Introduction

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Although EGF acting via its tyrosine kinase receptor is widely recognized for its mitogenic and acid-inhibitory activity (Cohen, 1962; Gregory, 1975), it is now appreciated that this peptide can also modulate the contractility of a variety of smooth muscle preparations, including those coming from vascular and stomach tissue (Berk et al., 1985; Muramatsu et al., 1985, 1988; Hollenberg, 1994a, b). Thrombin, best known for its role in the coagulation cascade, is now also known to cause a variety of responses in target tissues ranging from platelets to the vasculature. Thrombin acts on its target tissues via a G-protein-coupled receptor (PAR₁: Vu et al., 1991; Rasmussen et al., 1991) and as with EGF, exhibits potent mitogenic activity in a variety of cell systems. Also as with EGF, thrombin can regulate gastric smooth muscle contractility via a signaling pathway that depends both on cyclooxygenase- and tyrosine kinase-mediated events (Hollenberg 1994a,b, 1996). Although not yet established, it is possible that the contractile actions caused by PAR₁ or EGF receptor activation in gastric or vascular smooth muscle may play a role in the settings of tissue injury or inflammation. The intriguing mechanism whereby thrombin activates its G-protein-coupled receptor involves the proteolytic unmasking of an N-terminal anchored receptor-activating ligand (Vu et al., 1991; Coughlin et al., 1992). Strikingly, short synthetic peptides, based on the proteolytically revealed receptor-activating sequence, can on their own activate the thrombin receptor, so as to mimic the actions of thrombin in many tissues, including not only platelets (Vu et al., 1991) but also vascular and gastric smooth muscle (Muramatsu et al., 1992; Simonet et al., 1992; DeBlois et al., 1992; Yang et al., 1992a). Structure-activity studies of the receptor-activating peptides in work by us (Hollenberg et al., 1997; Kawabata et al., 1997) and by others (Blackhart et al., 1996) have led to the development of peptides that activate selectively either the thrombin receptor (PAR₁APs) or the closely related receptor (PAR₂) that is activated by trypsin, but not thrombin (Nystedt et al., 1994, 1995). In our own work (Hollenberg et al., 1997), the PAR₁AP, TF has been found to activate PAR₁ selectively compared with PAR₂ both in intact tissue and in isolated cell assays; and new data indicate that this peptide will not activate the recently described thrombin receptor, PAR₃, which as with PAR₁ is activated specifically by thrombin but not by trypsin (Ishihara et al., 1997).

In our initial work with PAR₁APs, we noted as outlined above, that in the gastric LM preparation obtained from either rats or guinea pigs, there are parallels in the contractile actions of the growth factor EGF and PAR₁-activating peptides, in that both contractile responses can be blocked by the cyclooxygenase inhibitor, indomethacin and the tyrosine kinase inhibitors, genistein and tyrphostin (Hollenberg et al., 1992). After the completion of that work, a number of issues have come to light that have prompted us to examine in more depth, the activation of the LM PAR₁ in comparison with the activation of the LM EGF receptor; and to compare more fully the growth factor signaling pathways that are involved in the contractile actions of the EGF and PAR₁ receptor systems in gastric LM tissue. First, it is now clear that the PAR₁AP, SFLLR-NH₂ used in our previous work with gastric LM tissue can activate both PAR₁ and PAR₂ (Blackhart et al., 1996; Hollenberg et al., 1997); these two receptors are both present in gastric LM tissue (Al-Ani et al., 1995). Second, it is now recognized that some actions of G-protein-coupled receptor agonists, including thrombin, may possibly result from transactivation of the EGF receptor (Daub, et al., 1996)., and we wished to rule out this mechanism for the contractile effect of PAR₁ activation in smooth muscle. It has been our working hypothesis, that because EGF and thrombin can both activate similar "growth factor" responses in fibroblast mitogenesis assays, the two receptor systems (i.e., PAR₁ and EGF receptor kinase) may also activate common "growth factor-"related signaling pathways to cause a rapid contractile response in smooth muscle systems. In view of the two concerns outlined in the above paragraph and in view of our working hypothesis that EGF and thrombin may activate common signaling pathways to cause a rapid contractile response in smooth muscle, we have used the PAR₁-selective agonist, TF, to activate PAR₁ in guinea pig gastric LM tissue for comparison with the activation of the EGF receptor. The aim of our work was to study the contractile response of the guinea pig LM preparation using several tyrosine kinase inhibitors, including the one targeted to src-family kinases (CP 118, 556 or PP1: Hanke et al., 1996), along with other signal pathway probes [PD153035, a high potency specific inhibitor of the EGF receptor kinase (Fry et al., 1994); PD98059, an inhibitor of mitogen-activated protein kinase kinase or MEK (Dudley et al., 1995); Wortmannin (WM) and LY294002 (inhibitors of PI3K) (Ui et al., 1995); the kinase C inhibitor GF109203X (Toullec et al., 1991) and the diacylglycerol lipase inhibitor, U57, 908 (Sutherland and Amin, 1982; Yang et al., 1991)]. These enzyme inhibitor reagents were used to compare in parallel the contractile activation pathways for PAR₁ and the EGF receptor. Further, in the context of contractile response both PAR₁ and EGF receptor-induced contractile responses, we evaluated increases in tissue phosphotyrosyl proteins and in the activity of the cellular nonreceptor tyrosine kinase, c-src. One major goal was to establish in further depth the signal pathways activated by PAR₁ in an intact smooth muscle system, and to compare these pathways with the signal pathways activated by EGF.

