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Endocrinology Vol. 140, No. 9 4198-4207
Copyright © 1999 by The Endocrine Society

ARTICLES

Inhibition of Atrial Wall Stretch-Induced Cardiac Hormone Secretion by Lavendustin A, a Potent Tyrosine Kinase Inhibitor1

Panu Taskinen, Miklos Toth, Olli Vuolteenaho, Jarkko Magga and Heikki Ruskoaho

Departments of Pharmacology and Toxicology and Physiology (O.V), Biocenter Oulu, University of Oulu, 90401 Oulu, Finland; and Department of Cardiovascular Surgery and First Department of Internal Medicine (M.T), Semmelwies University Medical School, 1102 Budapest, Hungary

Address all correspondence and requests for reprints to: Heikki Ruskoaho, M.D, Ph.D., Department of Pharmacology and Toxicology, University of Oulu, P.O. Box 5000, 90401 Oulu, Finland. E-mail: heikki.ruskoaho{at}.oulu.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The cellular processes linking mechanical wall stretch to atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) secretion from the heart are unclear. In the present study, a paced perfused rat heart preparation was used to study the signaling mechanisms of atrial wall stretch-induced secretion of ANP and BNP. Vehicle or drugs were infused into the perfusate for 40 min and right atrial wall stretch was superimposed for 10 min after 25-min drug infusions by elevating the level of the pulmonary artery cannula tip. Lavendustin A, a potent inhibitor of protein tyrosine kinases, at the concentrations of 0.5 and 1.3 µM decreased atrial wall stretch-induced ANP secretion (53% and 68%, respectively, P < 0.001) in the perfused rat heart preparation, whereas no difference in the hemodynamic variables (heart rate, contractile force and perfusion pressure) were noted between groups. Lavendustin A also completely abolished the wall stretch-induced secretion of BNP. Several other protein kinase inhibitors including staurosporine (protein kinase C inhibitor), ML-9 (myosin light chain kinase inhibitor), KN-62 (Ca2+/calmodulin-dependent protein kinase II inhibitor) and H-89 (protein kinase A inhibitor) had no significant effect on atrial wall stretch-stimulated ANP secretion. In a separate series of experiments, in which the right atria were stretched for 2 h, administration of lavendustin A (1 µM) but not staurosporine (30 nM) significantly decreased sustained wall stretch-induced ANP secretion. Okadaic acid, a potent protein phosphatase A2 (PPA2) and PP1 inhibitor, at the concentration of 100 nM had no effect on basal ANP secretion but significantly accelerated the ANP secretory response to atrial wall stretch (P < 0.05). In conclusion, the findings that inhibitors of protein tyrosine kinase and protein phosphatase selectively modulated atrial wall stretch-induced ANP secretion suggest a new mechanism involving endogenous protein tyrosine activity in the regulation of natriuretic peptide exocytosis from cardiac myocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MECHANICAL stretch alters the structure and function of many different cell types including myocytes, endothelial cells and fibroblasts. Molecular structures that may mediate the effects of mechanical stretch include mechanosensitive ion channels, Na+H+-exchanger, adenylate cyclase, enzymes involved in phosphatidylinositol turnover and cytoskeleton (1, 2, 3). In the heart, the inositol-3,4,5-trisphosphate(IP3)/diacylglycerol (DAG) signaling pathway may be important because distension of the right atria in vitro stimulates phosphatidylinositol turnover and formation of inositol phosphates within minutes (4). In neonatal ventricular cardiac myocytes, mechanical strain has been reported to increase DAG and IP3 content as well as protein kinase C activity and activate phospholipase C, D, and A2 (5). Cultured cardiac myocytes respond to mechanical stretch also by a marked increase in the activity of protein tyrosine and mitogen-activated protein (MAP) kinases (5). Because the mechanical stretch-induced c-fos activation was suppressed by protein tyrosine kinase and protein kinase C inhibitors (5), these protein kinases may be necessary for the stretch response in the cardiac myocytes. However, mechanical stress-induced signal transduction is characterized by simultaneous activation of several other second messenger systems including p21ras, raf-1, and c-Jun N-terminal protein kinase (JNK) (6), and thus, controversy exists as to which of these plays the dominant role in triggering different stretch-induced physiological and pathophysiological responses in the cardiac myocytes.

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (brain natriuretic peptide, BNP) are members of cardiac natriuretic peptide hormone family involved in the regulation of blood pressure and fluid homeostasis (7, 8). Atrial wall stretching is the predominant stimulus for the secretion of ANP (9, 10). Wall stretch appears also to be a potent stimulus for ventricular ANP release (11) and for the secretion of BNP from the ventricles (12) and atria (13) in vitro. However, the molecules converting the mechanical stress signal into biochemical events that regulate cardiac hormone secretion are unclear. Several studies using different experimental models of cardiac peptide hormone release have indicated that ion channels, cellular calcium homeostasis, and protein kinase C may be involved in stretch-induced ANP secretion in atrial myocytes (8, 14). In isolated perfused rat hearts, phorbol esters, which activate protein kinase C directly, have an additive effect upon stretch-activated ANP release in isolated perfused rat heart preparation, suggesting that protein kinase C may be important for wall stretch-induced ANP secretion (15). However, there are no reports of the role of protein tyrosine kinases in mechanical stretch-induced cardiac hormone secretion.

The aim of the present study was to examine the role of protein tyrosine kinases in the signal transduction pathways of the stretch-induced cardiac hormone secretion by using a modified perfused paced rat heart preparation (15, 16). This model allows to apply a simple and controlled mechanical stimulus to cardiac myocytes to analyze the signal pathways of mechanotransduction in the adult heart. Our present results show that atrial wall stretch-induced ANP and BNP secretion is selectively modulated by lavendustin A, a potent protein tyrosine kinase inhibitor (17, 18, 19), indicating that protein tyrosine kinase activity is an important regulator of cardiac hormone exocytosis from atrial myocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Drugs used in this study were: lavendustin A methyl ester (Research Biochemicals International Inc., Natick, MA); KN-62 (1-[N,0-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phynylpiperazine) and H-89 (N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide) (Seikagaku Corp. Co., Tokyo, Japan): ML-9 (1-[5-iodonaphthalene-1-sulfonyl]-H-hexahydro-1,4-diazepine), staurosporine, okadaic acid and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (Sigma Chemical Co., St. Louis, MO); and heparin (Leiras, Turku, Finland). All other reagents were purchased from Sigma Chemical Co.. Staurosporine, H-89, okadaic acid, TPA, and lavendustin A were dissolved in dimethyl sulfoxide (DMSO), KN-62 in DMSO and HCl, and ML-9 in ethanol. The final concentration of each organic solvent was less than 0.03%.

