Prostaglandins play a significant role in the physiology and pathophysiology of the digestive system by affecting water and electrolyte transport, mucus secretion, blood flow, and motility (1-4). Prostaglandin E is synthesized in large amounts and widely distributed throughout the gastrointestinal tract (5-7), particularly in the smooth muscle layers of the stomach and small intestine (8).

PGE2 can interact with at least four cell surface prostaglandin type E receptors (EP1, EP2, EP3 and EP4), which triggers a variety of intracellular responses depending on the G protein to which the cell surface receptors are coupled (9-11). Pharmacological studies suggest that PGE2 relaxes intestinal smooth muscle through the EP2 and EP4 receptors, which couple to cyclic-AMP generation. In contrast, PGE2 constricts intestinal and vascular smooth muscle through EP3 receptors, which inhibit cAMP generation via Gi protein, or EP1 receptors, which increase the mobilisation of Ca2+ (1,4,10,12).

The phosphorylation of tyrosine residues in signalling proteins is an important mechanism in the contraction of smooth muscle (13,14). Agonists, such as epidermal growth factor and platelet-derived growth factor, act on tyrosine kinase receptors to induce contraction (15). In addition, certain G protein-coupled receptor agonists, such as angiotensin II, vasopressin, bombesin, and endothelins can act through the stimulation of non-receptor tyrosine kinases (13,15). Biochemical studies demonstrate that both types of receptor activation (i.e., G protein-coupled and receptor tyrosine kinase) can lead to the phosphorylation of tyrosine residues of various cellular proteins (15). Protein tyrosine phosphorylation can participate in the regulation of mechanisms that couple receptor activation and increase intracellular calcium concentrations (16,17).

Vanadate has been reported to induce contraction in several smooth muscle preparations (14,16,18-22). Various mechanisms for the contractile effect of vanadate have been reported, including inhibition of Na+-K+-ATPase, Ca2+-ATPase, and tyrosine phosphatase (14,20).

The aim of this study was to examine the role of tyrosine phosphorylation in PGE2, vanadate and carbachol-evoked contractions in the longitudinal smooth muscle of rabbit duodenum.


MATERIALS AND METHODS
The equipment used, and the handling and sacrifice of animals were in accordance with the European Council legislation 86/609/EEC concerning the protection of experimental animals. Male New Zealand rabbits that weighed 2-2.5 kg were maintained at a constant room temperature (22 °C) with free access to water and standard rabbit fodder. All experimental protocols were approved by the Ethical Committee of the University of Zaragoza (Spain).

Preparation of smooth muscle segments

After 24 h of fasting, the animals were humanely killed by a blow to the head. Pieces of rabbit duodenum (1-6 cm distal to the pylorus) were removed, washed, freed from mesenteric attachment, and cut into smaller segments. Whole-thickness segments (10 mm long and 5 mm wide) were suspended in the direction of longitudinal smooth muscle fibres in a thermostatically controlled (at 37 °C) organ bath (10 mL capacity) containing Krebs solution and, continuously gassed with 95% O2 and 5% CO2. Each segment of duodenum was connected to an isometric force transducer (Pioden UF1, Graham Bell House, Canterbury, U.K.) and stretched passively to an initial tension of 20 mN. Signal output of the mechanical activity was amplified (The Mac Lab Bridge Amp, AD Instruments Inc., Milford MA, USA) with a range of 2 mV, recorded on a computer for later analysis using The Mac Lab System/8e computer program (AD Instruments Inc., Milford MA, U.S.A) and digitized at two samples per second per channel. Before testing, segments were allowed to equilibrate in Krebs solution for 60 min. During that time, the nutrient solution was changed every 20 min.

Experimental protocols

After the equilibration period, we discarded segments that did not show spontaneous activity. Each experimental protocol was systematically performed on two or three segments of duodenum taken from the same rabbit and repeated in three or four different animals. Thus, each preparation served as its own control.

PGE2 (10-7 M, 3 min), vanadate (10-3 M, 3 min) and carbachol (10-4 M, 3 min) singly were added to the bath. In order to avoid the desensitisation of muscle to those substances, they were added to the bath for 3 min, at 20 min intervals. Following the application of PGE2, vanadate or carbachol, the preparations were washed three times.