	Methods

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Tissue preparations for bioassay. The guinea pig gastric LM and CM preparations were prepared as described previously (Muramatsu et al., 1988; Yang et al., 1993) from male albino Hartley strain animals weighing about 350 g. Animals were cared for in accordance with the Canadian Council on Animal Care and were killed by rapid cervical dislocation, followed by exsanguination via the common carotid arteries and excision of the stomach tissue. The stomach was stripped carefully of overlying muscosa under a dissecting microscope after which the CM and LM preparations were prepared by cutting along (CM tissue) or at right angles (LM tissue) to the visible circular muscle bundles. This procedure permits the measurements of the contraction of either the LM or CM elements in the same tissue sample (Muramatsu et al., 1988). The width and length of the preparations were approximately 3 × 10 mm, respectively. The tissue preparations were mounted vertically in a plastic cuvette thermostated at 37°C, containing 4 ml of gassed (95% O₂/5% CO₂) Krebs-Henseleit solution pH 7.4 of the following composition (millimolar): NaCl, 118; KCl, 4.7; CaCl₂ 2.5; MgCl₂, 1.2; NaHCO₃, 25; KH₂PO₄, 1.2 and glucose, 10; in distilled deionized water. A tension of 1 g was applied and the tissue was allowed to equilibrate at 37°C for about 1 hr. Contractile responses were recorded isometrically through force-displacement transducers (either Statham UTC2 or Grass). Routinely, the integrity of each preparation was assessed by monitoring the contractile responses to 50 mM KCl and 1 µM Cch. For concentration-response curves, the contractile actions of increasing concentrations of TFLLR-NH₂ in the LM preparation (measured at the peak of the contractile response, e.g., fig. 2A) were expressed as a percentage (% KCl) of the contraction caused by 50 mM KCl (1.2 ± 0.3 g for n = 25) in the same tissue preparation. The assay of peptide activity was done in the presence of amastatin (10 µM) to inhibit aminopeptidase activity.

Western blot analysis and assay of Src-tyrosine kinase activity. LM tissue strips to be used for Western blot analysis and for the assay of Src-tyrosine kinase activity were prepared and treated with agonists (17 nM EGF or 1 µM TF) exactly as for the LM bioassay and were removed from the organ bath for processing at a time corresponding to the peak of tissue contraction, as detailed in previous work from this laboratory (Yang et al., 1992b, 1993). For Western blot analysis, tissue was quick-frozen on solid CO₂ and stored at -70°C for further analysis. Tissue was quick-thawed and homogenized in a 50 mM Tris HCl buffer, pH 7.4, containing protease inhibitors (0.2 mg/ml benzamidine, 0.1 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin and 0.3 mM PMSF) and supplemented with 1 mM EDTA, 2 mM MgCl₂ and 0.2 mM Na₂VO₄. Debris was removed from the homogenate, by low speed centrifugation (3000 RPM × 30 min at 4°C; Beckman TI65 rotor). Equal aliquots of protein extract (corrected for buffer blank: Bradford Reagent (Bio-Rad, Missisauga, Ontario, Canada) were combined with an equal volume of 2x concentrated immunoprecipitation buffer pH 7.4, that at 1x concentration contained 50 mM Tris HCl, 1 mM EGTA, 0.2 mM NO₂VO₄, 0.2 mM PMSF, 150 mM NaCl, 0.5% v/v NP-40 and 1% v/v triton X-100. For each tissue sample, equal amounts of protein extract were subjected to immunobead purification using monoclonal antiphosphotyrosine antibody (6D9) prepared according to Glenney et al. (1988) and coupled to Sepharose beads. The final washed bead pellet was solubilized in boiling electrophoresis sample buffer (Laemmli, 1971) in preparation for polyacrylamide gel electrophoresis (70 × 100 × 1.5 mm, 8% gel) and transfer to nitrocellulose (0.45 µm; Bio-Rad, Richmond, CA) for Western blot detection of protein. The same monoclonal antiphosphotyrosine antibody (6D9) coupled directly to horseradish peroxidase was used for visualization of membrane-bound proteins using enhanced luminescence detection (Amersham, Oakville, Ontario, Canada). Control experiments demonstrated that the phosphotyrosyl protein signal could be quenched by pretreatment of the antibody with a phosphotyrosyl hapten (either 12.5 mM phenylphosphate or 5 mM phosphotyrosine). Tissue to be analysed for Src-kinase activity was extracted with 0.5% NP-40/1% Triton x-100 as described above for immunoprecipitation. For each tissue sample, equal amounts of protein in the clarified detergent-containing tissue extract were supplemented with monoclonal anti-Src antibody 327 (Oncogene Sciences, Manhasset, NY) followed 1.5 hr thereafter by the addition of rabbit anti mouse immunoglobin (5 µg/ml, 1.5 hr at 4°C) and assayed for Src-kinase activity, using the cdc2 (6-20) peptide as a substrate (KVEKIGEGTYGVVYK-NH₂) essentially as described previously (Cheng et al., 1992). Before the assay, the immune complex was washed 3x with the immunoprecipitation buffer (above) and once with the kinase assay buffer (below). Assays were performed at 30°C over a 20-min interval in a 50 µl final reaction volume, using a kinase assay buffer, pH 7.0, comprising 50 mM Tris-HCl, 50 mM MgCl₂, 10 mM MnCl₂, 50 µM Na₃VO₄, 7 mg/ml p-nitrophenylphosphate (2 mM) and 100 µM  ³²P-ATP (range of specific activity, 300-1000 CPM/pmol). The cdc2 peptide substrate was at a concentration of 300 µM. The reaction was started by the addition of  ³²P-ATP and terminated 20 min thereafter by the addition of 25 µl of 50% (v/v in H₂O) acetic acid. The phosphorylated substrate was recovered by spotting 60-µl aliquots of the acidified reaction mixture on Whatman P81 phosphocellulose paper. cdc2-peptide-free reaction mixtures were used as a "background" blank. The washed [3x with 0.5% (v/v in H₂O) H₃PO₄] filter papers were acetone-extracted and air-dried before the measurement of incorporated radioactivity by scintillation counting. Under these conditions, it has been established (Cheng et al., 1992) that phosphate incorporation is linear over at least 30 min and that less than 3% of the substrate is consumed in the reaction. Routinely, the enzyme activity was expressed as cpm ³²PO₄ incorporated per sample over a 20-min interval, corrected for the non-substrate-containing "blank."

