© 2000 American Heart Association, Inc.
Cellular Biology |
Signaling Properties and Functions of Two Distinct Cardiomyocyte Protease-Activated Receptors
From the Departments of Pharmacology (A.S., G.M., H.Z., E.P., S.F.S.) and Medicine (S.F.S.), Columbia University, New York, NY, and the R.W. Johnson Pharmaceutical Research Institute (A.D., P.A.-G.), Spring House, Pa.
Correspondence to Susan F. Steinberg, MD, Associate Professor of Pharmacology and Medicine, Department of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 W 168 St, New York, NY 10032. E-mail sfs1{at}columbia.edu
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Abstract |
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Abstract—Previous studies have established that cardiomyocytes express protease-activated receptor (PAR)-1, a high-affinity receptor for thrombin, which is also activated by the tethered-ligand domain sequence (SFLLRN) and which promotes inositol trisphosphate accumulation, stimulates extracellular signal–regulated protein kinase, and modulates contractile function. A single previous report identified PAR-1 as a hypertrophic stimulus, but there have been no subsequent investigations of the mechanism. This study reveals the coexpression of PAR-1 and PAR-2 (a second PAR, which is activated by trypsin/tryptase but not thrombin) by Northern blot analysis and compares their signaling properties in neonatal rat ventricular cardiomyocytes. SFLLRN and SLIGRL (an agonist peptide for PAR-2) promote inositol trisphosphate accumulation, stimulate mitogen-activated protein kinases (extracellular signal–regulated protein kinase and p38-mitogen-activated protein kinase), elevate calcium concentration, and increase spontaneous automaticity. SFLLRN (but not SLIGRL) also activates c-Jun NH2-terminal kinase and AKT. In keeping with their linkage to pathways that have been associated with growth and/or survival, SFLLRN and SLIGRL both induce hypertrophy. However, PAR agonists promote cell elongation, a morphology that is distinct from the uniform increase in cell dimension induced by
1-adrenergic receptor
activation. These studies provide novel evidence that
cardiomyocytes coexpress 2 functional PARs, which link to a
common set of signals that culminate in changes in contractile function
and hypertrophic growth. PAR actions may assume clinical importance in
the border zone surrounding an infarction, where local proteolysis of
PARs by serine proteases generated during inflammatory or thrombogenic
pathways would elevate calcium concentration (setting the stage for
arrhythmias), promote hypertrophic growth, and/or influence
cardiomyocyte survival.
Key Words: thrombin • inositol trisphosphate • mitogen-activated protein kinases • Ca2+ • hypertrophy
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Introduction |
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Thrombin is a serine protease formed at the site of vascular injury with well-characterized roles in hemostasis, inflammation, and proliferative processes. Cellular responses to thrombin are mediated by a family of G-protein–coupled protease-activated receptors (PARs), of which PAR-1 is the prototype. PAR-1 is activated on cleavage of its N-terminal exodomain by thrombin, thus exposing a new amino terminal sequence (SFLLRN) that functions as a tethered peptide ligand.1 PAR-1 is also activated, independent of proteolysis, by SFLLRN.
Additional PAR family members have been identified. PAR-2 has 30% sequence identity to PAR-1 and is detected by Northern blot analysis in several tissues, including (at low levels) the heart.2 PAR-2 is activated by limited proteolysis of its amino-terminal exodomain by trypsin (but not thrombin) or by SLIGRL, the PAR-2 tethered-ligand sequence. Cleavage by trypsin likely represents the relevant PAR-2 activation mechanism in the gastrointestinal tract and in airway epithelium (where PAR-2 potently inhibits bronchoconstriction3 ). At sites not exposed to trypsin, serine proteases generated during inflammation, fibrinolysis, or thrombosis may be more relevant. Mast cell tryptase readily cleaves cell surface PAR-2 (but not PAR-1) and may be a pathophysiologically important activator of PAR-2 in the vasculature, where PAR-2 induces endothelium-dependent relaxation of arterial rings and mitogenesis.1 2 Mast cell infiltration and elevated tissue tryptase levels also are characteristic of certain cardiac syndromes,4 5 but a potential role for PAR-2 in the heart has never been considered. PAR-3 and PAR-4 are more recently identified PAR family members. PAR-3 and PAR-4 have been studied exclusively in the context of platelet aggregation; potential functions in other tissues remain completely unexplored.