To study the role of tyrosine kinase on PGE2-, vanadate- and carbachol-evoked contractions, we used genistein (10-5 M), tyrphostin B44 (10-5 M), tyrphostin 47 (10-5 M), all of them tyrosine kinase inhibitors, daidzein (10-5 M), an inactive negative control for genistein, and tyrphostin 1 (10-5 M), an inactive negative control for tyrphostin. These agents were preincubated 5 min before the PGE2 (10-7 M), vanadate (10-3 M) or carbachol (10-4 M) addition. Previous studies showed that genistein, tyrphostin B44, tyrphostin 47, tyrphostin 1 or daidzein at 10-5 M did not have an effect per se on spontaneous contractions in duodenum.

We studied, in the same way, the effect of vanadate (10-3 M) and U-73122 (10-7 M), a phosphatidyl-inositol-dependent phospholipase C inhibitor (PI-PLC) on PGE2-evoked contractions. Furthermore, we examined the actions of indomethacin (10-6 M), an inhibitor of the cyclooxygenase pathway, and U-73122 (10-7 M) on vanadate-evoked contractions in the rabbit duodenum.

To determine the effect of Ca2+ on the contractions evoked by vanadate, segments were exposed to verapamil (a voltage-dependent Ca2+ channel antagonist) or Ca2+-free solutions containing 0.5 mM EGTA, which were added 5 min before vanadate.

Analysis of data

All of the intestinal segments included in the analyses showed spontaneous contractions. PGE2-, vanadate- and carbachol-evoked responses were assessed using the integrated mechanical activity (IMA) per second, expressed as mN s-1 (milinewtons per second) (4,23), and normalised per square millimetre of cross-sectional area (CSA; mm2). IMA = A1- A0, where A1 is the integrated area per second in the first 3 min of the response to different agents, and A0 is the integrated area per second of spontaneous motility 3 min before the addition of the different agents. CSA was determined for each muscle strip using the equation "CSA (mm2) = mass (mg) [length (mm) density (mg mm-3)]-1," where rabbit intestinal muscle density was assumed to be 1.05 mg mm-3; and the length and mass (wet weight) of each segment were noted on completion of experiments. The integrated mechanical activity was expressed as a percentage of the control values (100%).

Values are expressed as mean ± SEM. Comparisons between means were made using one-way analysis of variance (ANOVA) tests and P-values were determined using the Scheffé F test. Differences with P-values <0.05 were considered statistically significant.

Solutions and substances

The composition of the normal Krebs solution was (mM): NaCl 120, KCl 4.7, CaCl2 2.4, MgSO4 1.2, NaHCO3 24.5, KH2PO4 1.0, glucose 5.6. Prostaglandin E2, sodium orthovanadate (Na3VO4), carbachol, indomethacin, verapamil, EGTA [ethylene-glycol-bis (ß-aminoethyl eter) N,N,N´,N´-tetraacetic acid], tyrphostin 1 [(4-methoxybenzylidene) malono-nitrile], tyrphostin 47 (3,4-Dihydroxy-alpha-cyanothiocinnamamide), tyrphostin B44 or tyrphostin AG 527 [(-)-(R)-N-(alpha-methylbenzyl)-3,4-dihydroxy-benzylidenecyanoacetamide], and daidzein (4',7-dihydroxyisoflavone) were purchased from Sigma (Madrid, Spain). Genistein (4',5,7-trihydroxyisoflavone) and U-73122 [1-[6-[[(17ß)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl-1H-pyrrole-2,5-dione] were obtained from Tocris (Bristol, UK). PGE2, tyrphostin B44, tyrphostin 47, tyrphostin 1, daidzein, genistein and U-73122 were dissolved in dimethyl sulfoxide, and indomethacin was dissolved in 5% sodium bicarbonate.


RESULTS
Effects of PGE2, vanadate and carbachol on spontaneous motility: PGE2, vanadate or carbachol induced contractile motor responses but they had different shape and time course. PGE2 (10-7 M) caused an increase in the amplitude and tone of spontaneous contractions (Fig. 1a). Vanadate (10-3 M) evoked a response consisting of an immediate decrease of the amplitude of spontaneous contractions followed by an increase of the amplitude of phasic contractions (Fig. 1b). Carbachol (10-4 M) induced a strong phasic contraction (Fig. 1c).

