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Am J Physiol Regul Integr Comp Physiol 285: R455-R462, 2003. First published April 3, 2003; doi:10.1152/ajpregu.00588.2002
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INFLAMMATION, CYTOKINES, AND TEMPERATURE REGULATION

Activated epidermal growth factor receptor (ErbB1) protects the heart against stress-induced injury in mice

Miguel Pareja, Olga Sánchez, Jordi Lorita, Maria Soley, and Ignasi Ramírez

Department of Biochemistry and Molecular Biology, University of Barcelona, E-08028 Barcelona, Spain

Submitted 20 September 2002 ; accepted in final form 28 March 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Acute, high-intensity stress induces necrotic lesions in the heart. We found that restraint-and-cold (4°C) exposure (RCE) raises plasma lactate dehydrogenase (LDH), creatine kinase (CK), and transaminase activity in a time-dependent manner, with a peak value 7 h after stimulus cessation. At 24 h, signs of necrotic lesions were observed in paraffin sections stained with hematoxylineosin: focal accumulation of mononuclear cells in subendocardial areas of the left ventricle wall and focal hemorrhage in papillary muscles. In contrast, intermale fighting (IF) did not increase plasma CK activity, although LDH and transaminase activities did increase. In IF, no histological evidence of heart injury was observed. Because IF, but not RCE, increased plasma epidermal growth factor (EGF) concentration by ~1,000-fold, we hypothesized that EGF receptor (ErbB1) activation may protect the heart against stress-induced injury. To examine this hypothesis, we injected the ErbB1 tyrosine kinase inhibitor tyrphostin AG-1478 (25 mg/kg ip) immediately before mice were exposed to IF. After 3 h, plasma activities of LDH-1 and CK increased. Plasma enzyme activities were as low in control mice (injected with vehicle alone) as in nonfighting mice. In the last experiment, we injected EGF (0.25 mg/kg ip) 20 min before exposing mice to RCE. After 7 h, plasma LDH-1 and CK activities were significantly lower in these animals than in mice injected with vehicle. The effect required ErbB1 activation, because simultaneous administration of AG-1478 completely abolished the effect of exogenous EGF. We conclude that activated ErbB1, by endogenous or exogenous ligands, may protect the heart against stress-induced injury.

aggressive behavior; mitogen-activated protein kinase; alanine aminotransferase; aspartate aminotransferase; corticosterone

CHRONICALLY ELEVATED STIMULATION of the {beta}-adrenergic system is toxic to the heart, leading to hypertrophy and heart failure. This was clearly observed in transgenic mice overexpressing {beta}1- or {beta}2-adrenergic receptors (14, 15). Studies in ventricular myocytes (11) and in whole animals (51) indicate that sustained {beta}-adrenergic stimulation may result in apoptotic cell death. Therefore, the hypertrophic response of myocytes to sustained catecholamine stimulation should be understood as a compensatory adaptation (15).

In addition to these long-term effects of catecholamines, it is known that acute stress episodes may also induce heart injury. Thus immobilization or restraint, alone or in combination with other stimuli, induces myocardial necrotic lesions in several species (13, 29, 35, 45). Stress-induced injury results in the release of cytosolic enzymes to the blood (3, 36). Not all stressful conditions lead to heart injury. We reported recently (48) that intermale fighting (IF) does not raise plasma creatine kinase (CK) activity, although it increases plasma transaminase and lactate dehydrogenase (LDH) activities. This suggests that the heart is protected in this stress model.

Catecholamines appear to be responsible for the effect of acute stress. Indeed, {beta}-blockers, but not cholinergic blockers, reduce the release of enzymes in the restraint-and-water immersion stress model (3). In humans, it was shown that {beta}-blockade reduced tissue injury (measured by the increase in plasma activity of the MB isoform of CK) and supraventricular tachycardia induced by severe head injury (12).

In addition to many other effects, catecholamines stimulate the release of epidermal growth factor (EGF) from submandibular salivary glands in mice (8, 22). Accordingly, EGF levels are elevated in plasma of stressed animals (40, 55), although the increase is higher in fighting than in restraint-stress models (48). The EGF receptor (also known as ErbB1) can dimerize with any member of the ErbB receptor family. ErbB1 binds EGF and other members of the EGF family (transforming growth factor-{alpha}, epiregulin, {beta}-cellulin, amphiregulin, and heparin-binding EGF-like growth factor) (62). It has been reported that ErbB1 ligands are cytoprotective in several systems (26, 27, 42, 44).

After the recent observation that EGF prevents some harmful effects of intense and sustained {beta}-adrenergic stimulation of the heart by interfering with {beta}-adrenergic signaling (34), we hypothesized that activated ErbB1 may be involved in protection of the heart in fighting mice. Here we test this hypothesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Animals. Adult Swiss-CD1 male mice (Interfauna, Barcelona, Spain; 2.5–3 mo old, 35–40 g) were fed ad libitum and maintained under a constant 12:12-h light-dark cycle (lights on at 0800) and controlled conditions of humidity (45–55%) and temperature (22 ± 1°C). All experimental procedures were approved by the Committee on Animal Care of the University of Barcelona.

Restraint and cold exposure. On the day of the experiment (starting at 0900), mice were introduced into small flat-bottom cylinders (restrainers) with adjustable head and tail gates (Panlab, Barcelona, Spain). This procedure resulted in an almost complete immobilization of the animals. Animals were maintained at 4°C for the indicated period of time and then killed (see below) or returned to individual cages at room temperature for recovery. During the recovery period, mice had free access to water and food.