Animals
Male Sprague Dawley rats (weighing 230–350 g) from the Centre for Experimental Animals at the University of Oulu, Finland, were used. The rats were housed in plastic cages in a room with controlled humidity of 40% and a temperature of 22 C. A 0600 h on and 1800 h off environmental light cycle was maintained. The experimental design was approved by the Animal Experimentation Committee of the University of Oulu. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

Isolated perfused rat hearts
The rat isolated perfused heart preparations used in this study were similar to those previously described (15, 20). Briefly, 20 min after ip injection of heparin (500 IU/kg), rats were killed by decapitation, hearts were quickly removed, cooled with perfusion fluid (4-10 C), and arranged for retrograde perfusion by the Langendorff technique. The hearts were perfused with a modified Krebs-Henseleit bicarbonate buffer, pH 7.40, equilibrated with 95% O2-5% CO2 at 37 C. The composition of the buffer was as follows (mmol/liter): NaCl 113.8, NaHCO3 22.0, KCl 4.7, KH2PO4 1.2, MgSO4 1.1, CaCl2 2.5 and glucose 11.0.

Variations in perfusion pressure arising from changes in coronary vascular resistance were recorded on a Grass polygraph (model 7DA, Grass Instrument Co., Quincy, MA) with a pressure transducer (model MP-15, Micron Instruments, Los Angeles, CA) situated on a side arm of the aortic cannula. Isometric force of contraction was recorded by a strain gauge transducer (Grass FT03) connected to the Grass polygraph. The output was damped to give a mean contractile force. Heart rate was counted from contractions by the Grass tachograph. The hearts were submitted to a resting tension of 2 g and the heart rate was increased 15–20% above the spontaneous beating rate by using a Grass stimulator (model S88, 10 V, 0.5 msec). During the equilibration period (60 min) the hearts were perfused with a peristaltic pump (Minipuls 3, model 312, Gilson, Villiers, France) using a flow rate of 7 ml/min and then switched to constant flow of 5 ml/min. The right atrial pressure was recorded on a Grass polygraph via a cannula (PE-60) in the inferior vena cava connected to a pressure transducer (model MP-15, Micron Instruments). A glass cannula was inserted into the pulmonary artery for the collection of perfusate. The right atrial pressure could be kept constant at any desired level by adjusting the level of the pulmonary artery cannula tip (15, 16).

Experimental design
After a 10-min control period, a continuous infusion of vehicle or drugs was made via the aortic perfusion cannula using an infusion pump (Secan PSA 55, Skyelectronics S.A., Grenoble, France) at a rate of 0.5 ml/min for 30 or 40 min. Atrial stretch was superimposed for 10 min after 25 min drug infusion by elevating the level of the pulmonary artery cannula tip. The concentrations of lavendustin A (0.5–1.3 µM) used in the present study have been shown to inhibit protein tyrosine kinase activity in A431 cells (17, 18, 19). The concentration of H-89 (100 nmol/liter) was chosen as this concentration has been shown to attenuate cAMP-dependent protein kinase activity in pheochromocytoma cells (21) and inhibit isoprenaline-induced increase in contractile force in isolated perfused rat heart preparation (22). KN-62 was infused at a concentration of 1.5 µM to avoid marked effect on cardiac contractility (20). The concentrations of staurosporine (30–100 nmol/liter), ML-9 (1–3 µM) and okadaic acid (100 nM) were chosen because these concentrations were shown to suppress protein kinase C-mediated responses in the isolated rat heart preparation (13), myosin light chain kinase activity in pancreatic ß cells (23, 24) and protein phosphatases in the heart (25), respectively. All hearts were used only for one experiment, and the study was conducted in a controlled and randomized manner, i.e. vehicle and drugs were run concomitantly and randomly. The coronary venous effluents were collected at 1 or 2 min intervals, placed immediately on dry ice and stored at -20 C until assayed. Control experiments were run with solvents DMSO, ethanol and HCl. Addition of an appropriate concentration of each solvent caused no significant change in hemodynamic variables or cardiac hormone secretion into the perfusate.

Assay of immunoreactive ANP and BNP in the perfusate
ANP and BNP were measured by the RIA as described earlier (12, 26). Perfusate BNP was extracted by Sep-Pak C18 cartridges. The BNP perfusate extracts and unextracted perfusate samples were incubated in duplicates of 100 µl with the rabbit ANP antiserum (final dilution, 1:100,000) or the rabbit BNP antiserum (final dilution, 1:50,000). Synthetic rat ANP99–126 and rat BNP51–95 (ranging from 0 to 160 fmol/tube) were used as standards. The ANP tracer was rat 125I-ANP99–126 and the BNP tracer was prepared by chloramine-T-iodination of synthetic rat Tyr0-BNP51–95, followed by Sephadex G-25 gel filtration and reverse phase HPLC purification. After incubation for 48 h at 8 C, the immunocomplexes were precipitated with goat antiserum against rabbit gammaglobulin in the presence of 8% polyethylene glycol, followed by centrifugation at 3000 x g for 30 min. The sensitivities of the ANP and BNP assays were 1.0 and 0.5 fmol/tube, respectively. The intraassay and interassay variations in both assays were less than 10% and 15%, respectively. Serial dilutions of the perfusate showed parallelism to the synthetic standards. The molecular forms of ANP-like and BNP-like immunoreactive material secreted by the perfused rat hearts were determinated by HPLC analyses as described earlier (12). The ANP and BNP immunoreactivities in the perfusate were almost completely due to processed, active ANP99–126 and BNP77–106 material, respectively (data not shown).

