in PBS for 2 min, the cells were placed in HBSS supplemented with Ca2+/Mg2+ and 10 mmol/l HEPES, pH 7.4, and placed in the incubator (37°C) for 15 min before treatment with agonists.
Immunoprecipitations and Western blotting. Cell lysates were thawed and cleared by centrifugation at 10,000 g. The supernatants were transferred to fresh microcentrifuge tubes and incubated with primary antibody for 90 min with rocking at 4°C. Washed protein A beads (25 µg; Pierce) were then added and incubated at 4°C with continued rocking overnight. The beads were recovered by centrifugation and washed three times with lysis buffer. Immunoprecipitated proteins were solubilized in 3× gel loading buffer and heated for 5 min at 95°C. The sample was cleared of beads by brief centrifugation, and the supernatant was loaded onto SDS-PAGE gels, transferred to either Nitro plus or PVDF-plus membranes (MSI). The membranes were blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) containing either 5% nonfat dry milk or 3% BSA. After being blocked, the membranes were incubated with the primary antibody for 1 h at room temperature or overnight at 4°C, washed three times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibody (1:1,500 dilution; Amersham) for 1 h at room temperature, followed by another three TBST washes. The membranes were developed using enhanced chemiluminescence substrate (Amersham) followed by exposure to ECL Hyper film (Amersham).
Antibodies.
Antibody to the phosphorylated sequence of CaM kinase II was obtained
through the services of HTI Bio-Products (Ramona, CA). Antigen peptide
was 283HRQET(P)VDCLKKF294,
corresponding to the Thr287 site in the
2-CaM kinase II subunit. The antibody was specific and reacted minimally with either unphosphorylated CaM kinase II or
other phosphorylated sequences from the kinase. Production and
specificity of the antibody for detection of
2-subunits
of CaM kinase II has been documented in previous publications
(18, 27). The polyclonal antibody specific for PYK2 and
the monoclonal antibody specific for tyrosine-phosphorylated protein
(4G10) were purchased from Upstate Biotechnology (Lake Placid, NY).
Polyclonal antibodies specific for the phosphorylated PYK2 were
purchased from Biosource International (Camarillo, CA). The polyclonal
antibody specific for the EGF receptor was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). The polyclonal antibodies for the
phosphorylated and unphosphorylated ERK1/2 were purchased from New
England Biolabs (Beverly, MA).
Materials. All tissue culture media were purchased from GIBCO BRL (Rockville, MD) unless otherwise specified. Tissue culture dishes and disposable plastic pipettes were purchased from Fisher Scientific (Pittsburgh, PA). KN-93 and KN-92 were purchased from Seikagaku America (Falmouth, MA). Ionomycin, phorbol 12,13-dibutyrate, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), AG-1478, and AG-1296 were obtained from Calbiochem (La Jolla, CA). All other chemicals were obtained from Sigma (St. Louis, MO).
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RESULTS |
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Ca2+-dependent activation of ERK1/2
is attenuated by inhibitors of CaM kinase II.
Once activated by Ca2+/calmodulin, CaM kinase II subunits
rapidly autophosphorylate on a conserved threonine residue
(Thr287 in the -subunit) (1).
Phosphorylation of Thr287 results in partial activation of
the kinase, even in the absence of Ca2+/calmodulin. Thus
either Ca2+/calmodulin-independent (autonomous) activity or
the degree of Thr287 phosphorylation can be used as an
index of CaM kinase II activation in situ. Previous work in this lab
established the in situ Ca2+ dependency for CaM kinase II
activation (by assaying generation of autonomous activity) in VSM cells
by using ionomycin, a Ca2+ ionophore, as a stimulus
(1). A selective inhibitor of CaM kinase II, KN-93,
attenuated this activation in situ with an IC50 of 14 µM
and maximal inhibition at 30 µM (2). Figure
1A shows Western blots of VSM
cell lysates using an antibody that specifically recognizes the peptide
sequence in CaM kinase II containing phosphorylated Thr287
(27). Both KN-93 and a peptide (AIP) modeled on the CaM
kinase II autoinhibitory domain, the latter introduced into the cells by reversible permeabilization, were found to inhibit
ionomycin-stimulated Thr287 autophosphorylation,
confirming their effectiveness as inhibitors of CaM kinase II
activity in the VSM cells. Ionomycin-induced activation of
ERK1/2 was blocked by both KN-93 and AIP (Fig. 1B), consistent with previous findings (2) that implicated CaM
kinase II as an intermediate in the Ca2+-dependent ERK1/2
signaling pathway. In contrast, EGF-induced ERK1/2 activation was
unaffected by pretreatment with KN-93 or AIP.