IF. After the period of adaptation to the animal housing facility, mice were maintained for 15 days in individual cages (14.02 dm2). On the day of the experiment (starting at 0900), each mouse (the intruder) was introduced into the cage of an older (and larger) mouse (the resident) that had been isolated in a smaller cage (7.56 dm2) for >=21 days. Residents had been tested for aggressiveness before the experiment. At the indicated time, the intruder was taken from the resident's cage and killed (see below) or returned to an individual cage for recovery. During the recovery period, mice had free access to water and food.

Sampling and analysis. After the mice were anesthetized with pentobarbital sodium (60 mg/kg), blood was collected into heparinized syringes from the inferior vena cava. Blood plasma was obtained by centrifugation. A sample was deproteinized and neutralized as indicated elsewhere (24) and used for glucose quantification (59). Another sample was processed for EGF quantification as indicated elsewhere (22). LDH, {alpha}-hydroxybutyrate dehydrogenase (LDH-1), CK, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activities in plasma were determined by standard procedures (LDH MPR1, HBDH MRP1, CK MRP1, AST MRP1, and ALT MPR1 assay kits, respectively, Roche, Mannheim, Germany). Plasma corticosterone was determined as indicated elsewhere (6). Immediately after collection of the blood, liver and submandibular salivary glands were excised, frozen in liquid nitrogen, and stored at -80°C until they were further processed (in <1 wk). Submandibular glands were homogenized in 10 ml of PBS. After centrifugation (100,000 g for 60 min at 4°C), the supernatant was stored at -40°C for EGF quantification (22). Liver glycogen was determined as indicated elsewhere (24).

In some experiments, the heart was immediately excised after collection of the blood, fixed in 4% paraformaldehyde, and embedded in paraffin to obtain 7-µm-thick sections. The sections were stained with hematoxylin-eosin and used for histological examination.

Administration of EGF and/or tyrphostin AG-1478. In some experiments, animals were injected with tyrphostin AG-1478 (Calbiochem, Merck Eurolab; 25 mg/kg ip dissolved in 10% dimethyl sulfoxide) and/or EGF (Roche; 0.25 mg/kg ip dissolved in PBS) 20 min before the stress experience. Control animals received an identical volume of vehicles.

PAGE. Liver samples were homogenized in lysis buffer (50 mM Tris, pH 7.5, 1 mM EGTA, 150 mM NaCl, 1 mM NaVO4, 1 mM NaF, 0.3 µM aprotinin, 1 µM leupeptin, 1 µM pepstatin, and 1 mM phenylmethylsulfonyl fluoride). After centrifugation (10 min, 1,000 g at 4°C), the supernatant was kept at -80°C until used for analysis, always in <1 mo. Twenty-five micrograms of tissue protein [total extracellular signal-regulated kinase (ERK)] or 50 µg (phospho-ERK) were run in 12% SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA) using standard procedures.

Analysis of total ERK1/2. After transfer, the membranes were soaked in blocking solution (5% defatted powdered milk in PBS) for 60 min at 37°C, rinsed [5 times for 5 min each in 100 ml of rinsing solution (MTP): 0.5% defatted powdered milk and 0.05% Tween 20 in PBS], and incubated overnight at 4°C with the primary antibody [rabbit anti-murine mitogen-activated protein (MAP) kinase (ERK1/2); catalog no. 06-182, Upstate, Barcelona, Spain] at 0.2 µg/ml in MTP. The membranes were rinsed as indicated above and incubated for 60 min at room temperature with the secondary antibody (GAR-IgG-HRP, Nordic, Tilburg, Netherlands; diluted 1:20,000 in MTP). After they were rinsed as described above, the membranes were developed with the ECL system (Amersham). Films were analyzed with Phoretix 1D Gel Analysis software after they were scanned (model GT-8500, Epson).

Analysis of phospho-ERK1/2. After transfer, the membranes were soaked in blocking solution [5% defatted powdered milk in Tris-buffered saline (TBS: 137 mM NaCl and 20 mM Tris, pH 7.6)] for 60 min at 37°C, rinsed [5 times for 5 min each in 100 ml of rinsing solution (MTT): 0.5% defatted powdered milk and 0.05% Tween 20 in TBS], and incubated overnight at 4°C with the primary antibody [mouse anti-murine phospho-MAP kinase (ERK1/2); catalog no. 05-841, Upstate] at 0.05 µg/ml in MTT. The membranes were rinsed as indicated above, incubated for 60 min at room temperature with the secondary antibody (GAM-IgG-HRP, Nordic; diluted 1:10,000 in MTT), and developed as described above.

Statistical analysis. To determine the significance of the differences in mean values, one- or two-way ANOVA and Tukey's post hoc test were performed with SPSS software (SPSS, Chicago, IL). When no homogeneity of variances was detected (plasma enzyme activities and EGF concentration) by Bartlett's test, logarithmic transformation was applied. If transformation did not homogenize variances, nonparametric Kruskal-Wallis one-way ANOVA was applied, and Dunn's post hoc test was used.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
We first studied the early responses to restraint- and-cold exposure (RCE) and IF (Fig. 1). RCE and IF increased plasma corticosterone concentration by 13- and 20-fold, respectively. Although the mean values were higher in animals subjected to IF than in those subjected to RCE, the difference was not significant. We determined liver glycogen content, because one of the most important effects of circulating catecholamines is stimulation of liver glycogen breakdown. RCE and IF decreased liver glycogen content. The decrease was higher in RCE than in IF, but the difference between groups was not significant. RCE neither decreased the content of submandibular salivary gland nor increased plasma EGF concentration. In contrast, IF decreased submandibular salivary gland EGF content by <50% and increased plasma EGF by 600-fold.