Studies during the past several years have identified cardiomyocytes as targets for the action of thrombin. In rat ventricular myocytes, thrombin stimulates phosphoinositide hydrolysis, activates the extracellular signal–regulated protein kinase (ERK), induces atrial natriuretic factor expression, modulates calcium homeostasis, increases automaticity, and hastens recovery from an imposed acid load by activating Na+-H+ exchange.6 7 8 9 Collectively, these (and likely other signaling events) profoundly alter electrophysiological properties and contractile behavior and induce cardiomyocyte hypertrophy. On the basis of the identification of PAR-1 mRNA and responses to SFLLRN, PAR-1 has been implicated in the actions of thrombin.7 8 9 However, SFLLRN typically elicits biochemical responses that are more robust than those elicited by thrombin.7 This could result from the actions of SFLLRN at both PAR-1 and PAR-2, because SFLLRN activates both, whereas thrombin is selective for PAR-1.10 Accordingly, the present study uses SLIGRL (a synthetic PAR-2 hexapeptide agonist, which activates PAR-2 but not PAR-110 ) to test the hypothesis that incremental responses to SFLLRN over thrombin are due to the copresence of PAR-1 and PAR-2. The results provide novel insights into the signaling and growth-regulatory properties of individual PARs in cardiomyocytes.
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Materials and Methods |
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Neonatal rat ventricular myocyte (NRVM) cultures were prepared according to methods described previously.7 For biochemical assays and measurements of calcium, myocytes were plated at a density of 3x104 cells/cm2 and cultured in DMEM supplemented with 10% FCS. This was followed by starvation in serum-free 1:1 DMEM/F-12 medium for 24 hours before assays on day 5 of culture. Cells were plated at 1.5x104 cells/cm2 for measurements of growth responses. Preliminary studies established that biochemical and calcium responses are not altered in a qualitative manner by differences in plating density. Cardiac fibroblast cultures were obtained from cells adherent to culture dishes during preplating.11
Northern blot hybridization was carried out with poly(A+)-enriched RNA and a fragment encompassing nucleotides 1 to 2123 of the human PAR-1 coding sequence12 or polymerase chain reaction products containing nucleotides 226 to 657 or 145 to 1364 of the murine PAR-2 or the human PAR-3 coding sequence.13 14 Probes were 32P-labeled by using the RadPrime labeling system (Life Technologies); hybridizations were carried out in Rapid-hyb buffer (Amersham) for 3 hours at 62°C. Wash stringency was 2x SSC and 0.1% SDS 2 times at 62°C, followed by 0.2x SSC and 0.1% SDS 2 times at 62°C.
Methods for the measurement of inositol phosphate (IP) accumulation and calcium in fura 2–loaded cultured NRVMs are published.7 Assessments of ERK and c-Jun NH2-terminal kinase (JNK) activities were by "in-gel" kinase assays with the use of 0.5 mg/mL myelin basic protein or glutathione S-transferase (GST)-c-Jun (purified by glutathione-Sepharose chromatography from Escherichia coli driven to express recombinant GST-c-Jun; plasmid was provided by Dr Peter Sugden, Imperial College School of Medicine, London, UK) as substrate, as described previously.7 Activation of p38-mitogen-activated protein kinase (p38-MAPK) was detected by immunoblot analysis with an antibody selective for the dually phosphorylated (activated) enzyme, according to the manufacturer’s instructions (New England Biolabs). Immunocomplex kinase assays for AKT were performed with histone H2B as substrate15 ; equal protein loading was validated by reprobing blots with anti-polyclonal AKT C-terminal.
Cell surface area was measured by digitized image analysis.
After exposure to agonist for 72 hours, 8 to 10 frames per dish were
captured at x40 magnification; 30 to 50 cells were analyzed
per treatment. For measurements of protein content, triplicate dishes
were stimulated in serum-free medium with agonists (or vehicle) for 48
hours at 37°C. The medium was supplemented with
[14C]phenylalanine (0.1 µCi/mL) plus 0.3
mmol/L nonradioactive phenylalanine during the final 24 hours of
stimulation. Cells were rinsed with PBS and incubated in ice-cold 10%
trichloroacetic acid for 30 minutes. Precipitates were washed twice
with ice-cold 10% trichloroacetic acid and solubilized in 1% SDS at
37°C for 1 hour. Duplicate aliquots from each sample were assayed for
radioactivity, protein, and DNA content. One hundred to 150 cells per
treatment group were scored (at x63 magnification) for sarcomeric
organization after incubation for 72 hours with the indicated agonists
and immunostaining with monoclonal anti–-sarcomeric
actinin.
