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NEUROPHARMACOLOGY
Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada
Received June 18, 2004; accepted October 18, 2004.
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Abstract |
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). These cotransmitters seem to be stored separately and released via independent mechanisms (Todorov et al., 1996, 1999). The ATP-induced smooth muscle contractile response is mediated via P2X1 receptors, which are ligand-gated cationic channels (Benham and Tsien, 1987; Bean, 1992). When activated, the P2X1 receptors produce excitatory junction potentials that summate allowing the opening of L-type voltage-dependent calcium channels (L-VDCC). The resulting influx of extracellular Ca2+ triggers action potentials and a twitch-like contraction. NE acts via 
1-adrenergic receptors to stimulate formation of inositol triphosphate, leading to a release of Ca2+ from intracellular stores, which contributes to the tonic phase of the contractile response.
It has been clearly shown that variations of the free intracellular calcium concentration regulates the contraction of the smooth muscle via a calcium-calmodulin stimulation of myosin light chain kinase and subsequent phosphorylation of myosin regulatory light chain (Kamm and Stull, 1989; Somlyo and Somlyo, 1994). There is a consensus that the signaling pathways involved in the process of smooth muscle contraction are driven by phosphorylation of proteins on serine and/or threonine. However, an accumulating body of evidence suggests that in addition to cell proliferation, differentiation, and migration (Ullrich and Schlessinger, 1990; Schieffer et al., 1997), signal transduction cascades that involve protein tyrosine phosphorylation may also regulate a number of processes leading to smooth muscle contraction (Hollenberg, 1994; Hughes and Wijetunge, 1998). For example, growth factors such as epidermal growth factor and platelet-derived growth factor, known activators of protein tyrosine kinases have been found to produce smooth muscle contraction (Burk et al., 1985, 1986). Moreover, tyrosine kinase inhibitors attenuate the contractile responses of vascular preparations to several G protein-coupled agonists, such as angiotensin-II, NE, and 5-hydroxytryptamine (Laniyonu et al., 1994; Jinsi et al., 1996; Watts et al., 1996). It should be emphasized that most of the studies linking protein tyrosine phosphorylation with smooth muscle contractility are indirect and in most part based on use of inhibitors of PTK. The specific PTK, protein tyrosine phosphatases (PTP) and their substrates involved in smooth muscle contraction, are currently unknown. Here, we provide evidence suggesting for the first time that protein tyrosine phosphorylation may be involved in the regulation of the amplitude of the smooth muscle contraction evoked by ATP.
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Materials and Methods |
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Superfusion. Tissue preparations were placed in a horizontally oriented organ chamber equipped with two platinum ring electrodes for producing electrical field stimulation (EFS). One end of the tissue was fixed in place, and the other was attached by silk surgical suture to a Grass force displacement transducer (FTO3). The tissues were superfused (2 ml/min) with oxygenated Krebs' solution (37°C). After 20 min of superfusion, an initial load of 0.5 g was applied to each tissue. The preparations were allowed to equilibrate for another 40 min before recording of stimulated contractile responses. The average of three control neurogenic responses evoked by a single electrical impulse with duration of 0.1 ms and supramaximal voltage were calculated and compared with that of neurogenic responses after pretreatment with drugs. In some experiments, the tissue preparations were subjected to a 20-s-long train of electrical impulses delivered at a frequency of 8 Hz.
Incubation. Vas deferens tissue preparations were fixed at one end to a holder and suspended vertically in an organ chamber filled with 5 ml of Krebs' solution (37°C). The other end of each tissue was attached to a force displacement transducer (Grass FTO3) by silk surgical suture. After 20 min of incubation, an initial load of 0.5 g was applied to each tissue by the means of micromanipulator, and the tissues were equilibrated for another 40 min. The Krebs' solution was replaced at 15-min intervals. Three control contractile responses to ATP (100 µM) or 3 µM ,
-methylene ATP (, mATP) were recorded at the beginning of each experiment. The average amplitude of these responses was compared with that of ATP and , mATP-induced contractile responses after treatment with drugs.
Recording and Processing of Data from Contraction Experiments. The signals from the force displacement transducers were recorded, displayed, and analyzed using a Power Lab system consisting of Power Lab Bridge Amplifier, Power Lab 8SP high-speed recorder, and a Pentium IV PC equipped with Power Lab Chart 4.2 software (ADInstruments Pty Ltd., Castle Hill, Australia). The areas under the waveforms were calculated and entered in spreadsheets of the Prism 3 (GraphPad Software Inc., San Diego, CA) software for statistical evaluation, pharmacological analysis, and graphic presentation. The results are presented as the mean ± S.E.M. One-way analysis of variance was applied to compare variables, and P values of less than 0.05 were considered to represent statistically significant differences.
Breakdown of Extracellular ATP by the Guinea Pig Vas Deferens. Guinea pig vas deferens tissue preparations were set up for recording contractile responses under incubation conditions as described above. After an equilibration period of 40 min, one of the vasa deferentia dissected from each animal was treated with phosphatase inhibitor cocktail 2 (Sigma-Aldrich, St. Louis, MO) diluted 1:100, whereas the other served as a control. The phosphatase inhibitor cocktail 2 contains sodium orthovanadate, sodium molybdate, sodium tartrate, and imidazole. The final concentration of sodium orthovanadate (SOV) in these experiments was 1 mM. After an incubation period of 15 min, the tissue chambers were drained and refilled with buffer containing the fluorescent analog of ATP, 1,N6-etheno-ATP (e-ATP) or buffer containing e-ATP and phosphatase inhibitor II, respectively. Ten minutes after the addition of the substrate, aliquots of 50 µl were taken, acidified with 100 µl of citric phosphate buffer (pH 4), and stored on ice. The tissue preparations were washed three times and left to equilibrate for another 20 min before repeating the same procedures with higher concentration of e-ATP (0.1-1000 µM). An HPLC-based assay was used to quantify the adenine nucleotides present in each of the samples.