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  		Fig. 1. Early responses to stress. Mice were subjected to restraint-and-cold (4°C) exposure (RCE) or intermale fighting (IF) for 30 min and immediately killed. C, unexposed animals were controls. CS, corticosterone; SMG, submandibular (salivary) glands. Values are means ± SE of 8–12 animals per group. Significant differences were determined by 1-way ANOVA, and Tukey's post hoc test was used to determine significance of differences vs. control: *P < 0.05; **P < 0.01; ***P < 0.001.

 

Several hours are required for observation of tissue injury measured by the release of cytosolic enzymes to the blood (3, 48). Therefore, in the next experiment (Fig. 2), we determined plasma enzyme activities in samples obtained 0, 3, 7, or 24 h after animals were subjected to RCE or IF (60 min). Control animals (not exposed to stress) maintained constant activities of LDH, CK, AST (also known as glutamic-oxaloacetic transaminase), and ALT (also known as glutamicpyruvic transaminase) in plasma. At the end of the stimulus, LDH, CK, AST, and ALT activities were increased in blood plasma of animals subjected to RCE. These activities continued to increase until 7 h after cessation of the stimulus and returned to control values by 24 h. At the end of the stimulus, in IF animals, LDH and AST activities were increased, CK activity was unchanged, and ALT activity was almost twice the control value, although the difference was not significant. LDH, AST, and ALT activities continued to increase until 3 h and decreased thereafter. CK activity was always the same as control.



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  		Fig. 2. Time course of plasma enzyme activities after RCE or IF stress. Mice were exposed for 60 min to RCE (stippled bars) or IF (solid bars). Control animals (open bars) were maintained in their cages. Then animals were returned to their cages with free access to food and water. At indicated times, animals were killed to determine plasma enzyme activities: lactate dehydrogenase (A), creatine kinase (B), aspartate aminotransaminase (C), and alanine aminotransaminase (D). Values are means ± SE of 6 animals per group. Significant differences were determined by 2-way ANOVA, and Tukey's post hoc test was used to determine significance of differences vs. corresponding control: *P < 0.05; **P < 0.01; ***P < 0.001.

 

Hearts from RCE and IF animals obtained 24 h after cessation of the stressful stimulus, as well as hearts from control animals, were processed for direct visualization of damage in the heart (Fig. 3). IF animals (Fig. 3B) showed no histological sign of necrotic lesions. The images were indistinguishable from those corresponding to control animals (Fig. 3A). In contrast, RCE animals showed focal accumulation of macrophage-like mononucleate cells surrounding damaged myocytes (Fig. 3C). Higher magnification revealed the lower eosinophilia of damaged myocytes (Fig. 3D). These alterations were observed in subendocardial areas of left ventricle wall. In some animals, focal hemorrhage was also observed in papillary muscles in the left ventricle (Fig. 3, E and F).



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  		Fig. 3. Morphological evidence of necrotic lesions in hearts of RCE mice. Transverse sections (7 µm thick) of paraffin-embedded hearts were stained with hematoxylin-eosin. Endocardial side of the left ventricle wall from nonstressed (control) mouse (A), IF mouse (B), or RCE mouse (C) is shown at x100 magnification. D: necrotic lesion in C shown at x500 magnification. E: transverse section of papillary muscles in the left ventricle shown at x100 magnification. F: hemorrhagic area in E shown at x500 magnification.

 

Next, we analyzed the ability of tyrphostin AG-478 to block EGF action in whole mice. In this experiment, we studied one of the best characterized effects of EGF, the induction of ERK1/2 phosphorylation (62). Although we analyzed liver samples, because the liver contains the greatest number of EGF receptors in nontransformed cells (41, 53), this effect is not cell specific. At 10 min after EGF administration, bands corresponding to phosphorylated ERK1 and ERK2 appeared in liver extracts (Fig. 4). EGF also reduced the electrophoretic mobility of total ERK1 and ERK2. Simultaneous administration of AG-1478 abolished the effect of EGF. When AG-1478 was administered 1 h before EGF, the blockade was only partial. When it was injected 6 h before EGF, the inhibitory effect was completely lost.



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  		Fig. 4. Effect of AG-1478 on EGF-induced extracellular signal-regulated kinase (ERK1/2) phosphorylation in liver. To determine effectiveness of tyrphostin AG-1478, mice were treated with EGF (0.25 mg/kg ip), with or without AG-1478 (25 mg/kg ip), and killed 10 min later. Control mice received vehicle. Liver samples were analyzed for phospho-ERK1/2 or total ERK1/2. A representative experiment is shown.

 

A new experiment in which half of the intruder mice received an intraperitoneal injection of AG-1478 before they were introduced into the resident's cage and the other half received vehicle was performed to study the involvement of ErbB1 in protection of the heart in the IF model (Fig. 5). IF increased plasma total LDH activity, but not activity of the cardiac isoform LDH-1 or CK. AG-1478 led to a further increase in plasma total LDH, LDH-1, and CK activities. Neither vehicle nor AG-1478 had any effect in nonfighting animals (data not shown).