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Results |
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Cardiomyocytes Coexpress PAR-1 and PAR-2
Figure 1
shows that a characteristic
size transcript for PAR-1 (3.5 kb) is readily detected in
cardiomyocytes and fibroblasts cultured from the neonatal
ventricle. The 3.5-kb PAR-1 band is also detected in the adult rat
ventricle, where it must be resolved by short exposure times from a
heterogeneous signal spanning from 
1 to 5 kb (identical
results were obtained with multiple RNA preparations and 3 different
PAR-1 random-primed cDNA probes as well as a uniformly labeled cRNA
probe). The diffuse nature of much of the PAR-1 signal is not the
result of a bulk mobility artifact, because PAR-2 and ß-actin are
detected as discrete bands in this sample. PAR-2 transcripts are
detected as a 3.0-kb band in adult ventricle and NRVMs; PAR-2
transcripts are at the limits of detection in cardiac fibroblasts.
PAR-3 is abundant in megakaryocytes (included as controls), but it is
not detected in cardiomyocytes or fibroblasts.
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PAR-1 and PAR-2 Stimulate Phosphoinositide Hydrolysis
Consistent with previous studies, thrombin and SFLLRN
induce rapid and transient increases in inositol trisphosphate
(IP3) and inositol bisphosphate
(IP2), which are followed by a more sustained
accumulation of inositol monophosphate (IP1).
IP1 accumulation in response to SFLLRN at 30
minutes is 2.3±0.2-fold greater than the response to thrombin
(P<0.05, n=6; Figure 2A). To
determine whether the incremental stimulation of
phosphoinositide hydrolysis by SFLLRN might be due to
its combined actions at PAR-1 and PAR-2, responses to trypsin and
SLIGRL were examined in parallel. Trypsin and SLIGRL promote the
sequential accumulation of IP3,
IP2, and IP1. Responses to
SFLLRN and SLIGRL display similar kinetics and
EC50 values (50 µmol/L; Figure 3B and Reference 7 ).
However, responses to SFLLRN are greater in magnitude that those
elicited by SLIGRL. FLLRN (500 µmol/L), a control peptide
lacking the N-terminal serine (does not activate PAR-1), does
not promote IP accumulation at 2 or 30 minutes (data not shown). This
result establishes that the stimulatory actions of SFLLRN and SLIGRL
are specific. Figure 2B demonstrates that SFLLRN
activates phospholipase C in cardiac fibroblasts but that
SLIGRL does not. Agonist peptide actions are consistent with
the Northern analysis of mRNAs from cardiac fibroblasts (Figure 1
) and indicate that SLIGRL-induced biochemical responses cannot
be attributed to robust PAR-2 signaling by a minor contaminating
fibroblast population in cardiomyocyte cultures.
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PAR Activation of MAPKs
The next experiments examined whether SLIGRL stimulates MAPKs. In
most experiments, ERK, JNK, and p38-MAPK activities were assayed in
parallel, on material from the identical culture preparation. This
permitted a rigorous analysis of the balance of ERK versus JNK
versus p38-MAPK activation by individual PARs, in line with our goal to
identify potential quantitative/qualitative differences in the
intensity of PAR signaling through individual MAPK cascades, which
might translate into distinct functional consequences.
Figure 3A shows that SLIGRL induces transient ERK activation;
both 42- and 44-kDa species of ERK are detectably activated by
2 minutes and are maximally activated by 5 minutes. The rapid
and transient kinetics of this response are similar to the kinetics
previously described for SFLLRN.7 ERK activation by SLIGRL
is concentration dependent (EC50 50
µmol/L). The concentration-response relationships for
SLIGRL-dependent activation of ERK and IP accumulation are similar
(Figure 3B). The magnitude of ERK activation by SFLLRN is
greater than the responses to equimolar SLIGRL or thrombin (Figure 3C). FLLRN (500 µmol/L for 5 minutes) does not
activate ERK, establishing the specificity of SFLLRN and SLIGRL
responses (data not shown).