Statistical analysis
The results are expressed as mean ± SEM. The data were analyzed with two- or one-way ANOVA. The statistical significance of the difference between two groups was determined with Student’s t test. Differences at the 95% level were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Atrial wall stretch-induced ANP secretion
In the present study, a modified perfused rat heart preparation was used as an experimental model to analyze the signal transduction pathways involved in atrial wall stretch-induced cardiac hormone secretion. Wall stretch was varied by manipulation of right atrial pressure by means of adjustment of the cannula leading into the pulmonary artery (15, 20). The mean concentration of IR-ANP in the perfusion fluid during control period was 131 ± 8 fmol/ml (n = 119). The basal heart rate was 311 ± 2 beats/min, the perfusion pressure 27 ± 1 mmHg, the contractile force 2.0 ± 0.1 g, and the right atrial pressure 1.5 ± 0.1 mmHg (n = 119). Table 1Go summarizes the basal values for perfusate IR-ANP concentration and hemodynamic variables in each group. When vehicle was infused for 40 min, the perfusion pressure, heart rate, contractile force and right atrial pressure remained constant (Fig. 1Go) showing that the preparation was stable during the period it was used in the studies.


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  		Table 1. Basal hemodynamic variables and the concentration of immunoreactive ANP (IR-ANP) in the perfusate in isolated rat hearts

 


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  		Figure 1. Effects of atrial stretch on hemodynamic variables and immunoreactive ANP (IR-ANP) secretion in the isolated perfused paced rat hearts. At the time 10 min, vehicle was added into the perfusion fluid for 40 min. The right atrium was distended for 10 min (horizontal lines) by elevating the pulmonary artery cannula tip 25 min after the start of vehicle infusion. {circ}, No stretch; •, atrial wall stretch. RAP, right atrial pressure. Values are expressed as mean ± SEM. For number of experiments in each group, see Table 1Go.

 
As shown in Fig. 1Go, when the level of pulmonary artery cannula tip was elevated, right atrial pressure increased immediately. Ten minutes of continuous wall stretch resulted in a significant increase in IR-ANP concentration in the coronary venous effluent of the perfused rat heart. After reduction of the right atrial pressure to its control value by lowering the level of pulmonary artery catheter tip, ANP secretion returned (within 5 min) to its basal level. The right atrial pressure increased similarly in all experimental groups during the stretch (5.3–5.8 mmHg, data not shown). The changes in the contractile force and the perfusion pressure were small (Fig. 1Go) and similar in all experimental groups during the stretch period (data not shown), except that perfusion pressure increased significantly in response to KN-62 infusion (from 33 ± 3 to 40 ± 5 mmHg, P < 0.05). Therefore, this experimental model allowed us to separate direct actions of compounds on ANP secretion from potential indirect secretory effects caused by chemically induced changes in the rate of contraction frequency and contractile force.

Lavendustin A inhibits atrial wall stretch-induced ANP and BNP secretion
To study the role of protein tyrosine kinases in ANP secretion, we infused vehicle or lavendustin A (17, 19) into the perfusion fluid. A 40% decrease in the perfusate IR-ANP concentration toward the end of the experiment was noted during 40-min vehicle infusion without stretch (Fig. 1Go), as previously described (15). Wall stretch during the vehicle infusion increased significantly the perfusate IR-ANP concentration when compared with the infusion of vehicle alone (from 75 ± 18 to 219 ± 43 fmol/ml, F = 8.1, P < 0.001 between stretch and vehicle group, two-way ANOVA) (Fig. 2AGo). Similar elevation in the right atrial pressure during lavendustin A infusion resulted in a significantly smaller increase in the perfusate IR-ANP concentration compared with the infusion of vehicle alone (0.5 µM, 53% reduction of the response, F = 2.6, P < 0.01: 1.3 µM, 68% reduction of the response, F = 3.8, P < 0.001) (Fig. 2AGo), whereas no difference in the hemodynamic variables were noted between the vehicle- and lavendustin A-treated groups (data not shown).



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  		Figure 2. A, Effect of lavendustin A on atrial wall stretch-induced secretion of immunoreactive ANP (IR-ANP) in the isolated perfused paced rat hearts. At the time 10 min, vehicle or lavendustin A was added into the perfusion fluid for 40 min. The right atrium was distended for 10 min (horizontal line) by elevating the pulmonary artery cannula tip 25 min after the start of vehicle infusion. {circ}, Vehicle plus stretch; {blacksquare}, lavendustin A 0.5 µM plus stretch; •, lavendustin A 1.3 µM plus stretch. B, The relation between the change in ANP secretion and the right atrial pressure (RAP) in vehicle and lavendustin A-treated groups. ANPstretch indicates ANP secretion (pmol/5 min) during the last 5 min of stretching, ANPcontrol before stretching (pmol/5 min). C, Effect of lavendustin A on atrial wall stretch-induced secretion of IR-ANP in the isolated perfused paced rat hearts. At the time 10 min, vehicle or lavendustin A were added into the perfusion fluid for 40 min. The right atrium was distended for 10 min (horizontal line) by elevating the pulmonary artery cannula tip 25 min after the start of vehicle infusion. The basal IR-BNP concentration in the perfusate was 1.0 ± 0.2 fmol/ml (n = 17). {circ}, Vehicle plus stretch; •, lavendustin A 1.3 µM plus stretch. D, Effect of lavendustin A on TPA induced increase in IR-ANP secretion in the isolated perfused paced rat hearts. At the time 10 min, as indicated by the horizontal line, vehicle ({circ}, n = 6), lavendustin A 1.3 µM (•, n = 6), TPA 46 nM (, n = 6), or lavendustin A 1.3 µM + TPA 46 nM ({blacksquare}, n = 7) were added into the perfusion fluid for 30 min. IR-ANP secretion is expressed as percentage changes ± SEM. For number of experiments in each group, see Table 1Go. *P < 0.05 (Student’s t test, unpaired).