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  		Fig. 5. Effect of AG-1478 on plasma enzyme activities in fighting mice. Mice were injected with AG-1478 (25 mg/kg ip) or vehicle alone immediately before they were introduced into a resident's cage. Plasma enzyme activities in intruder mice were determined after 3 h. Values are means ± SE of 8 animals per group. Statistical comparisons were made by nonparametric Kruskal-Wallis 1-way ANOVA, and Dunn's post hoc test was used to determine significance of differences between pairs of values: *P < 0.05; **P < 0.01; ***P < 0.001 vs. control. LDH, lactate dehydrogenase; LDH-1, {alpha}-hydroxybutyrate dehydrogenase; CK, creatine kinase.

 

In the last experiment, we addressed whether the lack of heart protection in the RCE model was due to the lack of EGF secretion from submandibular salivary glands. In this experiment, one-third of the mice received an intraperitoneal injection of EGF 20 min before they were subjected to RCE. Another third of the animals received combined doses of EGF and AG-1478. The rest of the animals were injected with vehicle. Samples were obtained 7 h after the mice were returned to their cages. RCE induced a large increase in plasma total LDH, LDH-1, and CK activities (Fig. 6). Previous EGF administration resulted in a significantly lower increase in total LDH, LDH-1, and CK activities. All these effects of EGF were abolished by simultaneous administration of AG-1478.



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  		Fig. 6. Effect of EGF on plasma enzyme activities in RCE mice. Mice were treated with EGF (0.25 mg/kg ip), with or without AG-1478 (25 mg/kg ip), 20 min before RCE for 60 min. Plasma enzyme activities in intruder mice were determined 7 h later. Values are means ± SE of 5 animals per group. Statistical comparisons were made by nonparametric Kruskal-Wallis 1-way ANOVA, and Dunn's post hoc test was used to determine significance of differences between pairs of values: *P < 0.05; **P < 0.01; ***P < 0.001 vs. control.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
RCE-induced cardiomyopathy. Acute stressful stimuli of variable intensity induce necrotic lesions in the heart of several animal species (20). Here we provide histopathological evidence of necrotic lesions in the heart from RCE mice. Our observations, small foci of cellular infiltrates in subendocardial areas of damaged myocytes in the left ventricle wall and hemorrhage in papillary muscles in this ventricle, coincide with other reports in rats (45), pigs (29), and rabbits (13). The appearance of mononucleate cells, similar to those observed after 24 h of the stressful stimulus, has been interpreted as part of the healing process (19). In a few days, these necrotic lesions in the ventricle wall evolve into nonspecific chronic lesions: accumulation of fiber and condensed fragments that correspond to remnants of myocardial cells (56).

The plasma enzyme profiles observed in RCE mice suggest heart injury. Thus we observed an increase in CK and LDH-1 activities. In addition, although both transaminase activities increased, the rise in AST activity was higher than the rise in ALT. Therefore, the ratio of AST to ALT increased (2.0 ± 0.1 and 5.0 ± 0.3 in control and RCE mice, respectively, P < 0.001). None of these results are exclusive markers of heart damage but, taken together, strongly suggest a cardiac origin of these enzymes. Indeed, this is consistent with the histological results discussed above. Enzyme activities offer an easier and faster way to quantify the extent of tissue damage.

There is strong evidence that catecholamines are responsible for acute stress-induced heart injury. First, catecholamine administration induces necrotic lesions similar to those induced by stress (1, 13, 47, 54). Second, adrenergic, but not cholinergic, blockers reduce the extent of stress-induced heart injury as measured by enzyme activities in plasma (3). Some authors suggested the involvement of {alpha}1-adrenergic receptors because of the effect of prazosin on norepinephrine-induced myocardial necrosis (31). Others pointed to {beta}-adrenergic receptors on the basis of the effect of {beta}-blockers (3) or isoproterenol (10, 57, 58). It is conceivable that both receptor types may be involved, and the balance between receptor signals would determine the final effect, as suggested for the effect of norepinephrine on apoptosis in cultured neonatal rat cardiac myocytes (28).

We propose that enhanced contractility (mediated by {beta}-adrenergic receptors), in combination with {alpha}1-adrenergic receptor-mediated coronary vasoconstriction (5), may induce functional hypoxia, which, together with accumulation of catecholamine metabolites (52), would result in sarcolemma damage, altered Ca2+ inflow, and, finally, cell death. This results in coagulative myocytolysis, which is the first morphological alteration to be observed after an intense catecholamine stimulation of the heart (58).

Heart protection in fighting mice. We reported previously that, after 3 h of cohabitation, plasma CK activity did not increase in intruder mice (48). Here we have extended the observation period to 24 h. The lack of rise in CK activity and the absence of necrotic lesions in the histological preparation suggest that the heart is protected in fighting mice. We did not find any reference in the MEDLINE database describing heart damage in rodents exposed to an aggressive male. At longer times, overcrowding appears to induce heart damage (17, 39, 60).

Aggressive behavior (isolation-induced fighting) is indeed a high-intensity stressor in rodents (30). We found that plasma corticosterone concentration was 20-fold higher in IF mice than in controls. This indicates a strong activation of the hypothalamus-pituitary-adrenal axis. In addition, liver glycogen content was decreased in IF mice. Glycogen degradation is an immediate effect of increased plasma catecholamine concentration. In rats, Sgoifo et al. (49) reported that defeat induced a higher increase in plasma catecholamine concentration than restraint. These authors reported that defeat resulted in a high risk of ventricular arrhythmias (49, 50). Therefore, the lack of heart injury in fighting mice may not be attributed to a lack of catecholamine stimulation of the heart. Rather, we suggest that, in this model, the heart is specifically protected.