Reagents. Human EGF was from Upstate Biotechnology Inc. (Lake Placid, NY); PD98059 and PD153035 were obtained from Parke Davis (Ann Arbor, MI), with the kind assistance of Drs. A. Saltiel and D. Fry. U57, 908 was a generous gift to Dr. D. L. Severson, originally from Dr. D. Morton (The Upjohn Co., Kalamazoo, MI). The following reagents were obtained from Sigma (St. Louis, MO): nifedipine, indomethacin and wortmannin. Calbiochem (La Jolla, CA) provided arachidonic acid, tyrphostin 47/AG213 and GF109203X, whereas the Src-selective kinase inhibitor, CP118, 556/PP1, was obtained through the courtesy of Dr. Hanke, Pfizer Central Research. Peptides (TFLLR-NH₂ and KVEKIGEGTYGVVYK) were synthesized by solid phase methodology with the assistance of Dr. D. McMaster of the Peptide Synthesis Facility at the University of Calgary, Faculty of Medicine (Calgary, AB, Canada). The concentration, purity and composition of peptide stock solutions (>95% purity) were determined by high-performance liquid chromatography, mass spectrometry and quantitative amino acid analysis.

Statistical analysis of data. Values (averages ± S.E.M.) expressing the contractile response (% KCl) relative to that of 50 mM KCl; or the degree of inhibition (% control) of contraction by a variety of agents, relative to the contractile responses observed in the absence of inhibitors were obtained from experiments done with 3 to 15 independently prepared tissue strips usually coming from two or more separate animals. The average values ± S.E.M., recorded in the tables and in the figures (error bars shown), comparing responses in inhibitor-treated vs. untreated tissues were assessed for statistical significance, where appropriate, using either paired or group Student's t tests.

	Results

Top Abstract Introduction Methods Results Discussion References

Sensitivity of the guinea pig LM and CM preparations to TFLLR-NH₂. Our previous work had established the PAR₁ (vs. PAR₂) selectivity of the PAR₁AP, TFLLR-NH₂, in both tissue and cell-based assays (Hollenberg et al., 1997). Nonetheless, we had yet to study the sensitivity of the guinea pig gastric smooth muscle preparations to TFLLR-NH₂. Therefore, we determined the concentration-response curve shown in figure 1, wherein an EC₅₀ of about 0.9 µM was observed. As with EGF, and in keeping with our observations with SFLLR-NH₂ (Hollenberg et al., 1992), TFLLR-NH₂ caused a transient contractile response that rose to a maximum within 5 min, and then declined toward baseline tension over a further 10- to 15-min time period (insert, fig. 1).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Concentration-effect curve for the contractile action of TFLLR-NH2 in LM tissue. The contractile responses of LM tissue strips to increasing concentrations of TFLLR-NH2 were measured and expressed as a percentage (% KCl) of the contraction caused in each tissue by 50 mM KCl. The inset shows a representative transient contractile response to 1 µM TFLLR-NH2 (TF-NH2, ). The scale for time and tension is shown to the right of the tracing; W (arrow) = tissue wash. Values represent the mean ± S.E.M. (error bars) for measurements done with four to six individual tissues taken from two to four different animals.