 
To compare ANP secretion during atrial wall stretch in the presence of lavendustin A or vehicle infusion, the ratio of IR-ANP secreted (pmol per 5 min) during the last 5 min of atrial stretch to the rate of IR-ANP secreted into the perfusate before atrial distension (pmol per 5 min) was calculated in each distension experiment. This change in IR-ANP secretion into the perfusion fluid was then related to increase in right atrial pressure. A 3-mmHg increase in the right atrial pressure induced a 2.77-fold increase in ANP secretion (Fig. 2BGo). During lavendustin A infusion, the relationship between changes in the perfusate IR-ANP concentration and right atrial pressure shifted to the right. The calculated increase in ANP secretion corresponding to the 3-mmHg increase in the right atrial pressure was 1.82-fold and 1.57-fold in the presence of 0.5 µM (P = 0.066) and 1.3 µM (P < 0.05) lavendustin A, respectively (Fig. 2BGo).

Next, we studied the effect of lavendustin A on the secretion of BNP stimulated by atrial wall stretch. Although BNP is mainly synthesized in the cardiac ventricles (27, 28), atrial wall stretch rapidly increases BNP gene expression and secretion from the atria in isolated perfused rat heart preparation (13) as well as in an isolated rat atria preparation (29). The time-course studies have further shown that ANP and BNP are secreted simultaneously from the right atria in response to atria wall stretch (13). In the present study, right atrial wall stretch induced a 2.2-fold increase in IR-BNP secretion from the perfused rat heart (from 1.2 ± 0.3 to 2.6 ± 0.6 fmol/ml) (Fig. 2CGo). Lavendustin A at the concentration of 1.3 µM significantly decreased the stretch-induced IR-BNP secretion (1.7-fold increase; from 0.9 ± 0.2 to 1.4 ± 0.5 fmol/ml; F = 2.2, P < 0.05) (Fig. 2CGo).

Specificity of lavendustin A as a protein tyrosine kinase inhibitor
Several natural and synthetic inhibitors of protein tyrosines kinase have been used to study signal transduction mechanism of growth factors. Some of the inhibitors have shown to affect other kinases such as protein kinase C as well (19, 24). To exclude the possibility that lavendustin A under these experimental conditions has an influence on protein kinase C, we studied the effects of lavendustin A and tumor promoting TPA both alone and in combination on ANP secretion. Vehicle or drugs were infused to the perfusion fluid for 30 min. As reported earlier (15), TPA at the concentration of 46 nM increased ANP secretion by 95% (P < 0.001) (Fig. 2DGo). Because lavendustin A at the concentration of 1.3 µM failed to decrease TPA-induced ANP secretion from the isolated perfused rat heart (Fig. 2DGo), these results indicate that lavendustin A at the concentrations used in this study has no effect on protein kinase C-mediated responses in the adult rat heart.

Okadaic acid enhances atrial wall stretch-induced ANP secretion
Reversible protein phosphorylation is a critical component of the signal transduction mechanisms by which extracellular signals regulate cellular processes. In addition to protein kinases, extracellular effectors act by modulating protein phosphatases (PP), which catalyze the dephosphorylation of proteins on their serine, threonine, and tyrosine residues (30). For example, several components of the MAP/ERK pathways are subject to regulation by protein phosphatase, PPA2, which causes inhibition of kinase activity (30). In the present study, okadaic acid, specific and potent PPA2, and PP1 inhibitor (31) had no effect on basal ANP secretion at the concentration of 100 nM but caused the ANP secretory response to right atrial wall stretch to appear significantly earlier (F = 3.2, P < 0.05, okadaic acid plus stretch vs. stretch, during the first 5 min of stretch) (Fig. 3Go). The calculated increase in ANP secretion corresponding to the 2-mmHg increase in the right atrial pressure during the first 3 min of wall stretch was 2.03-fold in the presence of okadaic acid and 1.45-fold in the vehicle group (P < 0.05). Okadaic acid had no statistically significant effect on the maximal wall stretch-induced ANP secretion (Fig. 3Go).



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  		Figure 3. Effect of okadaic acid on atrial wall stretch-induced secretion of immunoreactive ANP (IR-ANP) in the isolated perfused paced rat hearts. A, At the time 10 min, vehicle or okadaic acid (100 nM) were added into the perfusion fluid for 40 min. The right atrium was distended for 10 min (horizontal line) by elevating the pulmonary artery cannula tip 25 min after the start of vehicle infusion. {circ}, Vehicle plus atrial wall stretch; •, okadaic acid plus atrial wall stretch. B, The relation between the change in ANP secretion and the right atrial pressure (RAP) in vehicle and okadaic acid-treated groups. ANPstretch indicates ANP secretion (pmol/3 min) during the first 3 min of stretching, ANPcontrol before stretching (pmol/5 min). For number of experiments in each group, see Table 1Go. *P < 0.05 (Student’s t test, unpaired).

 
Effect of inhibitors of PKC, protein kinase A, calcium/calmodulin and myosin light chain kinase on wall stretch-induced ANP secretion
Because it is possible that there is a hierarchy in the activation of different second messenger pathways, the inhibition of atrial wall stretch-induced ANP secretion by lavendustin A may be due to the inhibition of stretch-induced phospholipase C activation, causing inhibition of phosphoinositol turnover, Ca2+ release and protein kinase C, which have been previously reported to influence cardiac hormone secretion (8). To further characterize the selectivity of the action of lavendustin A on wall stretch-induced ANP secretion, we infused the protein kinase C inhibitor staurosporine (100 nM), Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62 (1.5 µM), protein kinase A inhibitor H-89 (100 nM) and myosin light chain kinase inhibitor ML-9 (1 µM) into the perfusate. None of these inhibitors was capable of decreasing statistically significantly the right atrial wall stretch-induced increase in IR-ANP secretion (Fig. 4Go). The calculated increase in ANP secretion corresponding to the 3 mmHg increase in right atrial pressure were 2.27-, 2.82-, 2.50-, and 2.38-fold for staurosporine, KN-62, H-89 and ML-9 groups (data not shown). Previously, we have shown that under these experimental conditions coronary vasoconstrictor and ANP secretory responses induced by phorbol ester TPA were completely abolished by staurosporine at the concentrations from 10 to 100 nM (12, 32).