To determine whether PARs activate JNK, lysates from
cardiomyocytes stimulated with PAR agonists for
variable intervals (2 to 30 minutes) were subjected to "in-gel"
kinase assays with GST-c-Jun as substrate (with sorbitol, which induces
a 10-fold increase in JNK activity, as control). Figure 4A shows that JNK is activated by
thrombin and SFLLRN (PAR-1). These responses display kinetics that are
rapid (peak at 5 to 10 minutes, data not shown) and contrast with the
more gradual and sustained activation of JNK by other
G-protein–coupled receptors (GPCRs) in cardiomyocytes. The
magnitude of JNK activation by thrombin and SFLLRN is similar. JNK is
not significantly activated by SLIGRL (PAR-2).
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-32P]ATP
incorporation into histone H2B substrate (0.1 mg/mL, C).
Representative experiments are illustrated on top, with
the magnitude of agonist-dependent activation quantified on the bottom
(n=3).
p38-MAPK becomes phosphorylated (activated) in
cells treated with SFLLRN, thrombin, and SLIGRL (Figure 4B); in
each case, the kinetics of p38-MAPK activation is relatively rapid
(responses detectable at 2 minutes and maximal by 5 minutes, data not
shown). p38-MAPK activation by SFLLRN tended to be somewhat greater
than activation by thrombin; both responses are much more robust than
the response to SLIGRL. Collectively, these studies identify distinct
patterns of MAPK activation by PARs. Both PARs activate ERK,
but only PAR-1 couples effectively to the activation of the
stress-activated protein kinases (JNK and p38 MAPK); activation
of stress-activated protein kinases by PAR-2 is weak (p38-MAPK)
or undetectable (JNK).
AKT Is Activated by PAR-1 but Not PAR-2
There is limited recent evidence for GPCR activation of the
phosphatidylinositol 3-kinase/AKT survival pathway in
noncardiomyocytes.15 Figure 4C shows
that AKT is activated by thrombin and SFLLRN, whereas
activation by SLIGRL is not detected. Nevertheless, these responses are
relatively modest compared with the 9-fold increase in AKT activity
induced by insulin-like growth factor (IGF)-1.
PAR-1 and PAR-2 Accelerate Automaticity and Elevate Calcium
Concentration
SFLLRN (300 µmol/L) accelerates the automatic beating rate
of cultured NRVMs (53±5%, from 45±4 to 69±8 bpm at the peak of the
response, which occurs 15 to 20 seconds after bolus administration of
peptide agonist; n=6). Parallel studies with equimolar SLIGRL establish
that PAR-2 activation also induces a positive chronotropic response
with identical kinetics. However, the magnitude of the SLIGRL-dependent
positive chronotropic response is more modest (28±4%, from 43±3 to
55±3 bpm; n=6).
To determine whether SLIGRL also influences calcium
concentration, independent of any effect on contractile rate, these
experiments were repeated in fura 2–loaded cells electrically driven
at 1 Hz (to maintain a constant beating rate during agonist exposure).
Figure 5 shows that SLIGRL mimics the
effect of SFLLRN to rapidly increase calcium (results are summarized in
Table 1). For both agonists, the
increase in calcium was dose dependent (maximum at 300
µmol/L) and was completely reversed during 3 to 4 minutes of washout.
However, the increase in calcium evoked by SFLLRN was larger than that
elicited by SLIGRL.
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PAR-1 and PAR-2 Promote Cardiomyocyte Hypertrophy
A single report by Glembotski et al8 in 1993
demonstrated that NRVMs grown in the presence of thrombin enlarge,
display highly organized sarcomeres, and express atrial
natriuretic factor. That study also reported that SFLLRN
promotes atrial natriuretic factor expression; its effects
on other indices of hypertrophy were not examined. Because
SFLLRN and SLIGRL both activate mechanisms implicated in
hypertrophic signaling, we examined their effects on various indices of
cell growth. Table 2 demonstrates
that SFLLRN and SLIGRL both induce significant increases in
[3H]phenylalanine incorporation, protein
content, cell size, and myofibrillar organization. Growth-stimulatory
effects of SFLLRN are more pronounced than those elicited by SLIGRL (by
all parameters measured). However, the morphological
features of cells treated with SFLLRN and SLIGRL are similar and quite
distinct from the morphology of cells grown in the presence of
norepinephrine (Figure 6).
1-Adrenergic receptor
(1-AR) activation by
norepinephrine leads to a uniform increase in cell
dimensions (length and width, typical of the morphological changes
observed in the setting of pressure-overload hypertrophy).