Effects of inhibitors of cyclooxygenase, tyrosine kinase and diacylglycerol-lipase. In keeping with our previous observations with the PAR₁/PAR₂ nonselective agonist, SFLLR-NH₂ (Hollenberg et al., 1992) in guinea pig and rat gastric tissue, the contractile action of the PAR₁-selective agonist, TFLLR-NH₂ was blocked by the cyclooxygenase inhibitor, indomethacin, but not by either the epoxygenase inhibitor, ketoconazole (5 µM) or the lipoxygenase inhibitor, nordihydroguairetic acid (30 µM) (fig. 2A and data not shown). The tyrosine kinase inhibitors, tyrphostin 47 (AG213) and genistein, both blocked the contractile response to TFLLR NH₂. As we had previously observed for EGF-stimulated contractions (fig. 2E and Yang et al., 1992b), maximal inhibition for TFLLR-NH₂-induced contractions was observe at 7.5 µM genistein and at 20 µM tyrphostin 47 (AG213); the inhibition caused by 7.5 µM genistein (100%) was the same as that caused by 20 µM tyrphostin 47 (AG213) (fig. 2D). The diacylglycerol lipase inhibitor, U57, 908, also blocked the contractile response (fig. 3). In contrast, the phospholipase A₂ inhibitor, mepracrine (3 µM) had no effect on the contractile response (not shown). At the concentrations of indomethacin, tyrphostin 47/AG213 and U57, 908 that were used, we had previously shown that these inhibitors were able to block the contractile actions of EGF (figs. 2 and 3) without affecting contractions caused by either bradykinin or carbachol (Yang et al., 1991, 1992b). In addition to the tyrosine kinase inhibitors, tyrphostin 47/AG213 and genistein, the Src-selective tyrosine kinase inhibitor, CP118, 556/PP1 (Hanke et al., 1996) was able to block contractions caused by both EGF and TFLLR-NH₂ with comparable IC₅₀s (50 to 80 nM) (fig. 4).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Comparative effects of indomethacin and tyrphostin-47 on the contractile actions of TFLLR-NH2, EGF and arachidonic acid in the LM preparation. After monitoring a control contractile response to TFLLR-NH2 (TF-NH2, 1µM, A and D), EGF (17 nM, B and E) and arachidonic acid (AA, 10 µM, C and F), tissues were washed (W, arrow) and pretreated for 20 min with either indomethacin (IND, 3 µM, A to C) or tyrphostin-47 (TP47, 20 µM, D to F) and the tissues were then rechallenged with the same agonists. The tracings are representative of results obtained with four to six tissue strips derived from three or more animals.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Effect of the diacylglycerol lipase inhibitor, U57, 908 on LM contractions caused by TFLLR-NH2 and EGF. After monitoring a control contraction in response to either TFLLR-NH2 (TF, 1 µM) or EGF (17 nM), tissues were washed and were incubated for 20 min with 20 µM U57, 908 (solid bars). The preparations were then rechallenged with the same agonists in the continued presence of U57, 908. The contractile response in the presence of the inhibitor (solid bar) was expressed as a percentage (% control) of the contractile response observed in the absence of inhibitor. The solid bar shows the average response (% control) ± S.E.M. (error bars) for data obtained with seven different tissues strips. **P < .001 compared with control.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of the Src-selective tyrosine kinase inhibitor, PP1, on contractions caused by TFLLR-NH2, EGF and arachidonic acid in the LM preparation. After monitoring control contractile response to TFLLR-NH2 (TF, 1 µM: ), EGF (17 nM, ) or arachidonic acid (AA, 10 µM: ), tissues were washed and preincubated for 20 min with increasing concentrations of PP1 (x-axis) before a rechallenge of each tissue with the same agonist in the continued presence of PP1. The contractile responses in the presence of PP1 were expressed as a percentage (% control) of the response observed in the absence of inhibitor. Data points represent means ± S.E.M. (bars) for measurements with four to six individual tissue strips for each concentration of PP1.

Role of extracellular calcium. We confirmed in the present study that, as previously published, EGF did not cause a contractile response in the absence of extracellular calcium (data not shown and Laniyonu et al., 1994). Similarly, a contraction in response to TFLLR-NH₂ was not observed in the absence of extracellular calcium (upper tracing, fig. 5); replenishing the buffer with extracellular calcium, in the continued presence of the receptor-activating peptide, resulted in a contractile response (upper tracing, fig. 5). For reasons we were unable to determine, the readdition of extracellular calcium to a tissue preactivated by TFLLR-NH₂ without Ca⁺⁺ resulted in a tonic, rather than a transient contractile response, as observed when TFLLR-NH₂ was added to the tissue in the concurrent presence of 2.5 mM Ca⁺⁺ (see insert, fig. 1). In accord with this result, the calcium channel antagonist, nifedipine (1 µM) essentially abolished the contractile response of the LM preparation to TFLLR-NH₂ (lower tracing, fig. 5). As expected, the contractile response to 50 mM KCl was also blocked by nifedipine.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Dependence of the contractile response of the LM preparation to TFLLR-NH2 on extracellular calcium. Upper, The preparation was first exposed to TFLLR-NH2 (TF, 1 µM: ) followed by washing (W, arrow) and resuspension in a calcium-free Krebs-Henseleit buffer containing 0.2 mM EGTA (Ca++-free). Twenty minutes thereafter, the tissue was again challenged in calcium-free buffer with TFLLR-NH2 (); the replenishment of the buffer with 2.5 mM CaCl2 (+) in the continued presence of TFLLR-NH2 resulted in a contractile response. (lower): After monitoring a control response to TFLLR-NH2, the tissue was washed and preincubated for 20 min with the voltage-dependent calcium channel blocker, nifedipine (NIF, 1 µM: ). TFLLR-NH2 was then added to the organ bath in the continued presence of nifedipine. The tracings are representative of experiments done with three or more tissue strips taken from at least two different animals.