Does activated ErbB1 protect the heart? Male mouse submandibular salivary glands accumulate as much as 30-fold more EGF than female glands (22). Because aggressive behavior induces acute secretion of EGF into blood plasma, and the result is a ~1,000-fold increase in plasma EGF concentration (40, 48), a role of EGF in response to this male-characteristic behavior was suggested but never proven.

To examine the role of the EGF receptor (ErbB1), we used the tyrphostin AG-1478, a highly specific inhibitor of ErbB1 tyrosine kinase activity. The IC50 for ErbB1 is 100,000 times lower than that for related tyrosine kinases (HER2-Neu or platelet-derived growth factor receptor) (32). This inhibitor was shown to delay A431 tumor growth in nude mice (7) and to decrease hydroxyproline accumulation in a rat model of pulmonary fibrosis (46). In these studies, a dose of 50 mg·kg-1·day-1 was used. We observed that AG-1478, at 25 mg/kg, completely and reversibly blocked EGF-induced ERK1/2 phosphorylation in mouse liver. An alternative to AG-1478 would be the administration of neutralizing antibodies directed to ErbB1 or ErbB2 [the preferred partner for heterodimerization (62)]. To our knowledge, available antibodies are directed to human receptors, and they do not (or are not reported to) react with murine receptors (25, 37). We thus decided to examine the role of ErbB1, acutely activated by secreted EGF, by means of the effect of the ErbB1 inhibitor AG-1478.

We conclude that activated ErbB1 protects the heart against stress-induced injury, because 1) IF raised plasma CK and LDH-1 activities in AG-1478-injected mice and 2) activation of ErbB1 by exogenous administration of EGF greatly reduced the RCE-induced rise in plasma CK and LDH-1 activities. We showed previously that EGF (0.25 mg/kg ip) raised plasma concentration by ~100-fold (18). The effect of exogenous EGF was dependent on ErbB1 activation, because it was completely blocked by simultaneous administration of AG-1478. Our results suggest that RCE mice were not protected against stress-induced heart injury because of the lack of EGF secretion from submandibular salivary glands.

Our results provide no insight into the mechanisms involved in the effect of EGF. However, several hypotheses can be outlined. If {beta}- and {alpha}1-adrenergic receptors are involved in catecholamine-induced heart injury, interference with signaling of either receptor would reduce the response to catecholamines and, hence, the effect of these hormones. We showed recently that EGF interferes with {beta}-adrenergic signaling in heart myocytes and with the inotropic and chronotropic responses to epinephrine in perfused hearts (34). One consequence of these effects of EGF was protection against some of the harmful effects of catecholamines: the arrhythmogenic effect of sustained infusion of epinephrine in perfused hearts and induction of the Bezold-Jarisch reflex by repeated administration of epinephrine in whole animals (34). Whether the interference with {beta}-adrenergic signaling also explains the protection of the heart against stress-induced injury remains to be established. In addition, a hypothetical effect of EGF on {alpha}1-adrenergic receptor signaling will have to be explored. It has been reported that EGF interferes with contraction of norepinephrine-induced canine helical mesenteric arterial strips, an {alpha}1-adrenergic receptor-mediated effect (38). However, interference with {alpha}1-adrenergic receptor signaling was not observed in other cell types (23).

Because catecholamine metabolites or other highly oxidative products may participate in the initial steps of sarcolemmal damage, the protective effect of EGF may target oxidative stress processes. In many systems, EGF or other ErbB1 ligands were shown to be protective against oxidative stress-induced damage or even cell death (2, 4, 9, 16, 33, 43, 61). Therefore, a number of possible mechanisms have to be explored to elucidate the molecular basis of the effect of EGF on stress-induced heart injury. Use of specific compounds to block different signaling pathways will require a new experimental model (perfused hearts or isolated myocytes), because, in the whole animal, these inhibitors would block not only ErbB1 but many other stimuli signaling as well.

Concluding remarks. Our results suggest that activation of ErbB1 by endogenous ligands, very likely EGF secreted from submandibular salivary glands, is involved in heart protection when male mice are exposed to an aggressive encounter. In addition, our results also suggest that exogenous intervention to activate ErbB1 may protect the heart against catecholamine-induced injury. These results, together with the recent observation that EGF prevents catecholamine-induced functional disturbances in perfused hearts and whole animals (34), suggest that it will be worth exploring the usefulness of external activation of ErbB1 receptor with EGF or other ligands of the EGF family to prevent reperfusion-induced injury after transient ischemia in which local release of catecholamines is involved (21). Indeed, further experiments in perfused hearts and isolated cardiomyocytes are required to demonstrate that activation of ErbB1 directly protects the heart.


   DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This study was supported by Dirección General de Enseñanza Superior e Investigación Cientófica Grant PB97-0936 from Ministerio de Educación y Ciencia, Spain.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. Ramírez, Dept. of Biochemistry and Molecular Biology, University of Barcelona, Avda. Diagonal 645, E-08028 Barcelona, Spain (E-mail: iramirez{at}ub.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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Analóczy Z. Role of catecholamines in stress-induced heart disease. In: Pathogenesis of Stress-Induced Heart Disease, edited by Beamish RE, Panagia V, and Dhalla NS. Boston, MA: Martinus Nijhoff, 1985, p. 213–227.
  2. Andreoli SP, Mallett CP, McAteer JA, Kempson SA, and Fineberg N. Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury. In Vitro Cell Dev Biol Anim 34: 824–830, 1998.[Web of Science][Medline]
  3. Arakawa H, Kodama H, Matsuoka N, and Yamaguchi I. Stress increases plasma enzyme activities in rats: differential effects of adrenergic and cholinergic blockades. J Pharmacol Exp Ther 280: 1296–1303, 1997.[Abstract/Free Full Text]
  4. Banan A, Choudhary S, Zhang Y, Fields JZ, and Keshavarzian A. Ethanol-induced barrier dysfunction and its prevention by growth factors in human intestinal monolayers: evidence for oxidative and cytoskeletal mechanisms. J Pharmacol Exp Ther 291: 1075–1085, 1999.[Abstract/Free Full Text]
  5. Beamish RE and Dhalla NS. Involvement of catecholamines in coronary spasm under stressful conditions. In: Stress and Heart Disease, edited by Beamish RE. Boston, MA: Martinus Nijhoff, 1985, p. 129–141.
  6. Benavides A, Siches M, and Llobera M. Circadian rhythms of lipoprotein lipase and hepatic lipase activities in intermediate metabolism of adult rat. Am J Physiol Regul Integr Comp Physiol 275: R811–R817, 1998.[Abstract/Free Full Text]
  7. Busse D, Doughty RS, Ramsey TT, Russell WE, Price JO, Flanagan WM, Shawver LK, and Arteaga CL. Reversible G1 arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27KIP1 independent of MAPK activity. J Biol Chem 275: 6987–6995, 2000.[Abstract/Free Full Text]
  8. Byyny RL, Orth DN, Cohen S, and Doyne ES. Epidermal growth factor: effects of androgen and adrenergic agents. Endocrinology 95: 776–782, 1974.[Abstract/Free Full Text]
  9. Caraglia M, Abbruzzese A, Leardi A, Pepe S, Budillon A, Baldassare G, Selleri C, De Lorenzo S, Fabbrocini A, Giuberti G, Vitale G, Lupoli G, Bianco AR, and Tagliaferri P. Interferon-{alpha} induces apoptosis in human KB cells through a stress-dependent mitogen-activated protein kinase pathway that is antagonized by epidermal growth factor. Cell Death Differ 6: 773–780, 1999.[Web of Science][Medline]
  10. Chagoya de Sánchez V, Hernández-Muñoz R, López-Barrera F, Yáñez L, Vidrio S, Suárez J, Cota-Garza MD, Aranda-Fraustro A, and Cruz D. Sequential changes of energy metabolism and mitochondrial function in myocardial infarction induced by isoproterenol in rats: a long-term and integrative study. Can J Physiol Pharmacol 75: 1300–1311, 1997.[Web of Science][Medline]
  11. Communal C, Singh K, Pimentel DR, and Colucci WS. Norepinephrine stimulates apoptosis in adult rat ventricular myocytes by activation of the {beta}-adrenergic pathway. Circulation 98: 1329–1334, 1998.[Abstract/Free Full Text]
  12. Cruickshank JM, Neil-Dwyer G, Degaute JP, Hayes Y, Kuurne T, Kytta J, Vincent JL, Carruthers ME, and Patel S. Reduction of stress/catecholamine-induced cardiac necrosis by {beta}1-selective blockade. Lancet 2: 585–589, 1987.[Web of Science][Medline]
  13. Downing SE and Chen V. Myocardial injury following endogenous catecholamine release in rabbits. J Mol Cell Cardiol 17: 377–387, 1985.[Web of Science][Medline]
  14. Du XJ, Gao XM, Wang BH, Jennings GL, Woodcock EA, and Dart AM. Age-dependent cardiomyopathy and heart failure phenotype in mice overexpressing {beta}2-adrenergic receptors in the heart. Cardiovasc Res 48: 448–454, 2000.[Abstract/Free Full Text]
  15. Engelhardt S, Hein L, Wiesmann F, and Lohse MJ. Progressive hypertrophy and heart failure in {beta}1-adrenergic receptor transgenic mice. Proc Natl Acad Sci USA 96: 7059–7064, 1999.[Abstract/Free Full Text]
  16. Engler JA, Gupta A, Li L, and Rao RK. Inhibition of DNA synthesis in Caco-2 cells by oxidative stress: amelioration by epidermal growth factor. Dig Dis Sci 44: 1902–1909, 1999.[Web of Science][Medline]
  17. Fani K, Jimenez FA, and De Soto F. Heart morphological changes in rats placed in a crowded environment. J Toxicol Environ Health 3: 421–429, 1977.[Web of Science][Medline]