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  		Figure 4. Effects of various protein kinase inhibitors on the atrial wall stretch-induced release of immunoreactive atrial natriuretic peptide (IR-ANP) in the isolated perfused paced rat hearts. At the time 10 min, vehicle ({circ}), staurosporine 100 nM (•), KN-62 1.5 µM (•), H-89 100 nM (•) or ML-9 1 µM (•) were added into the perfusion fluid for 40 min. The right atrium was distended for 10 min (horizontal line) by elevating the pulmonary artery cannula tip 25 min after the start of vehicle or drug infusion. The values are expressed as percentage changes ± SEM. For number of experiments in each group, see Table 1Go.

 
Lavendustin A but not staurosporine inhibits ANP secretion induced by sustained increase in atrial wall stretch
Finally, in a separate series of the experiments, the effects of inhibitors of protein tyrosine kinase and protein kinase C on ANP secretion stimulated by sustained increase in atrial wall stretch were studied. The right atria were stretched for 2 h during infusion of lavendustin A, staurosporine, or their combination. Hemodynamic variables did not differ between the vehicle- and drug-infused groups (data not shown). During continuous atrial wall stretch, the maximum increase of ANP secretion was seen after 10 min distension. Thereafter, the peptide levels gradually decreased toward the end of the experimental period but remained elevated compared with those in the unstretched hearts (at 2 h, P < 0.05). Administration of staurosporine at the concentration of 30 nM had no effect on the wall stretch-induced ANP secretion but lavendustin A (1 µM) either alone or in combination with staurosporine (30 nM) inhibited significantly sustained stretch-induced increase in perfusate ANP levels (Fig. 5Go) (F = 1.9, ns., lavendustin A plus stretch vs. vehicle plus stretch). Thus, under these experimental conditions, lavendustin A consistently inhibited ANP secretion in response to both acute and sustained increase in right atrial wall stretch.



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  		Figure 5. Effects of lavendustin A (1 µM), staurosporine (30 nM), or their combination on sustained right atrial wall stretch-induced secretion of immunoreactive ANP (IR-ANP) in the isolated perfused paced rat hearts. After a 10-min control period, vehicle or protein kinase inhibitors were added into the perfusate for 2 h (horizontal lines) during stretch of the right atria using pressure level of 5 mmHg. {circ}, no stretch; •, stretch. Each point represents the mean ± SEM of six experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mechanical stretch of cardiac myocytes in vitro causes an activation of multiple second messenger systems that are very similar to growth-factor induced cell signaling systems. A number of peptide growth factors including transforming growth factor-ß1, acidic, and basic fibroblast growth factors have been identified in the adult heart and shown to induce myocyte hypertrophy and modulate cardiac gene expression (33). Peptide growth factors act by binding to and activating specific receptors with intrinsic protein tyrosine kinase activity (34, 35). Receptor tyrosine kinases have transmembrane segments, and some of the nonreceptor-type tyrosine kinases, such as Src family tyrosine kinases, are anchored to the inner surface of cell membranes. Thus, it is possible that membrane stretch directly causes conformational change of tyrosine kinases, thereby activating them. Our present study shows that right atrial wall stretch-induced ANP and BNP secretion was markedly decreased by lavandustin A, a potent protein tyrosine kinase inhibitor. This dose-dependent inhibition of wall stretch-induced increase in cardiac hormone secretion occurred at the concentrations similar to or even below those shown to inhibit the activities of protein tyrosine kinase in vitro (17, 18).

Several natural and synthetic inhibitors of protein tyrosine kinases have been used to study the physiological and pathophysiological role of protein tyrosine kinases. These inhibitors at micromolar concentrations have been shown to inhibit cell proliferation, DNA synthesis, proto-oncogene gene expression, and phosphatidylinositol turnover caused by several growth factors (19, 36, 37, 38). However, some of them have shown to inhibit other protein kinases including protein kinase C (19, 24) and they may have effects other than direct inhibition of protein tyrosine kinase activity. For example, genistein, which in neonatal cardiac myocytes suppresses mechanical stretch-induced c-fos activation (5), increased cardiac contractile force and basal ANP secretion in isolated perfused rat heart preparation (20). In the present study, we used lavendustin A, a competitive inhibitor of ATP binding to the catalytic domain of tyrosine kinases (17), to study the potential role of protein tyrosine kinases in regulation of wall stretch-induced ANP and BNP secretion. It is unlikely the effects of lavendustin A were nonspecific because at the concentration used in the present study (maximally 1.3 µM) lavendustin A had no effects on basal cardiac hormone secretion or cardiac function. Indeed, a substantially higher concentration (26 µM) of lavendustin A was necessary to influence basal ANP secretion and hemodynamics in the isolated perfused rat heart (unpublished observation, Taskinen and Ruskoaho). Lavendustin A also failed to decrease TPA-induced ANP secretion, indicating that lavendustin A has no influence on protein kinase C-mediated responses. Because lavendustin A has been shown to be a selective protein tyrosine kinase inhibitor and does not show activity on cAMP-dependent protein kinase, myosin light chain kinase or serine kinases (17, 19, 24), our results indicate that protein tyrosine kinases are involved in the regulation of wall stretch-induced cardiac hormone secretion.