In contrast, PAR activation leads to cell elongation, with less of an
increase in cell width (Table 2). This morphologically distinct
form of hypertrophy is more characteristic of the changes
induced by the mechanical stimulus of volume overload or, in the in
vitro culture model, cardiotrophin-1 (an interleukin-6 family member
that signals through gp13017 ). Thus, the
qualitative/quantitative differences in signaling molecule activation
by individual PARs may contribute to differences in the degree of cell
enlargement, but they do not impart specificity with respect to cell
morphology. Rather, the distinct morphological phenotypes
appear to be dictated by differences in signaling pathways recruited by
PARs versus 1-ARs.
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Discussion |
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The profound cardiac actions of thrombin were first identified almost a decade ago. In the ensuing years, there has been considerable progress defining signaling pathways that contribute to the cardiomyocyte hypertrophic growth program. However, the initial observations on the actions of thrombin were not extended in the context of current concepts of cardiomyocyte hypertrophic signaling mechanisms, and the research completely neglected to consider whether cardiomyocytes also express other PARs with similar or overlapping signaling properties. The present study demonstrates that cardiomyocytes express PAR-2; PAR-2 mRNA is detected by Northern blot analysis, and the PAR-2 agonist peptide SLIGRL activates a spectrum of biochemical signaling pathways and functional responses (phospholipase C, ERK, p38-MAPK, calcium, spontaneous automaticity, and hypertrophic growth). Responses to SLIGRL report the specific actions of PAR-2; there is no evidence that SLIGRL can activate any other known PAR.10 The present study also demonstrates for the first time that PAR-1 activates JNK, p38-MAPK, and AKT in cardiomyocytes and that PARs (PAR-1 and PAR-2) promote a form of cardiomyocyte hypertrophy that is morphologically distinct from that elicited by the prototypical
1-adrenergic GPCR. The observation that prolonged stimulation of either PAR-1 or PAR-2 culminates in hypertrophic growth is consistent with the literature implicating many of the signaling mechanisms common to PAR-1 and PAR-2 in cardiomyocyte hypertrophy. For example, PAR-1 and PAR-2 both effectively activate the ERK cascade. This mechanism originally was implicated in hypertrophic signaling and more recently has been identified as a mediator of cardiomyocyte survival.16 18 PAR-1 also activates the stress-activated protein kinases, whereas PAR-2 does not increase JNK activity and stimulates p38-MAPK weakly. Because PAR-2 induces cardiomyocyte growth, these results argue that the stress-activated protein kinase pathways are not absolutely required for a growth response (although the absence of these additional signals could underlie the more modest hypertrophic growth response induced by PAR-2, relative to PAR-1).
PAR-1 and PAR-2 both couple to increases in spontaneous automaticity
and elevations in calcium. These responses would tend to predispose to
arrhythmias, but they also are predicted to promote
hypertrophic growth, at least in part through mediators such as the
B isoform of calmodulin kinase
II19 and the calcium/calmodulin-dependent
phosphoprotein phosphatase, calcineurin.20 Of note, PARs
and 1-ARs promote morphologically distinct
forms of hypertrophy; these GPCRs also differ in their
ability to modulate calcium in NRVMs. PARs induce a robust increase in
calcium, whereas calcium is not detectably modulated by
1-ARs (data not shown). Although increases in
calcium that are localized or are below the limits of detection with
the use of fura 2 as indicator cannot be excluded, the failure to
detect prominent calcium responses to 1-AR
agonist activation is consistent with published
literature.21 These results suggest that calcium-dependent
signals might play a particularly prominent role in hypertrophic
signaling by PARs, relative to 1-ARs. The
relative contribution(s) of calcium versus other signaling mechanisms
(Rho/Rho kinase, Src) in the induction of morphologically distinct
forms of hypertrophy by PARs and
1-ARs requires further study.
Results reported in the present study demonstrate that AKT is activated by PAR-1 in cardiomyocytes; to our knowledge, this represents the first evidence that a GPCR activates AKT in cardiomyocytes. The significance of the relatively low level of PAR-1–dependent AKT activation (relative to IGF-1) is uncertain. Known downstream targets of AKT (that also are activated by PARs) should be the focus of future investigations. These would include S6 kinase (which has been implicated in cardiomyocyte hypertrophy22 ) and endothelial NO synthase (which has emerged as a pivotal regulator of cardiomyocyte contractile function and growth responses23 ). AKT is a critical mediator of growth factor–dependent survival in neurons; a similar process may impact on cell loss during injury/infarction in the heart.