  18. Fernández G, Mena MP, Arnau A, Sánchez O, Soley M, and Ramírez I. Immobilization stress induces c-Fos accumulation in liver. Cell Stress Chaperones 5: 306–312, 2000.[Web of Science][Medline]
  19. Ferrans VJ and Van Vleet JF. Morphological aspects of myocardial lesions associated with stress. In: Stress and Heart Disease, edited by Beamish RE. Boston, MA: Martinus Nijhoff, 1985, p. 211–227.
  20. Goldstein DS. Stress and catecholaminergic function in cardiovascular diseases: cardiac necrosis and cardiomyopathy. In: Stress, Catecholamines, and Cardiovascular Disease. Oxford, UK: Oxford University Press, 1995, p. 379–382.
  21. Goldstein DS. Stress and catecholaminergic function in cardiovascular diseases: coronary heart disease. In: Stress, Catecholamines, and Cardiovascular Disease. Oxford, UK: Oxford University Press, 1995, p. 360–370.
  22. Grau M, Rodríguez C, Soley M, and Ramírez I. Relationship between epidermal growth factor in mouse submandibular glands, plasma, and bile: effects of catecholamines and fasting. Endocrinology 135: 1854–1862, 1994.[Abstract]
  23. Grau M, Soley M, and Ramírez I. Interaction between adrenaline and epidermal growth factor in the control of liver glycogenolysis in mouse. Endocrinology 138: 2601–2609, 1997.[Abstract/Free Full Text]
  24. Grau M, Tebar F, Ramírez I, and Soley M. Epidermal growth factor administration decreases liver glycogen and causes mild hyperglycaemia in mice. Biochem J 315: 289–293, 1996.
  25. Herbst RS and Shin DM. Monoclonal antibodies to target epidermal growth factor receptor-positive tumors: a new paradigm for cancer therapy. Cancer 94: 1593–1611, 2002.[Web of Science][Medline]
  26. Hoffmann P, Zeeh JM, Lakshmanan J, Wu VS, Procaccino F, Reinshagen M, McRoberts JA, and Eysselein VE. Increased expression of transforming growth factor-{alpha} precursors in acute experimental colitis in rats. Gut 41: 195–202, 1997.[Abstract/Free Full Text]
  27. Ishikawa S, Cepinskas G, Specian RD, Itoh M, and Kvietys PR. Epidermal growth factor attenuates jejunal mucosal injury induced by oleic acid: role of mucus. Am J Physiol Gastrointest Liver Physiol 267: G1067–G1077, 1994.[Abstract/Free Full Text]
  28. Iwai-Kanai E, Hasegawa K, Araki M, Kakita T, Morimoto T, and Sasayama S. {alpha}- and {beta}-adrenergic pathways differentially regulate cell type-specific apoptosis in rat cardiac myocytes. Circulation 100: 305–311, 1999.[Abstract/Free Full Text]
  29. Jönsson L and Johansson G. Cardiac muscle cell damage induced by restraint stress. Virchows Arch B Cell Pathol 17: 1–12, 1974.[Medline]
  30. Koolhaas JM. Aggressive behavior. In: Encyclopedia of Stress, edited by Fink G. San Diego, CA: Academic, 2000, p. 100–104.
  31. Lee JC and Sponenberg P. The contribution of the {alpha}-adrenoceptor system to the pathogenesis of norepinephrine cardiomyopathy. In: Pathogenesis of Stress-Induced Heart Disease, edited by Beamish RE, Panagia V, and Dhalla NS. Boston, MA: Martinus Nijhoff, 1985, p. 251–260.
  32. Levitzki A and Gazit A. Tyrosine kinase inhibition: an approach to drug development. Science 267: 1782–1788, 1995.[Abstract/Free Full Text]
  33. Lieberthal W, Triaca V, Koh JS, Pagano PJ, and Levine JS. Role of superoxide in apoptosis induced by growth factor withdrawal. Am J Physiol Renal Physiol 275: F691–F702, 1998.[Abstract/Free Full Text]
  34. Lorita J, Escalona N, Faraudo S, Soley M, and Ramírez I. Effects of epidermal growth factor on epinephrine-stimulated heart function in rodents. Am J Physiol Heart Circ Physiol 283: H1887–H1895, 2002.[Abstract/Free Full Text]
  35. Matsuoka N, Arakawa H, Kodama H, and Yamaguchi I. Characterization of stress-induced sudden death in cardiomyopathic hamsters. J Pharmacol Exp Ther 284: 125–135, 1998.[Abstract/Free Full Text]
  36. Meltzer HY. Plasma creatine phosphokinase activity, hypothermia, and stress. Am J Physiol 221: 896–901, 1971.[Free Full Text]
  37. Mendelsohn J and Baselga J. The EGF receptor family as targets for cancer therapy. Oncogene 19: 6550–6565, 2000.[Web of Science][Medline]
  38. Muramatsu I, Hollenberg MD, and Lederis K. Modulation by epidermal growth factor-urogastrone of contraction in isolated canine helical mesenteric arterial strips. Can J Physiol Pharmacol 64: 1561–1565, 1986.[Web of Science][Medline]
  39. Nagaraja HS and Jeganathan PS. Influence of different types of stress on selected cardiovascular parameters in rats. Indian J Physiol Pharmacol 43: 296–304, 1999.[Medline]
  40. Nexo E, Hollenberg MD, and Bing J. Aggressive behaviour in mice provokes a marked increase in both plasma epidermal growth factor and renin. Acta Physiol Scand 111: 367–371, 1981.[Web of Science][Medline]
  41. O'Connor-McCourt M, Soley M, Hayden LJ, and Hollenberg MD. Receptors for epidermal growth factor (urogastrone) and insulin in primary cultures of rat hepatocytes maintained in serum-free medium. Biochem Cell Biol 64: 803–810, 1986.[Web of Science][Medline]