In support for the involvement of protein tyrosine kinases in regulating cardiac hormone secretion, wall stretch-induced ANP secretion was specifically inhibited by lavendustin A but not by protein kinase C and several other protein kinase inhibitors. Previously, inhibitors of protein kinase C have been reported to either decrease (39) or have no effect (40) on stretch-induced ANP secretion in isolated rat atria preparations. Yet, staurosporine at a low concentration (10 nM) was potent inhibitor of ANP secretion produced by passive left ventricular wall stretch suggesting that protein kinase C pathway may play an important role in the regulation of ventricular stretch-stimulated ANP secretion (12). In agreement with our present results, hypotonic-swelling induced c-fos gene expression was abolished by tyrosine kinase inhibitors but not by inhibitors of protein kinase C and phospholipase C (41). In contrast, mechanical stretch-induced c-fos gene induction was inhibited by inhibitors of tyrosine kinases, protein kinase C, and phospholipase C (5). Protein kinase C and protein tyrosine kinase activities may both be also involved in coupling cardiac overload to alterations in atrial BNP synthesis because lavendustin A and staurosporine inhibited stretch-induced increase in atrial BNP concentrations in perfused rat hearts (42). Thus, although mechanical stretch activates multiple signaling mechanisms in the heart, specific protein kinase pathways seems to be important for different cellular process, and of those pathways, protein tyrosine kinase activity appears to be required for wall stretch-induced ANP and BNP secretion. It remains to be determined, however, which tyrosine kinase is responsible for wall stretch-induced cardiac hormone secretion. Mechanical stretch caused activation of Src within 5 min in fetal lung cells (43) and an increase in tyrosine phoshorylation of focal adhesion kinase in mesangial cells (44).

The mechanism of tyrosine kinase activation by mechanical wall stress as well as the following activation of downstream signaling pathways are yet unclear. Yet, it is noteworthy that mechanical stress activates tyrosine kinase very rapidly. In cardiac myocytes, mechanical stretch causes a significant increase in phosphotyrosine content of proteins, such as p42 and p44, within 1 min (5). In cultured neonatal myocytes phorbol esters, endothelin-1 (45) and mechanical stretch (5) have been shown to stimulate tyrosine phosphorylation of MAP kinases, a family of related serine/threonine kinases whose activities are dependent on phosphorylation of both tyrosine and threonine residues (46). These dual specificity protein kinases (MAPK) are in turn activated by serine phosphorylation by MAPKK kinase. Several components of this ERK1/ERK2 pathways are subject to regulation by protein phosphatase, PPA2, which causes dephosphorylation of threonine and inhibition of kinase activity. Our finding that okadaic acid, a potent inhibitor of PP2A2 and a strong inhibitor of PP1 (31), can accelerate wall stretch-induced ANP secretion suggest that wall stretch-induced ANP hormone secretion may involve activation of protein tyrosine kinase pathway modulated by MAP/ERK pathways, although many other possibilities also exists. Nevertheless, because the only targets of okadaic acid are the catalytic subunits of PPA2 and PP1 (30, 31), these enzymes appear to play a significant role in atrial wall stretch-induced ANP secretion. Furthermore, the findings that okadaic acid enhanced and lavendustin decreased significantly ANP secretion show that a precise balance of protein tyrosine kinase and protein phosphatase activity plays a major role in mechanical stretch-induced ANP secretion.

Although lavendustin A inhibited ANP secretion, the effect may not be direct but mediated by an stimulator of protein tyrosine kinases, which can be activated or released locally by mechanical stretch. Indeed, previous studies have shown that mechanical stretch causes release of factor(s) into the culture medium, which in turn induces c-fos expression and activates MAP kinases (5). Endogenous paracrine/autocrine factors such as angiotensin II and endothelin-1 liberated in response to mechanical stretch rather than direct stretch appear to be responsible for the activation of cardiac gene expression in neonatal ventricular myocytes (6, 47). Several studies have demonstrated that endothelin-1 and angiotensin II signal through the protein tyrosine kinase-dependent mechanism (48, 49, 50). However, because the release of ANP by mechanical stretch takes place in the presence of treatment with an angiotensin II type 1 antagonist losartan (51, 52), it is unlikely that angiotensin II is involved in mediating the wall stretch-induced ANP secretion observed in the present study. Furthermore, a mixed ETA/ETB receptor antagonist bosentan did not modulate the atrial wall stretch-induced ANP secretion under these experimental conditions (Taskinen, P., O. Vuolteenaho, and H. Ruskoaho, unpublished observation) showing that endothelin-1, which is the most potent ANP secretatogue in the isolated perfused rat heart prepation yet identified (8), is not directly involved in regulating atrial wall stretch-induced ANP secretion. These experiments, however, do not exclude the possibility that other autocrine and/or paracrine factors are released, which may be capable of stimulating protein tyrosine kinase activity and result in wall stretch-induced cardiac hormone secretion. Thus, elucidation of the mechanism of mechanical stretch-induced tyrosine kinase activation seems essential to determine whether protein tyrosine kinase may be a direct mechanosensor for cardiac hormone secretion.

In conclusion, we have shown for the first time that stimulation of ANP and BNP secretion in vitro by atrial wall stretch is inhibited by lavendustin A, at the concentrations similar to or below those shown to inhibit the activities of protein tyrosine kinases. In contrast, inhibitors of protein kinase C, Ca2+/calmodulin-dependent protein kinases, protein kinase A and myosin light chain protein kinase failed to decrease stretch-stimulated ANP secretion showing an important regulatory role of protein tyrosine kinase in ANP secretion. The finding that okadaic acid enhanced ANP secretion suggests that protein phosphatases may play a regulatory role in mechanical stretch-induced cardiac hormone exocytosis from atrial myocytes, possibly by dephosphorylating signaling molecules activated by protein tyrosine kinases.


   Acknowledgments
 
We thank Mrs. Marja-Leena Vainikka for expert technical assistance.