Fig. 1. Responses of PGE2 (10-7 M), vanadate (10-3 M) and carbachol (10-4 M), in the absence or presence of different tyrosine kinase inhibitors on spontaneous contractions from rabbit duodenum. (a) and (d) response of PGE2 (10-7 M) in the absence or presence of tyrphostin B44 (10-5 M); (b) and (e) response of vanadate (10-3 M) in the absence or presence of tyrphostin 47 (10-5 M); (c) and (f) response of carbachol (10-4 M) in the absence or presence of genistein (10-5 M). W: Washing.

Effect of tyrosine kinase inhibitors and vanadate on PGE2-evoked contractions: PGE2-evoked contractions (10-7 M) decreased in the presence of genistein (10-5 M, 5 min, n=6), or tyrphostin B44 (10-5 M, 5 min, n=6), inhibitors of tyrosine protein kinase (Fig. 1d and 2). On the contrary, neither tyrphostin 47 (10-5 M, 5 min, n=8) nor daidzein (10-5 M, 5 min, n=8), nor tyrphostin 1 (10-5 M, 5 min, n=6) influenced PGE2-evoked contractions (10-7 M) significantly (Fig.2). However, PGE2-evoked contractions (10-7 M) increased by 120% in the presence of vanadate (10-3 M, for 5 min, P<0.01, n= 6), and the effect of vanadate on PGE2 was additive.

Fig. 2. Effects of genistein (Gen, 10-5 M), tyrphostin B44 (Tyr B44, 10-5 M), tyrphostin 47 (Tyr 47, 10-5 M), daidzein (10-5 M), and tyrphostin 1 (Tyr 1, 10-5 M) for 5 min on PGE2-evoked contractions (10-7 M) of longitudinal smooth muscle in rabbit duodenum. Columns are mean percentage values (from three or four animals) of integrated mechanical activity with respect to PGE2-evoked contractions (100%). Vertical bars indicate SE. * p<0.05; *** p<0.001.

Effect of tyrosine kinase inhibitors on vanadate-evoked contractions: Vanadate-evoked contractions (10-3 M) decreased in the presence of genistein (10-5 M, 5 min, n=6). In contrast, tyrphostin 47 (10-5 M, 5 min, n=6) increased vanadate-evoked contractions (Fig. 1e and 3). Neither tyrphostin B44 (10-5 M, 5 min, n=11), nor daidzein (10-5 M, 5 min, n=9) nor tyrphostin 1 (10-5 M, 5 min, n=6) influenced vanadate-evoked contractions (10-3 M) significantly (Fig. 3).

Fig. 3. Effects of genistein (Gen, 10-5 M), tyrphostin B44 (Tyr B44, 10-5 M), tyrphostin 47 (Tyr 47, 10-5 M), daidzein (10-5 M), and tyrphostin 1 (Tyr 1, 10-5 M) on vanadate-evoked contractions (10-3 M) in longitudinal smooth muscle from rabbit duodenum. Columns are mean percentage values (from three or four animals) of integrated mechanical activity with respect to vanadate-evoked contractions (100%). Vertical bars indicate SE. * p<0.05.

Role of extracellular calcium in vanadate-evoked contractions: Vanadate-evoked contractions (10-3 M) were reduced by 51% in the presence of verapamil for 5 min (10-7 M, P<0.001, n=6) and by 88% in Ca2+-free solutions containing 0.5 mM EGTA for 5 min (P<0.001, n=6).

Sensitivity of vanadate-evoked contractions to indomethacin: Indomethacin (10-6 M, 5 min) decreased vanadate-evoked contractions (10-3 M, 3 min, P<0.05, n=8) by 16%.

Role of inositol-1,4,5-trisphosphate (IP3) in vanadate and PGE2-evoked contractions: U-73122 (10-7 M, 5 min) did not influence vanadate-evoked contractions (10-3 M, 3 min, n=10), but PGE2-evoked contractions with a decrease of 21% (10-7 M, 3 min, P<0.05, n=7).