Effects of inhibitors of PKC, PI3K and MEK. We had not previously, in the guinea pig LM preparation, assessed potential roles for PKC, phosphatidylinositol 3' kinase and MAP-kinase-kinase in the contractile actions for the PAR₁/PAR₂-activating peptides. Although we had recently reported that inhibitors of these three enzymes could block the contractile action of EGF in the guinea pig LM tissue (Zheng and Hollenberg, 1997; Zheng et al., 1997), we believed it necessary to reevaluate in more detail, with comparable tissue samples from the same series of animals, the effects of the inhibitors on the actions of TFLLR-NH₂ and EGF. The PKC inhibitor, GF109203X, at 1 µM was found to block completely contractions elicited in the LM preparation by the PKC activator, phorbol dibutyrate (not shown). This same concentration of GF109203X was also able to inhibit (90 ± 5%, mean ± S.E.M. for n = 5) the EGF-mediated contractile response; but in contrast, GF109203X was not able to block more than 50% of the contractile response caused by TFLLR-NH₂ (inhibition: 50 ± 5%, mean ± S.E.M. for n = 6) (fig. 6). Similarly, the PI3 kinase inhibitors wortmannin (0.1 µM) and LY294002 (2.5 µM) were able to block the contractile response to EGF almost completely (90 ± 5% inhibition, mean ± S.E.M. for n = 5), but were only partially effective (58 ± 5% inhibition: mean ± S.E.M. for n = 6) in blocking the contractile response caused by TFLLR-NH₂ (fig. 7). These concentrations of Wortmannin and LY294002 were at the respective plateaus of their concentration-inhibition curves (not shown). The IC₅₀ for Wortmannin was approximately 70 nM, in keeping with the potency of this reagent to inhibit PI3 kinase. When added together, the PKC inhibitor, GF109203X (1 µM) and Wortmannin (0.1 µM) were able to block the TFLLR-NH₂-induced contraction completely (fig. 6). In contrast, the contractile responses to 1 µM carbachol or 50 mM KCl were unaffected by either 0.1 µM Wortmannin or 1 µM GF109203X (data not shown). The selective MEK inhibitor, PD98059 was able to block completely contractions caused both by EGF (IC₅₀, approx. 0.1 µM) and by TFLLR-NH₂ (IC₅₀, approx. 0.2 µM) (fig. 8). The MEK inhibitor had no effect on the contractile response elicited by 1 µM carbachol (not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effects of the protein kinase C inhibitor GF109203X on the contractile responses of LM tissue. LM tissue strips were first exposed to EGF (17 nM), TFLLR-NH2 (TF, 1 µM) and arachidonic acid (AA, 10 µM). The control responses (open bars) were recorded, followed by a tissue wash and incubation of each tissue with 1 µM GF109203X (GF) for 20 min. The tissue responses to the same contractile agonists were subsequently recorded in the continued presence of 1 µM GF (solid bar); these responses were expressed as the percentage of the previous contraction observed in the absence of inhibitor (Y-axis). For TF-stimulated contractions, the combined effects of 1 µM GF plus .1 µM wortmannin (WM/bar) were also monitored (hatched bar). Data represent the mean ± S.E.M. (error bars) for measurements done with seven individual tissue strips for each experimental group. Student's t test, **P < .01, compared with control responses.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Effects of inhibitors of PI3-kinase on agonist-induced contractions in the LM preparation. After monitoring the control contractile responses (empty bars) in LM tissues to EGF (17 nM), TFLLR-NH2 (TF, 1 µM) and arachidonic acid (AA, 10 µM) (shown on x-axis), followed by a tissue wash, the tissues were preincubated for 20 min with the PI3-kinase inhibitors, either wortmannin (WM, solid bar, 0.1 µM) or LY294002 (LY, hatched bar, 2.5 µM). The contractile responses to various agonists obtained in the presence of the PI3-kinase inhibitors were expressed as the percentage of the control responses (y-axis). Data represent the mean ± S.E.M. (error bars) for measurements done with seven individual tissue strips for each experimental group. **P < .01, compared with control responses.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Concentration-effect curves for the inhibition of contraction by the MEK inhibitor, PD98059. The gastric longitudinal muscle (LM) preparations were exposed to arachidonic acid (AA 10 µM: ), EGF (17 nM: ) or TFLLR-NH2 (TF, 1 µM: ) to obtain the control responses, followed by a tissue wash. The tissues were then preincubated with various concentrations of the MEK inhibitor, PD98059 (x-axis), for 20 min, after which the responses to the same concentrations of AA, EGF and TFLLR-NH2 were recorded and expressed as the percentage of the corresponding control responses (y-axis). Data represent the mean ± S.E.M. (error bars) for measurements done with nine individual tissue strips for each concentration point.

Effect of the EGF receptor kinase inhibitor, PD153035. Because previous work had shown that the EGF receptor could be potentially transactivated by thrombin in cultured Rat-1 fibroblasts, we wished to demonstrate that the contractile action of TFLLR-NH₂ in the LM preparation was not due to the concurrent activation of the EGF receptor. To deal with this possibility, we used the potent and selective EGF receptor kinase inhibitor, PD153035, that is not believed to affect other kinases, such as kinase C or src-kinase (Fry et al., 1994). As shown in figure 9, the receptor kinase inhibitor was able to abolish the contractile action of EGF in the LM preparation, with an IC₅₀ of about 50 nM, without affecting the contractile action of TFLLR-NH₂ at inhibitor concentrations as high as 1 µM. Unfortunately, a selective inhibitor of PAR₁ activation is not yet available, and it was not possible to assess whether the reciprocal mechanism (i.e., trans-activation of PAR₁ by the EGF receptor kinase) might occur.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Concentration-dependent effect of the EGF receptor kinase inhibitor, PD153035, on the contractile responses caused by EGF and TFLLR-NH2. The responses of lognitudinal muscle (LM) preparations to EGF (17 nM: ) or TFLLR-NH2 (TF, 1 µM: ) were first obtained and recorded as the control. Tissues were then washed and preincubated with various concentrations of PD153035 (x-axis), followed by exposure to the same concentrations of EGF and TFLLR-NH2. The contractile responses obtained in the presence of PD153035 were expressed as the percentage of the control response observed in the absence of this inhibitor (y-axis). Data represent the mean ± S.E.M. (error bars) for measurements done with eight individual tissue strips for each concentration point.