   Footnotes
 
1 This study was supported by the Medical Research Council of the Academy of Finland, Sigfrid Juselius Foundation, the Finnish Foundation for Cardiovascular Research, Ida Montin Foundation and Finnish Cultural Society. Back

Received January 21, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Watson PA 1991 Function follows form: generation of intracellular signals by cell deformation. FASEB J 5:2013–2019[Abstract]
  2. Vandenburgh HH 1992 Mechanical forces and their second messengers in stimulating cell growth in vitro. Am J Physiol 262:R350–R355
  3. Davies PF, Tripathi SC 1993 Mechanical stress mechanism and the cell. Circ Res 72:239–245[Abstract/Free Full Text]
  4. von Harsdorf R, Lang RE, Fullerton M, Woodcock EA 1989 Myocardial stretch stimulates phosphatidylinositol turnover. Circ Res 65:494–501[Abstract/Free Full Text]
  5. Sadoshima JI, Izumo S 1993 Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism. EMBO J 12:1681–1692[Medline]
  6. Sadoshima J, Izumo S 1997 The cellular and molecular response of cardiac myocytes to mechanical stress. Annu Rev Physiol 59:551–571[CrossRef][Medline]
  7. Nakao K, Ogawa Y, Suga SI, Imura H 1992 Molecular biology and biochemistry of the natriuretic peptide system. I: Natriuretic peptides. J Hypertens 10:907–912[Medline]
  8. Ruskoaho H 1992 Atrial natriuretic peptide: synthesis, release, and metabolism. Pharmacol Rev 44:479–602[Medline]
  9. Lang RE, Thölken H, Ganten D, Luft FC, Ruskoaho H, Unger T 1985 Atrial natriuretic factor-a circulating hormone stimulated by volume loading. Nature 314:264–266[CrossRef][Medline]
  10. Ruskoaho H, Tholken H, Lang R 1986 Increase in atrial pressure releases atrial natriuretic peptide from isolated perfused rat hearts. Pfluegers Arch 407:170–174[CrossRef][Medline]
  11. Kinnunen P, Vuolteenaho O, Uusimaa P, Ruskoaho H 1992 Passive mechanical stretch releases atrial natriuretic peptide from rat ventricular myocardium. Circ Res 70:1244–1253[Abstract/Free Full Text]
  12. Kinnunen P, Vuolteenaho O, Ruskoaho H 1993 Mechanism of the atrial and brain natriuretic peptide release from rat ventricular myocardium: effect of stretching. Endocrinology 132:1691–1970
  13. Mäntymaa P, Vuolteenaho O, Marttila M, Ruskoaho H 1993 Atrial stretch induces rapid increase in brain natriuretic peptide but not in natriuretic peptide gene expression in vitro. Endocrinology 133:1470–1473[Abstract/Free Full Text]
  14. de Bold AJ, Bruneau BG, Kuroski de Bold ML 1996 Mechanical and neuroendocrine regulation of the endocrine heart. Cardiovasc Res 31:7–18[CrossRef][Medline]
  15. Ruskoaho H, Vuolteenaho O, Leppäluoto J 1990 Phorbol esters enhance stretch-induced atrial natriuretic peptide secretion. Endocrinology 127:2445–2455[Abstract/Free Full Text]
  16. Taskinen P, Ruskoaho H 1996 Stretch-induced increase in atrial natriuretic peptide secretion is blocked by thapsigargin. Eur J Pharmacol 308:295–300[CrossRef][Medline]
  17. OnodaT, Iinuma H, Sasaki Y, Hamada M, Isshiki K, Naganawa H, Takeuchi T, Tatsuta K, Umezawa K 1989 Isolation of a novel tyrosine kinase inhibitor, lavendustin A, from Streptomyces griseolavendus. J Nat Prod 52:1252–1257[CrossRef][Medline]
  18. Onoda T, Isshiki K, Takeuchi T, Umezawa K 1990 Inhibition of tyrosine kinase and epidermal growth factor receptor internalization by lavendustin A methyl ester in cultured A431 cells. Drugs Exp Clin Res 16:249–253[Medline]
  19. Levitzki A 1992 Tyrphostins: tyrosine kinase blockers as novel antiproliferative agents and dissectors of signal transduction. FASEB J 6:3275–3282[Abstract]
  20. Taskinen P, Toth M, Ruskoaho H 1994 Effect of genistein on cardiac contractile force and atrial natriuretic peptide release in the isolated perfused rat heart. Eur J Pharmacol 256:251–261[CrossRef][Medline]
  21. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H 1990 Inhibition of forskolin-induced neurite outgrowth and protein phosphorulation by a newly synthesized selective inhibitor of cyclic AMP dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:5267–5272[Abstract/Free Full Text]
  22. Szokodi I, Kinnunen P, Tavi P, Weckström M, Toth M, Ruskoaho H 1998 Evidence for cAMP-independent mechanisms mediating the effects of adrenomedullin, a new inotropic peptide. Circulation 97:1062–1070[Abstract/Free Full Text]
  23. Niki I, Okazaki K, Saitoh M, Niki A, Niki H, Tamagawa T, Iguchi A, Hidaka H 1993 Presence and possible involvement of Ca/calmodulin-dependent protein kinase in insulin release from the rat pancreatic ß cells. Biochem Biophys Res Commun 191:255–261[CrossRef][Medline]
  24. Hidaka H, Kobayashi R 1992 Pharmacology of protein kinase inhibitors. Annu Rev Pharmacol Toxicol 32:377–397[CrossRef][Medline]
  25. Neumann J, Boknik P, Herzig S, Schmitz W, Scholz H, Gupta RC, Watanabe AM 1993 Evidence for physiological functions of protein phosphatases in the heart: evaluation with akadaic acid. Am J Physiol 265:H257–H266
  26. Vuolteenaho O, Arjamaa O, Ling N 1985 Atrial natriuretic polypeptides (ANP): rat atria store high molecular weight precursor but secrete processed peptides of 25–35 amino acids. Biochem Biophys Res Commun 129:82–88[CrossRef][Medline]
  27. Ogawa Y, Nakao K, Mukoyama M, Hosoda K, Shirakami G, Arai H, Saito Y, Suga S-I, Jougasaki M, Imura H 1991 Natriuretic peptides as cardiac hormones in normotensive and spontaneously hypertensive rats. The ventricle is a major site of synthesis and secretion of brain natriuretic peptide. Circ Res 69:491–500[Abstract/Free Full Text]