Effect of tyrosine kinase inhibitors on carbachol-evoked contractions: Carbachol-evoked contractions (10-4 M) which decreased in the presence of genistein (10-5 M, 5 min, n=8) (Figures 1f and 4), tyrphostin B44 (10-5 M, 5 min, n=6) or tyrphostin 47 (10-5 M, 5 min, n=7) (Fig. 4). Neither daidzein (10-5 M, 5 min, n=8) nor tyrphostin 1 (10-5 M, 5 min, n=8) influenced carbachol-evoked contractions (10-4 M) significantly (Fig. 4).

Fig. 4. Effects of genistein (Gen, 10-5 M), tyrphostin B44 (Tyr B44, 10-5 M), tyrphostin 47 (Tyr 47, 10-5 M), daidzein (10-5 M), and tyrphostin 1 (Tyr 1, 10-5 M) on carbachol-evoked contractions (10-4 M) in longitudinal smooth muscle in rabbit duodenum. Columns are mean percentage values (from three or four animals) of integrated mechanical activity with respect to carbachol-evoked contractions (100%). Vertical bars indicate SE. * p<0.05; *** p<0.001.

DISCUSSION
Prostaglandins play an important role in communication between the gastrointestinal immune system and the enteric nervous system. In the intestinal inflammation, prostaglandins are released from a variety of cell types, including sympathetic nerve terminals. These have been attributed to be inflammatory mediators (3,4). PGE2 causes a contractile influence on longitudinal muscle and an inhibitory influence on circular muscle preparations of the gut (11,24-32). The contractile effect of PGE2 on smooth muscle is likely to be due to interactions with the contractile EP1 or EP3 receptors because EP2 and EP4 receptors are relaxant (1,12-33).

In the present study, the PGE2-evoked contractions, on longitudinal smooth muscle of rabbit duodenum in vitro, decreased in the presence of genistein or tyrphostin B44. However, tyrphostin 47, daidzein or tyrphostin 1 did not modify them. These results demonstrate that PGE2-evoked contractions were acting through the activation of protein tyrosine phosphorylation. Similarly, Aoki et al. (34) found that those tyrosine kinase inhibitors blocked the neurite retraction and cell rounding induced by the activation of the EP3 receptor. PGE2 evokes contractions in the gastrointestinal tract through EP1 and EP3 receptors coupled to G proteins. In that way, several G-protein-linked agonists, such as carbachol, angiotensin II, arginine vasopressin, epidermal growth factor-urogastrone, noradrenaline, PGF2alpha, serotonin, endothelin-1 and thromboxane A2, evoke contractions in vascular or intestinal preparations that were inhibited by genistein or tyrphostin (21,35,36). Those inhibitors block the stimulatory effects of PGF2alpha on IP3 accumulation, [Ca2+]i mobilization and contraction in iris sphincter smooth muscle of cat (22,37). These data support the hypothesis that certain G-protein-linked contractile agonists may act in part through the stimulation of cytosolic nonreceptor tyrosine kinases (13,15,36).

Those tyrosine kinases might stimulate Ca2+ entry, activate phospholipase C (PLC) and, through the phosphorylation of unknown substrates, evoke contractions. In addition, the activation of PLC causes the production of diacylglycerol (DAG) and IP3. DAG might act as a substrate for DAG lipase and produce contractile prostaglandins, and IP3 would release intracellular calcium (15). Our results show that U-73122, a phosphatidyl-inositol-dependent phospholipase C inhibitor (PI-PLC), decreased PGE2-evoked contractions, which indicates that the IP3 generated because of PLC activation appears to be involved in the contractile response. Narumiya et al. (10) demonstrated that the EP3 receptor is coupled to several G proteins (Gi, Gs, Gq) and can evoke an IP3 response.

Vanadate is a potent inhibitor of phosphotyrosine phosphatase activity and, consequently, increases the protein tyrosine phosphorylation of several substrates (17). Tyrosine phosphorylation is an important mechanism for regulating smooth muscle contractions. Since tyrosine phosphorylation depends on the balance between the activity of protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP), it has been postulated that the inhibition of tyrosine phosphatase activity should increase protein tyrosine phosphorylation in smooth muscle and, thereby, induce contractions (14,17). Furthermore, vanadate contracts several vascular, bronchial and gastrointestinal smooth muscle preparations (16,18-22,38,39).