Effects of inhibitors on the contractile effect of exogenously added arachidonic acid. For both EGF and TFLLR-NH₂, the inhibitory actions of indomethacin and U57, 908 indicated that the LM contractile response itself was due to a presumed cyclooxygenase metabolite of AA, most likely acting via a G-protein-coupled receptor distinct from PAR₁. Thus, to distinguish between the effects of the various inhibitors on the PAR₁ signaling pathway from possible effects on the receptor(s) activated by the arachidonic acid metabolite(s), it was necessary to evaluate the effects of the various inhibitors on the contractile action of AA itself; we presumed that AA would be converted to one or more contractile metabolites by the LM tissue. Preliminary work showed that 10 µM of arachidonic acid added to the organ bath yielded a contractile response equivalent to the response caused by either 1 µM TFLLR-NH₂ or 17 nM EGF (fig. 2). Thus, further work was done using this concentration of arachidonate. In keeping with our working hypothesis that a cyclooxygenase product of arachidonate was responsible for the observed contractile effect caused by EGF and TFLLR-NH₂, 3 µM indomethacin completely blocked the contractile action of added arachidonate (fig. 2C). The contractile response to arachidonate also required the presence of extracellular calcium and was attenuated by the calcium channel blocker, nifedipine (not shown; and table 1). However, unlike the actions of EGF and TFLLR-NH₂, the contractile action of arachidonate was not affected by the other inhibitors we tested (see table 1), including the tyrosine kinase inhibitors tyrphostin 47/AG213 and CP118, 556/PP1 (figs. 2 and 4), the MEK inhibitor PD98059 (fig. 8), the PI3-kinase inhibitors Wortmannin and LY294002 (fig. 7) and the protein kinase C inhibitor, GF109203X (fig. 6). In this manner, it was possible to distinguish clearly between the common signal pathways activated by PAR₁ and the EGF receptor kinase, and the contractile signal pathway activated by the metabolite(s) of AA. Table 1 summarizes the effects of all of the inhibitors we tested on the actions of EGF, TFLLR-NH₂ and arachidonate.

Phosphotyrosyl proteins and Src-kinase activity. Because the Src-selective inhibitor, CP118, 556/PP1 blocked the contractile action of EGF and TFLLR-NH₂, we wished to assess the presence of phosphotyrosyl proteins in the LM tissue that had been contracted by either EGF or TFLLR-NH₂; and we wished to determine if Src-kinase activity was indeed elevated during the contractile process. As shown in table 2 and figure 10, EGF and TFLLR-NH₂ both caused increases in Src-kinase activity, as determined after immunoprecipitation using the Src-targeted cdc2 peptide as a substrate; and both contractile agonists caused an increase in phosphotyrosyl proteins that were harvested by antiphosphotyrosine immunobeads, as detected by Western blotting with horseradish peroxidase-coupled monoclonal antiphosphotyrosine antibody. Nonetheless, the degree of increase of phosphotyrosyl proteins detected after agonist treatment (A to F in fig. 10) appeared to differ for EGF and TFLLR-NH₂. For instance, desensitometry of the phosphotyrosyl protein bands C and E (fig. 10) showed that there were comparable increases (about 1.3-fold, relative to control) caused by either TF or EGF; but, for constituents B and D, EGF caused a more pronounced increase in tyrosine phosphorylation (about 2.5-fold for B; 2.1-fold for D) compared with TF (about 1.5-fold for B; 1.3-fold fold D). These possible differences in the tyrosine phosphorylation triggered by the two agonists will require further study using antibodies directed against specific signal pathway components such as the 85-kDa regulatory subunit of PI-3K (possibly, fig. 10C) that can become tyrosine phosphorylated in the course of EGF action.

View larger version (47K): [in this window] [in a new window]   Fig. 10.   Increase in phosphotyrosyl protein content stimulated by EGF and TFLLR-NH2. Tissues (LM preparation) were mounted in the organ bath and were equilibrated as for a bioassay. In triplicate, tissue strips were either untreated, or were exposed to EGF (17 nM) or TFLLR-NH2 (1 µM). After observing a reproducible contraction to either EGF or TFLLR-NH2, followed by washing and reequilibration, the preparations were again exposed to each agonist and were harvested at the point when tension was about 80% of the plateau level (at about 2 min after adding agonist). Control tissues were harvested after the same equilibration/washing procedures, but without the addition of agonists. The triplicate tissues were pooled, rapidly frozen on solid CO2 and processed for Western blot analysis and chemiluminescence detection of phosphotyrosyl proteins, as outlined in "Methods." Protein bands A to F (dots on right) were found to exhibit an increased luminescence signal upon treatment with either EGF (E) or TFLLR-NH2 (T) compared with control tissues (C). Increases of luminescence over control bands were estimated by quantitative densitometry, as recorded in the text for bands B to E. The position of the molecular weight markers (kDa) are shown on the left; migration was toward the anode (arrow).