  42. Olsen PS, Poulsen SS, Kirkegaard P, and Nexo E. Role of submandibular saliva and epidermal growth factor in gastric cytoprotection. Gastroenterology 87: 103–108, 1984.[Web of Science][Medline]
  43. Peng H, Wen TC, Tanaka J, Maeda N, Matsuda S, Desaki J, Sudo S, Zhang B, and Sakanaka M. Epidermal growth factor protects neuronal cells in vivo and in vitro against transient forebrain ischemia- and free radical-induced injuries. J Cereb Blood Flow Metab 18: 349–360, 1998.[Web of Science][Medline]
  44. Pillai SB, Hinman CE, Luquette MH, Nowicki PT, and Besner GE. Heparin-binding epidermal growth factor-like growth factor protects rat intestine from ischemia/reperfusion injury. J Surg Res 87: 225–231, 1999.[Web of Science][Medline]
  45. Raab W, Chaplin JP, and Bajusz E. Myocardial necroses produced in domesticated rats and in wild rats by sensory and emotional stresses. Proc Soc Exp Biol Med 116: 665–669, 1964.[Medline]
  46. Rice AB, Moomaw CR, Morgan DL, and Bonner JC. Specific inhibitors of platelet-derived growth factor or epidermal growth factor receptor tyrosine kinase reduce pulmonary fibrosis in rats. Am J Pathol 155: 213–221, 1999.[Abstract/Free Full Text]
  47. Rona G. Involvement of catecholamines in the development of myocardial cell damage. In: Pathogenesis of Stress-Induced Heart Disease, edited by Beamish RE, Panagia V, and Dhalla NS. Boston, MA: Martinus Nijhoff, 1985, p. 228–236.
  48. Sánchez O, Arnau A, Pareja M, Poch E, Ramírez I, and Soley M. Acute stress-induced tissue injury in mice: differences between emotional and social stress. Cell Stress Chaperones 7: 36–46, 2002.[Web of Science][Medline]
  49. Sgoifo A, De Boer SF, Westenbroek C, Maes FW, Beldhuis H, Suzuki T, and Koolhaas JM. Incidence of arrhythmias and heart rate variability in wild-type rats exposed to social stress. Am J Physiol Heart Circ Physiol 273: H1754–H1760, 1997.[Abstract/Free Full Text]
  50. Sgoifo A, Stilli D, De Boer SF, Koolhaas JM, and Musso E. Acute social stress and cardiac electrical activity in rats. Aggressive Behav 24: 287–296, 1998.
  51. Shizukuda Y, Buttrick PM, Geenen DL, Borczuk AC, Kitsis RN, and Sonnenblick EH. {beta}-Adrenergic stimulation causes cardiocyte apoptosis: influence of tachycardia and hypertrophy. Am J Physiol Heart Circ Physiol 275: H961–H968, 1998.[Abstract/Free Full Text]
  52. Singal PK, Kapur N, Beamish RE, Das PK, and Dhalla NS. Antioxidant protection against epinephrine-induced arrhythmias. In: Stress and Heart Disease, edited by Beamish RE. Boston, MA: Martinus Nijhoff, 1985, p. 190–201.
  53. Soley M and Hollenberg MD. Epidermal growth factor (urogastrone)-stimulated gluconeogenesis in isolated mouse hepatocytes. Arch Biochem Biophys 255: 136–146, 1987.[Web of Science][Medline]
  54. Szakacs JE and Cannon AI. Norepinephrine-myocarditis. Am J Clin Pathol 30: 425–434, 1958.[Web of Science][Medline]
  55. Tebar F, Grau M, Mena MP, Arnau A, Soley M, and Ramírez I. Epidermal growth factor secreted from submandibular salivary glands interferes with the lipolytic effect of adrenaline in mice. Endocrinology 141: 876–882, 2000.[Abstract/Free Full Text]
  56. Todd GL. Morphological correlates of catecholamine-induced heart cell damage. In: Pathogenesis of Stress-Induced Heart Disease, edited by Beamish RE, Panagia V, and Dhalla NS. Boston, MA: Martinus Nijhoff, 1985, p. 327–250.
  57. Todd GL, Baroldi G, Pieper GM, Clayton FC, and Eliot RS. Experimental catecholamine-induced myocardial necrosis. I. Morphology, quantification and regional distribution of acute contraction band lesions. J Mol Cell Cardiol 17: 317–338, 1985.[Web of Science][Medline]
  58. Todd GL, Baroldi G, Pieper GM, Clayton FC, and Eliot RS. Experimental catecholamine-induced myocardial necrosis. II. Temporal development of isoproterenol-induced contraction band lesions correlated with ECG, hemodynamic and biochemical changes. J Mol Cell Cardiol 17: 647–656, 1985.[Web of Science][Medline]
  59. Trinder P. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Biochem 6: 24–27, 1969.
  60. Weber HW and Van Der Walt JJ. Cardiomyopathy in crowded rabbits. Recent Adv Stud Card Struct Metab 6: 471–477, 1975.
  61. Yamada M, Enokido Y, Ikeuchi T, and Hatanaka H. Epidermal growth factor prevents oxygen-triggered apoptosis and induces sustained signalling in cultured rat cerebral cortical neurons. Eur J Neurosci 7: 2130–2138, 1995.[Web of Science][Medline]
  62. Yarden Y and Sliwkowski MX. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2: 127–137, 2001.[Web of Science][Medline]



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