  28. Yasue H, Yoshimura M, Sumida H, Kikuta K, Kugiyama K, Jougasaki M, Ogawa H, Okumura K, Mukoyama M, Nakao K 1994 Localization and mechanism of secretion of B-type natriuretic peptide in comparison with those of A-type natriuretic peptide in normal subjects and patients with heart failure. Circulation 90:195–203[Abstract/Free Full Text]
  29. Bruneau BG, Piazza LA, de Bold AJ 1996 Alpha 1-adrenergic stimulation of isolated rat atria results in discoordinate increases in natriuretic peptide secretion and gene expression and enhances Egr-1 and c-Myc expression. Endocrinology 137:137–143[Abstract]
  30. Hunter T 1995 Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80:225–236[CrossRef][Medline]
  31. Cohen P, Holmes CFB, Tsukitani Y 1990 Okadaic acid, a new probe for the study of cellular regulation. Trends Biochem Sci 15:98–102[CrossRef][Medline]
  32. Pitkänen M, Mäntymaa P, Ruskoaho H 1991 Staurosporine, a protein kinase C inhibitor, inhibits atrial natriuretic peptide secretion induced by sarafotoxin, endothelin and phorbol ester. Eur J Pharmacol 195:307–315[CrossRef][Medline]
  33. Schneider MD, Parker TG 1990 Cardiac myocytes as targets for the action of peptide growth factors. Circulation 81:1443–1456[Free Full Text]
  34. Ullrich A, Schlessinger J 1990 Signal transduction by receptors with tyrosine kinase activity. Cell 61:203–212[CrossRef][Medline]
  35. Van der Geer P, Hunter T 1994 Receptor protein-tyrosine kinases and their signal transduction pathways. Annu Rev Cell Biol 10:251–337[CrossRef]
  36. Lyall RM, Zilberstein A, Gazit A, Gilon C, Levitzki A, Schlessinger J 1989 Tyrphostins inhibit epidermal growth factor (EGF)-receptor tyrosine kinase activity in living cells and EGF-stimulated cell proliferation. J Biol Chem 264:14503–14509[Abstract/Free Full Text]
  37. Margolis B, Rhee SG, Felder S, Mervic M, Lyall R, Levitzki A, Ullrich A, Zilberstein A, Schlessinger J 1989 EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism of EGF receptor signalling. Cell 57:1101–1107[CrossRef][Medline]
  38. Hill TD, Dean NM, Mordan LJ, Lau AF, Kanemitsu MY, Boynton AL 1990 PDGF-induced activation of phospholipase C is not required for induction of DNA synthesis. Science 248:1660–1663[Abstract/Free Full Text]
  39. Page E, Goings GE, Power B, Upshaw Earley J 1991 Basal and stretch-augmented natriuretic peptide secretion by quiescent rat atria. Am J Physiol 259:C801–C818
  40. Ishida A, Tanahashi T, Okumura K, Hashimoto H, Ito T, Ogawa K, Satake T 1988 A calmodulin antagonist (W-7) and protein kinase C inhibitor (H-7) have no effect on atrial natriuretic peptide release induced by atrial stretch. Life Sci 42:1659–1667[CrossRef][Medline]
  41. Sadoshima J, Qiu Z, Morgan JP, Izumo S 1996 Tyrosine kinase activation is an immediate and essential step in hypotonic cell swelling-induced ERK activation and c-fos gene expression in cardiac myocytes. EMBO J 15:5535–5546[Medline]
  42. Magga J, Vuolteenaho O, Tokola H, Marttila M, Ruskoaho H 1997 Involvement of transcriptional and posttranscriptional mechanisms in cardiac overload-induced increase of B-type natriuretic peptide gene expression. Circ Res 81:694–702[Abstract/Free Full Text]
  43. Liu M, Qin Y, Liu J, Tanswell AK, Post M 1996 Mechanical strain induces pp60src activation and translocation to cytoskeleton in fetal rat lung cells. J Biol Chem 271:7066–7071[Abstract/Free Full Text]
  44. Hamasaki K, Mimura T, Furuya H, Morino N, Yamazaki T, Komuro I, Yazaki Y, Nojima Y 1995 Stretching mesangial cells stimulates tyrosine phosphorylation of focal adhesion kinase pp125FAK. Biochem Biophys Res Commun 212:544–549[CrossRef][Medline]
  45. Bogoyevitch MA, Glennon PE, Sugden PH 1993 Endothelin-1, phorbol esters and phenylephrine stimulate MAP kinase activities in ventricular cardiomyocytes. FEBS Lett 3:271–275
  46. Anderson NG, Maller JL, Tonks NK, Sturgill TW 1990 Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature 324:651–653[CrossRef]
  47. Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I, Hiroi Y, Mizuno T, Maemura K, Kurihara H, Aikawa R, Takano H, Yazaki Y 1996 Endothelin-1 is involved in mechanical stress-induced cardiomyocyte hypertrophy. J Biol Chem 271:3221–3228[Abstract/Free Full Text]
  48. Force T, Kyriakis JM, Avruch J, Bonventre JV 1991 Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells. J Biol Chem 266:6650–6656[Abstract/Free Full Text]
  49. Molloy CJ, Taylor DS, Weber H 1993 Angiotensin II stimulation of rapid protein tyrosine phosphorylation, and protein kinase activation in rat aortic smooth muscle cells. J Biol Chem 268:7338–7345[Abstract/Free Full Text]
  50. Simonson MS, Herman WH 1993 Protein kinase C and protein tyrosine kinase activity contribute to mitogenic signalling by endothelin-1. J Biol Chem 268:9347–9357[Abstract/Free Full Text]
  51. Leskinen H, Vuolteenaho O, Ruskoaho H 1997 Combined inhibition of endothelin and angiotensin II receptors blocks volume load-induced cardiac hormone release. Circ Res 80:114–123[Abstract/Free Full Text]
  52. Magga J, Vuolteenaho O, Marttila M, Ruskoaho H 1997 Endothelin-1 is involved in stretch-induced early activation of B-type natriuretic peptide gene expression in atrial but not in ventricular myocytes. Circulation 1997:3053–3062



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