	Discussion

Top Abstract Introduction Methods Results Discussion References

The main finding of our study was that there is a remarkable parallel between the signaling pathways activated by PAR₁ and by the EGF receptor kinase to cause a contractile response in the guinea pig LM tissue. These parallels would appear to be upstream of the steps (phospholipase activation and diacylglycerol lipase action) leading to the formation of the contractile AA metabolite. Based on our own recent data (Hollenberg et al., 1997) and on work by others (Blackhart et al., 1996; Ishihara et al., 1997), we are confident that our results, using the receptor-activating peptide TFLLR-NH₂, reflect the selective activation of PAR₁ and not either PAR₂ or PAR₃. The parallels between PAR₁ and EGF receptor kinase signaling pathways included the activation of Src-tyrosine kinase activity and a sensitivity of the tissue response toward inhibitors of PI3-kinase and MEK. In contrast, the contractile action of the G protein-coupled agonist, carbachol and of the (presumably) G-protein-coupled cyclooxygenase product of arachidonic acid were unaffected by the "growth factor" signal pathway probes that were used (e.g., the Src-kinase inhibitors, CP118, 556/PP1; the PI3 kinase inhibitor wortmannin; and the MEK inhibitor, PD98059) (summarized in table 1). Further, the lack of effect of the potent EGF receptor-kinase inhibitor, PD153035, to block the contractile action of TFLLR-NH₂, showed that PAR₁ stimulation was not due to transactivation by the EGF-receptor tyrosine kinase, as appears to be the case in cultured rat-1 fibroblasts (Daub et al., 1996). Unfortunately, it was not possible to rule out the converse possibility, that activation of the EGF receptor kinase might have trans-activated PAR₁. It can be noted that for the contractile responses elicited by EGF and TFLLR-NH₂, there may be an unusual "double" role for diacylglycerol, acting both "conventionally" as an activator of kinase C (contractions attenuated by the kinase C inhibitor GF109203X) and "unconventionally" as a substrate for diacylglycerol lipase (contractions blocked by U57, 908). It is quite likely that such a "metabolic" role for diacylglycerol, in addition to its kinase C-activating role, may occur in the course of the action of a number of agonists, as we have previously documented for angiotensin-II (Yang et al., 1993). In the context of our results showing that U57,908 (formerly known as RHC80267) blocked the contractile action of TF, it is important to note that this compound can also inhibit the effects of thrombin on guinea pig platelets (Amin et al., 1986). To date, we have not been able to determine whether the diacylglycerol is produced directly via the activation of a phospholipase C isoform; or alternatively via the combined activation of phospholipase D followed by phosphatidate phosphohydrolase.

Despite the many parallels between the actions of TFLLR-NH₂ and EGF, there were some minor but significant differences in terms of the sensitivity of the contractile responses to the various inhibitors (table 1). For instance, although the contractile response to EGF was essentially abolished by the kinase C inhibitor, GF109203X and by the PI3-kinase inhibitors, Wortmannin and LY294002, the response to TFLLR-NH₂ was not completely blocked by these reagents. Yet, when added together, a kinase C inhibitor and a PI3 kinase inhibitor abolished the contractile response to PAR₁ activation (fig. 6 and table 1). Thus, in the guinea pig LM tissue, PKC would appear to play a major role in the contractile action of EGF. It is possible that EGF acts first via an upstream activation by PI3-kinase (Toker and Cantley, 1997; see also discussion below), followed sequentially by a kinase C-mediated activation of the MEK/MAP kinase pathway (Ueda et al., 1996). However, the activation pathway triggered by TFLLR-NH₂ would appear to involve both a PKC-dependent and a PKC-independent pathway that involves the activation of both PI3-kinase and MEK in the course of the contractile event. Previously, the activation of the MAP kinase pathway via both a protein kinase C-dependent and independent pathway possibly involving tyrosine phosphorylation has been observed for the action of thyrotropin-releasing hormone (Ohmichi et al., 1994). It is admittedly difficult, in the context of intact tissue studies (as opposed to studies with cultured cell systems), to place PKC activation as being unequivocally upstream or downstream of the activation of either the nonreceptor tyrosine kinase or Ras (both of which we believe are involved in the TFLLR-NH₂-mediated the contractile response). One possibility that must be considered (Toker and Cantley, 1997) is that phosphoinositides resulting from PI3-kinase action [e.g., Ptd Ins (3, 4)P₂ and Ptd Ins (3, 4, 5)P₃] could in turn activate PKC isoforms in the LM tissue, placing PI3-kinase upstream of PKC in the contractile process. Further studies will be required to resolve these issues.