The present study establishes that PAR-1 and PAR-2 can mediate protease-dependent alterations in cardiomyocyte function. Ultimately, the significance of these findings rides on the identification of natural activators of endogenous cardiomyocyte PARs. The actions of thrombin are predicted to be important in the setting of hemorrhagic infarction, in which the endothelial barrier is broken and myocytes come into direct contact with blood-borne substances. Although cardiomyocytes typically do not encounter trypsin, 5 to 10 homologues of trypsin have been identified in the mammalian genome; one of these trypsin-like enzymes is expressed ubiquitously, including in the heart.24 Its activity as a proteolytic agonist for PARs must be tested. Mast cell tryptase is another protease that activates PAR-2.25 Mast cells can be identified between muscle fibers in normal ventricles and (in increased density) in idiopathic and dilated cardiomyopathies.5 Mast cells degranulate and release biologically relevant concentrations of tryptase during acute insults. Thus, it is reasonable to speculate that at least some of the derangements during myocardial infarction could result from PAR-2 activation by tryptase. Cardiomyocyte PAR-1 and/or PAR-2 activation in border zone regions adjacent to areas of myocardial necrosis could constitute critical signals that influence gene expression, promote hypertrophy/apoptosis, and/or change contractile function.
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Acknowledgments |
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This study was supported by US Public Health Service, National Heart, Lung, and Blood Institute grant HL-49537. We thank Ema Stasko for preparing myocyte cultures.
Received November 24, 1999; accepted March 30, 2000.
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N. Tyagi, K. C. Sedoris, M. Steed, A. V. Ovechkin, K. S. Moshal, and S. C. Tyagi Mechanisms of homocysteine-induced oxidative stress Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2649 - H2656. [Abstract] [Full Text] [PDF]
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K. S. Moshal, N. Tyagi, B. Henderson, A. V. Ovechkin, and S. C. Tyagi Protease-activated receptor and endothelial-myocyte uncoupling in chronic heart failure Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H2770 - H2777. [Abstract] [Full Text] [PDF]
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S. Weidinger, A. Mayerhofer, L. Kunz, M. Albrecht, M. Sbornik, E. Wunn, R. Hollweck, J. Ring, and F.M. Kohn Tryptase inhibits motility of human spermatozoa mainly by activation of the mitogen-activated protein kinase pathway Hum. Reprod., February 1, 2005; 20(2): 456 - 461. [Abstract] [Full Text] [PDF]
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S. F. Steinberg The Cardiovascular Actions of Protease-Activated Receptors Mol. Pharmacol., January 1, 2005; 67(1): 2 - 11. [Abstract] [Full Text] [PDF]
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C. S. Moreno, S. Ramachandran, D. G. Ashby, N. Laycock, C. A. Plattner, W. Chen, W. C. Hahn, and D. C. Pallas Signaling and Transcriptional Changes Critical for Transformation of Human Cells by Simian Virus 40 Small Tumor Antigen or Protein Phosphatase 2A B56{gamma} Knockdown Cancer Res., October 1, 2004; 64(19): 6978 - 6988. [Abstract] [Full Text] [PDF]
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 A. Zhao and T. Shea-Donohue PAR-2 agonists induce contraction of murine small intestine through neurokinin receptors Am J Physiol Gastrointest Liver Physiol, October 1, 2003; 285(4): G696 - G703. [Abstract] [Full Text] [PDF]
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A. Sabri, J. Guo, H. Elouardighi, A. L. Darrow, P. Andrade-Gordon, and S. F. Steinberg Mechanisms of Protease-activated Receptor-4 Actions in Cardiomyocytes. ROLE OF Src TYROSINE KINASE J. Biol. Chem., March 21, 2003; 278(13): 11714 - 11720. [Abstract] [Full Text] [PDF]
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D. E. Montgomery, B. M. Wolska, W. G. Pyle, B. B. Roman, J. C. Dowell, P. M. Buttrick, A. P. Koretsky, P. Del Nido, and R. J. Solaro alpha -Adrenergic response and myofilament activity in mouse hearts lacking PKC phosphorylation sites on cardiac TnI Am J Physiol Heart Circ Physiol, June 1, 2002; 282(6): H2397 - H2405. [Abstract] [Full Text] [PDF]
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