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Tyrosine kinases are intermediates in the
Ca2+-dependent activation of ERK1/2.
Activation of ERK1/2 in response to GPCR agonists depends on activation
of the nonreceptor tyrosine kinases, PYK2 and src, which
results in the tyrosine phosphorylation and activation of EGF receptors
(11, 12). To determine if the Ca2+-dependent
activation of ERK1/2 specifically required activation of src
and the EGF receptor tyrosine kinase, VSM cells were pretreated with
PP2, a src family-selective tyrosine kinase inhibitor, or AG-1478, a selective EGF receptor tyrosine kinase inhibitor. Both PP2
(Fig. 2, B and C)
and AG-1478 (Fig. 2) inhibited ERK1/2 activation in response to
addition of ionomycin. Transactivation of PDGF receptor following
stimulation with GPCR agonists has also been linked to ERK1/2
activation (15). However, pretreatment of VSM cells with
the PDGF receptor tyrosine kinase inhibitor AG-1296, which inhibited
control PDGF-induced activation of ERK1/2, had no effect on
ionomycin-induced activation of ERK1/2 (Fig. 2A). To rule
out nonspecific effects of these tyrosine kinase inhibitors on CaM
kinase II activation, the effects of AG-1478 and PP2 on CaM kinase II
autophosphorylation were determined. Neither drug inhibited the peak
activation-dependent autophosphorylation of the kinase after 30-s
stimulation with ionomycin (Fig. 3).
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Ca2+-dependent activation of the EGF
receptor is inhibited by KN-93, Ca2+ chelators, and
inhibitors of src family kinases.
Inhibition of ionomycin-stimulated ERK1/2 activation by AG-1478
implicates the EGF receptor tyrosine kinase as an intermediate in the
Ca2+-dependent signaling pathway. As shown in Fig.
4, this was confirmed by directly
documenting tyrosine phosphorylation of the EGF receptor in response to
addition of ionomycin. Tyrosine phosphorylation of the EGF receptor was
maximal 3 min after ionomycin treatment (Fig. 4A),
temporally lagging peak CaM kinase II activation at 30 s (Fig. 3)
but preceding peak ERK1/2 activation at 5 min (Fig. 2B).
KN-93 blocked ionomycin-stimulated EGF receptor tyrosine phosphorylation but had no effect on phosphorylation stimulated by EGF
binding. Activation of CaM kinase II is dependent on increases in
intracellular Ca2+. Chelation of free intracellular
Ca2+ with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), introduced into the cells by addition of the cell-permeable BAPTA-AM, prevented the ionomycin-dependent activation of CaM kinase II
(data not shown) and EGF receptor (Fig. 4B), confirming the
Ca2+ dependence of the response.
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Activation of PYK2 is dependent on CaM kinase II.
Previous studies have implicated the nonreceptor tyrosine kinase PYK2
in the Ca2+-dependent pathway leading to EGF receptor
transactivation and ERK1/2 activation. Addition of ionomycin to VSM
cells resulted in the rapid (within 1-3 min) tyrosine
phosphorylation of PYK2 (Fig.
6A) that has been previously
shown to correlate with PYK2 activation (22). Pretreatment
of the cells with the CaM kinase II inhibitors KN-93 or AIP (Fig. 6)
significantly attenuated ionomycin-induced PYK2 tyrosine
phosphorylation. Chelation of intracellular free Ca2+ with
BAPTA (Fig. 7A) also inhibited
the ionomycin-dependent activation of PYK2. The physiological stimuli
ATP and PDGF have also been shown to cause an increase in intracellular
Ca2+ as well as to activate CaM kinase II in VSM cells
(27, 28). Treatment with these agonists resulted in the
rapid activation of PYK2 in the VSM cells, responses that were partly
inhibited by pretreatment with KN-93 (Fig. 7B).