The analysis of the signal transduction pathways triggered by EGF and TFLLR-NH₂ to cause a contractile response is challenging, since both of these agonists act via a "cascade" process, wherein it is the indomethacin-sensitive cyclooxygenase product of (presumably) arachidonic acid that ultimately drives the contraction. Thus, in our study reported here, one must consider two tiers of signal pathway activation: 1) the signal process triggered by PAR₁ and EGF receptor systems leading to the activation of a phospholipase, that yields diacylglycerol and 2) the signal process activated by the (presumed) prostanoid agonist via its own G protein-coupled mechanisms to cause contraction. To sort out these two tiers of signaling it was essential to examine the effects of the various signal pathway probes on the contractile action of added arachidonate itself. We fully realized the possibility that the exogenous addition of arachidonate to the tissue might yield contractile agonists in addition to the one(s) liberated in the course of EGF and TFLLR-NH₂ action. Despite these reservations, an analysis of the data summarized in table 1 can permit some conclusions to be made. First, the contractile response to 10 µM AA was completely blocked by indomethacin. Contractions caused by 10 µM AA were equivalent in magnitude to those caused by TF and EGF. Thus, we believe secure in our hypothesis that it is a cyclooxygenase product that is responsible for the PAR₁-driven contractile response, and not an epoxygenase product, as we have previously documented for the action of angiotensin-II in gastric CM tissue (Yang et al., 1993). Second, because none of the "growth factor" signal pathway probes we used attenuated the response to added arachidonate (e.g., CP118,556/PP1, GF109203X, wortmannin/LY294002, or PD98059), whereas all of these compounds affected the contractile actions of both EGF and TFLLR-NH₂, we conclude that for both TFLLR-NH₂ and EGF the tyrosine kinase/PI3 kinase/kinase C/MEK signal pathways are upstream of the process of activation of a phospholipase C or D isoform that yields diacylglycerol as a substrate for the ensuing production of the arachidonate metabolite. A further concern that the results resolve is the possibility that one or more of the signal pathway inhibitors might have, as with indomethacin, blocked the action cyclooxygenase to yield the contractile AA metabolite(s). The role of both extracellular and intracellular calcium in the overall process is unfortunately difficult to dissect, because the response of the tissue not only to EGF and TFLLR-NH₂ but also to added arachidonate appears to require the influx of extracellular calcium. Finally, it can be pointed out that the signal process activated by either EGF or TFLLR-NH₂ (but not arachidonate) appears to require the simultaneous (or sequential) activation of both PI3-kinase and MEK (and presumably, MAP-kinase).

The involvement of both PI3-kinase and MEK in the contractile actions of EGF and TFLLR-NH₂ to cause the contractile response is in some ways puzzling, because in other circumstances, such as the "growth factor" signal pathways activated by insulin, the downstream events promoted concurrently by either PI3-kinase activation (e.g., insulin-stimulated glucose transport) or by MEK (insulin-mediated activation of ribosomal S6 kinase) can occur quite independently of one another. For instance, inhibition of MEK does not affect insulin-regulated glucose transport, but abrogates p90^{rsk1, 2} activation; and under comparable circumstances, inhibition of PI3-kinase blocks insulin-mediated glucose uptake, but does not affect insulin-mediated p90^{rsk1, 2} activation, (an event that is downstream of MEK/MAP-kinase stimulation). It is thus possible that for PAR₁ and EGF receptor stimulation of the LM tissue, there may be a linear link between a specific isoform of PI3-kinase and activation of MEK. This situation for PAR₁ activation in the LM tissue would be akin to the PI3-kinase  link between G-protein receptors and MAP-kinase signaling reported recently by Lopez-Llasaca and coworkers (Lopez-Llasaca et al., 1997). One scenario to be considered for EGF is that PI3-kinase may be upstream of the activation of protein kinase C (see above discussion) and that the PKC-mediated activation of raf-1 could in turn cause the activation of MEK (Ueda et al., 1996). Further work is thus indicated with the LM tissue to determine if a unique PI3-kinase isoform is involved in the EGF/TFLLR-NH₂ signaling process.

A final issue that merits discussion is the potential role that Src may play in regulating smooth muscle tension. In cell transfection systems, it now appears that G-protein-coupled agonists such as LPA, via the participation of the G-protein subunit, can cause an activation of Src; and that this activation of Src is essential for a downstream activation of MAP-kinase (Luttrell et al., 1996). Further, it has been suggested that the EGF receptor itself may play a "scaffolding" role in such a process without acting directly as a kinase (Luttrell et al., 1997). A similar situation might take place in the LM tissue preparation. However, the role of PI3-kinase in such a potential scheme has yet to be elucidated; and it can be pointed out that interactions between Src itself and PI3-kinase must also be considered in such a scheme (Pleiman et al., 1994). Our data demonstrated that Src was indeed activated in the course of the contractile response driven by either EGF or TFLLR-NH₂; and that the Src-selective tyrosine kinase inhibitor, CP118,556/PP1 blocked the contractile response to both agonists. However, CP118,556/PP1 is not completely selective for Src. Additionally, two tyrosine kinase inhibitors (tyrphostin 47/AG213 and genistein) that are poor inhibitors of Src-kinase activity (K_is in the range > 100 µM: M.D. Hollenberg and B. Renaux, unpublished data) were, nonetheless quite good inhibitors of the contractile response in the 8 to 40 µM range. Our data thus imply that, quite apart from the activation of Src itself, other nonreceptor tyrosine kinases may be involved in the tissue response to EGF and TFLLR-NH₂. Ongoing work in our laboratory is aimed at identifying those tyrosine kinases in addition to Src that may play a role in the rapid regulation of smooth muscle function.