Our results show that vanadate evoked a biphasic response in longitudinal smooth muscle of rabbit duodenum. That result is similar to those observed in rabbit ileum (38). These authors have suggested that the inhibitory response was due to release of catecholamines from sympathetic nerve endings or enterochromaffin cells in the wall of the intestine; and the excitatory response was due to the inhibition of a Ca2+-ATPase that controls intracellular Ca2+ levels in intestine.

The vanadate-evoked contractions in rabbit duodenum decreased in the presence of genistein, increased with tyrphostin 47 and were not modified with tyrphostin B44, daidzein or tyrphostin 1 (Fig 3). That result is similar to those observed in rat gastric muscle strips, aortic rings, and in gallbladder smooth muscle, indicating that protein tyrosine phosphorylation mediates vanadate-evoked contractions (14,18). These authors suggest that it might be due to the phosphorylation of proteins associated with the contractile mechanism, and this potentiation might reflect complex and reciprocal regulatory relationships between protein tyrosine kinases and tyrosine phosphatases. However, Zhou et al. (20) concluded that tyrosine phosphorylation is not involved in the vanadate-induced contractions in rat aorta, and Cortijo et al. (19) suggested that protein tyrosine phosphorylation is only of limited importance in mediating the spasmogenic effects of vanadate in human bronchus. Since Src-related kinases are negatively regulated by phosphorylation of specific tyrosine residues, the inhibition of tyrosine kinase activity by these tyrphostin compounds could enhance the activity of these proteins, which would be increased in the presence of phosphatase inhibitors such as vanadate. Furthermore, genistein and tyrphostin have different mechanisms of action. Genistein inhibits tyrosine kinase activity by interacting with the ATP-binding site, and tyrphostin inhibits enzymatic activity by interacting with the phosphotyrosine substrate-binding site (14).

Previous studies conclude that vanadate-evoked gastrointestinal muscle contractions are mainly dependent on extracellular Ca2+ (13,14,16,17,40). The tyrosine-phosphorylated substrates might increase the conductance of Ca2+ channels in the sarcolemma and permit the influx of extracellular Ca2+. Our results are consistent with those studies because Ca2+-free solutions and verapamil, a voltage-dependent Ca2+ channel antagonist, reduced the contractile response of vanadate. However, we found that U-73122 did not influence vanadate-evoked contractions, which indicates that the IP3 generated because of PLC activation did not appear to be involved in the contractile response evoked by vanadate in rabbit duodenum. This finding is similar to those obtained in the gallbladder (14).

In our study, vanadate-evoked contractions were reduced in the presence of the COX inhibitor, indomethacin, indicating the involvement of prostaglandins in the vanadate-induced contractions. In many types of smooth muscles, tyrosine kinase-dependent contractions are known to be sensitive to indomethacin (14,15,41) and there is a relationship between cytosolic tyrosine phosphorylation and the release of prostaglandins. Furthermore, it has been reported that the DAG-lipase pathway for arachidonic acid formation may be involved in the contractile response to vanadate in rat aortae (20). That might explain the additive effect of the PGE2 response in the presence of vanadate.

Carbachol is a contractile agent and has been considered a G-protein linked agonist that acts through the tyrosine kinase pathway (35). Our results showed that the carbachol-evoked contractions in rabbit duodenum were decreased in the presence of genistein, tyrphostin B44 or tyrphostin 47, but were not modified with daidzein or tyrphostin 1. Similar results were obtained with genistein and tyrphostin on carbachol-induced contractions in guinea pig taenia coli (35).

Our results suggest that PGE2, vanadate and carbachol-evoked contractions are mediated by protein tyrosine phosphorylation. Protein tyrosine phosphorylation might cause an increase in calcium influx through voltage-dependent channels and the release of prostaglandins in the longitudinal smooth muscle of the rabbit duodenum.

Acknowledgements: We thank Dr. M Teresa Muin~o for her valuable comments.
This research was supported by the Spanish Ministry of Science and Technology (Dirección General de Investigación AGL2000-1228, AGL2003-03291 and ERDF). A personal grant to Laura Grasa was provided by Government of Aragon and European Social Fund (B010/2003, Spain).


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