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DISCUSSION |
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Agonists for growth factor receptors and GPCR (8),
membrane depolarization (28), and mechanosensitive
mechanisms (19) are among the diverse stimuli that have
been reported to result in an activation of MAP kinases, including
ERK1/2. In the case of stimuli acting through GPCR, there appear to be
at least three general pathways leading to ERK1/2 activation; a G
protein -subunit-dependent pathway, a protein kinase C-dependent
pathway, and a Ca2+-dependent pathway. Elucidation of the
precise mechanisms involved for any one of these or the relative
importance of each for a given agonist is complicated by the fact that
the pathways may be redundant and activated in both an agonist- and
cell type-specific manner. For example, in VSM cells and
cardiac fibroblasts, ANG II-induced ERK1/2 activation occurs through
redundant protein kinase C- and Ca2+-dependent pathways.
Inhibition of both pathways is required to block ANG II-stimulated
ERK1/2 activation (4). To specifically study the
Ca2+-dependent pathways leading to ERK1/2 activation in VSM
cells, we have relied on the Ca2+ ionophore, ionomycin, to
selectively activate these pathways independent of heterotrimeric G
protein activation or protein kinase C. Results from previous studies
have implicated the multifunctional serine/threonine kinase CaM kinase
II as an intermediate in the Ca2+-dependent pathway leading
to ERK1/2 (2). In the present study, we have narrowed down
the likely site of CaM kinase II involvement to a point proximal to
activation of the nonreceptor tyrosine kinases PYK2 and src.
There is a substantial body of evidence implicating growth factor receptors, such as the EGF receptor, as intermediates in the activation of ERK1/2 following GPCR activation in a number of cell types, including VSM (9, 10, 12, 13, 15, 22). This circuitous pathway requires activation of PYK2 and src or src family kinases to initiate EGF receptor "transactivation" (10) or to stimulate a protease that releases latent EGF from the extracellular matrix (9, 13, 20, 21) with consequent EGF receptor activation leading to ras-dependent activation of the MAP kinase signaling cascade. The results shown here, indicating essentially complete inhibition of ionomycin-stimulated ERK activation with an EGF receptor tyrosine kinase inhibitor (AG-1478) or a src family tyrosine kinase inhibitor (PP2), suggest a requirement for these intermediate tyrosine kinases in the Ca2+-dependent pathways leading to ERK1/2 activation in VSM cells. However, because of potential specificity problems with the tyrosine kinase inhibitor, it is still not possible to conclude with certainty which src family tyrosine kinases are involved or whether they act proximal to, coincident with, and/or distally to PYK2 activation.
In VSM cells, transactivation of both the PDGF and EGF receptors has been implicated in ANG II-dependent activation of ERK1/2 (10, 15). However, in the present study we found that treatment with AG-1296, a selective PDGF receptor tyrosine kinase inhibitor, had no effect on ionomycin-stimulated ERK1/2. Together, these results suggest that even within the same cell type there can be differences in signaling pathways, possibly due to the source of the cells, differences in primary cell culture, or relative abundance or differential cellular compartmentalization of receptor types. It is also possible that PDGF receptor transactivation, although not Ca2+ dependent, may be activated by alternative pathways stimulated by GPCR agonists, for example involving protein kinase C-dependent activation of PYK2/src.
Because the primary approach used in this study was pharmacological, we were careful to document the intended effect of the CaM kinase II inhibitors on CaM kinase II activation and to establish their relative specificity by demonstrating a lack of effect of the inhibitors on steps downstream of CaM kinase II or on pathways independent of Ca2+/calmodulin. Two chemically and mechanistically distinct inhibitors of CaM kinase II were used in the studies with similar results. KN-93 prevents activation of CaM kinase II by interfering with calmodulin-dependent activation of the kinase (24). On the other hand, AIP is a peptide modeled on the autoinhibitory domain of CaM kinase II and appears to act as a competitive inhibitor of CaM kinase II substrates (16). Treatment of VSM cells with either drug inhibited ionomycin-stimulated CaM kinase II and ERK1/2 activation with similar potencies. Although maximal concentrations of KN-93 and AIP strongly inhibited Ca2+-dependent activation of PYK2, EGF receptor tyrosine kinase, and ERK1/2, no effects were observed on the same responses stimulated by phorbol 12,13-dibutyrate or EGF. KN-92, the inactive analog of KN-93, also had no effect on ionomycin-stimulated PYK2 activation. Conversely, the two tyrosine kinase inhibitors (AG-1478 and PP2), which effectively blocked ionomycin-stimulated ERK1/2 activation, had no effect on activation of CaM kinase II. Collectively, these controls support the conclusions that are based on the specificity of these drugs.
Recently, it was reported that membrane depolarization stimulated phosphorylation and activation of PYK2, the EGF receptor, and ERK1/2 in PC12 cells, and, on the basis of experiments that used a calmodulin antagonist (W-7) and a CaM kinase II inhibitor (KN-62), it was concluded that these responses were CaM kinase II dependent (17). Interestingly, activation of these responses by either bradykinin or ionomycin was unaffected by the CaM kinase II inhibitors. It is possible to rationalize the lack of effect of the inhibitors on bradykinin-induced responses on the basis of activation of redundant signaling pathways, for example, involving protein kinase C. However, without a direct demonstration of the efficacy of the inhibitors on activation of CaM kinase II in the PC12 cells, it is difficult to reconcile their lack of effect on ionomycin-induced EGF receptor activation. Previously, we reported that the activation of CaM kinase II in VSM cells in response to ionomycin was due to a release of Ca2+ from intracellular pools (14). Activation of CaM kinase II in PC12 cells by KCl is due primarily to influx of Ca2+ due to membrane depolarization. Thus another possible explanation for the results in PC12 cells may be a relative selectivity of the inhibitors used for CaM kinase II activated via Ca2+ influx as opposed to release of intracellular Ca2+.
Activated src has been shown to physically associate with both PYK2 (10) and EGF receptor (12). However, a number of molecular studies using active and dominant-negative PYK2 constructs have implicated PYK2 activation as a proximal step in the transactivation of the EGF receptor (3, 28). The data presented in this study support a model of Ca2+/calmodulin-dependent ERK1/2 activation that is dependent on a CaM kinase II at a step proximal to activation of PYK2 and src and, consequently, the EGF receptor tyrosine kinase (Fig. 9). Although the exact mechanisms by which CaM kinase II leads to activation of nonreceptor tyrosine kinases such as PYK2 or src family kinases remains to be determined, the model potentially explains the Ca2+/calmodulin dependency for PYK2 activation and suggests that CaM kinase II and protein kinase C may act via a common substrate or set of substrates proximal to PYK2 and src family kinases. The requirement for PYK2 and src family kinases in the GPCR-induced transactivation of the EGF receptors has recently been established using genetic (knockout) approaches (3). Importantly, these studies also established the requirement for src family kinases in the GPCR-induced activation of PYK2. This suggests that future studies aimed at elucidating the intermediate steps that couple activation of CaM kinase II, and possibly protein kinase C, to PYK2 might be directed toward proteins known to be involved in regulating src family kinases.
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ACKNOWLEDGEMENTS |
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We thank Wendy Hobb for expert assistance in the preparation of the manuscript and Dr. Kevin Pumiglia and Paul Pfleiderer for helpful discussions of the results.
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FOOTNOTES |
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This work was supported by a Trustee Scholarship from Albany Medical Center to R. Ginnan and grants to H. Singer from the National Heart, Lung, and Blood Institute (HL-49426 and HL-40992).
Address for reprint requests and other correspondence: H. A. Singer, Center for Cardiovascular Sciences, Albany Medical College (MC-8), 47 New Scotland Ave., Albany, NY 12208 (E-mail: singerh{at}mail.amc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00335.2001
Received 20 July 2001; accepted in final form 27 November 2001.
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