HPLC-Based Assay for Nucleotidase Activity. The fluorescent 1,N6-etheno analog of ATP was used as substrate in these experiments designed to study the breakdown of ATP by the guinea pig vas deferens. The rationale of this assay is based on the fact that a sequential dephosphorylation of e-ATP results in formation of 1,N6-etheno ADP followed by formation of 1,N6-etheno AMP, and finally formation of 1,N6-etheno adenosine. The substrate and the resulting metabolites were simultaneously quantified by HPLC with fluorescence detection.
HPLC Technique. The e-purines were separated on a gradient HPLC system equipped with a Waters Resolve radial pack cartridge (8NV Ph 4 µm; 8 x 10 mm). The amount of each compound was quantified by means of a Waters 474 fluorescent detector at an excitation wavelength of 230 nm and an emission wavelength of 420 nm. Buffer solutions consisted of 0.1 M phosphate (KH2PO4), pH 6.0 (buffer A) and 75% 0.1 M phosphate and 25% methanol (buffer B). The nucleotides and adenosine were separated using a gradient in which the concentration of buffer B was increased from 0 to 100% in 8 min according to Waters gradient profile 7. Identification of individual peaks was by comparison with the retention times of known e-purines standards, and the concentration was determined by peak area per picomole compared with standards. Standards were run with each set of samples. The HPLC equipment was controlled by, and data collected by, a Pentium II-based desktop computer equipped with an LAC/E card and Millennium 2010 Chromatography Manager software from Waters (Milford, MA). Results were normalized for volume and tissue weight and the data calculated as picomoles per milligram.
Protein Separation and Western Blots Experiments. Guinea pig vasa deferentia were set up for experiments under incubation conditions, and the contractile responses of each of the tissues to a bolus injection of ATP (100 µM) were recorded as described above. The tissue preparations were divided in three groups: 1) preparations treated only with ATP (control), 2) preparations treated for 15 min with SOV (300 µM) before the application of ATP, and 3) preparations treated for 15 min with genistein (100 µM) before the application of SOV and ATP. Immediately after recording the contractile responses, the tissues were lifted by their holders, dipped in ice-cold Ca2+-free Krebs' solution, containing 1 mM SOV and 5 mM EDTA, and immediately frozen in liquid nitrogen. The frozen tissues were crushed to powder using a Cryo-Press device (Microtec, Chiba, Japan) chilled in liquid nitrogen. The powder from each tissue was transferred in ice-cold sucrose buffer (250 mM sucrose, 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 mM 4-(2-aminoethyl) benzenesulphonyl fluoride hydrochloride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM SOV, 10 mM NaF, and 0.5% Triton X-100) and centrifuged at 14,000g for 10 min at 4°C. Supernatants were collected and protein concentrations were determined using the bicinchoninic acid protein assay (Pierce Chemical, Rockford, IL). Samples containing equal amounts of protein were solubilized in equal volume of SDS-Laemmli buffer and boiled for 5 min at 90°C. The proteins (20 µg) were separated by electrophoresis on 10% Tris-SDS polyacrylamide gels (Ready-Gel; Bio-Rad, Hercules, CA) and electrically transferred onto Hybond nitrocellulose membranes (Amersham Biosciences Inc., Piscataway, NJ). The membranes were blocked using the Odyssey blocking buffer (Li-COR, Lincoln, NE) and incubated for 2 h with the mouse anti-phosphotyrosine antibody (PY-99; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) as well as the rabbit anti-ERK1/2 polyclonal antibody (Promega, San Obispo, CA). The membranes were further incubated for 1 h with the goat anti-mouse antibody coupled to Alexa-Flour 680 (Molecular Probes, Eugene, OR) as well as the goat anti-rabbit antibody coupled to IRDye 800 (Rockland, Gilbertsville, PA) diluted at 1:100,000. During laser scanning of the membranes, direct fluorescence from labeled proteins was simultaneously detected by two separate infrared detection channels of the Imaging System Odyssey (LI-COR). Proteins phosphorylated on tyrosine (Alexa 680) were detected in red, and total ERK protein was detected in green (IRDye 800). Estimation of molecular size of protein bands was made by extrapolation from standard curve of broad-range prestained SDS-polyacrylamide gel electrophoresis standards (Bio-Rad) loaded on each gel. To eliminate any variability of the amount of the loaded total protein, we normalized the phosphotyrosine signal intensities in each lane against ERK, and the relative intensity ratios (in arbitrary units) have been plotted in Fig. 6B using GraphPad Prism software.
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Chemicals Used. The following chemicals were purchased from Sigma-Aldrich: adenosine 5' triphosphate disodium salt; , methylene adenosine 5' triphosphate; butein; daidzein; dephostatin; 2,5-dihydroxycinnamic acid methyl ester; genistein; levandustin C; norepinephrine hydrochloride; ouabain; phosphatase inhibitor cocktail 2; sodium orthovanadate; thyrphostin 23; thyrphostin 1. The chemicals used for the preparation of the Krebs' solution which were of analytical grade were also purchased from Sigma-Aldrich.
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Results |
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; Sneddon and Westfall, 1984). Accordingly, this contractile response is mimicked by exogenously applied ATP (Fig. 1). Pretreatment of the tissue with SOV caused a robust increase of the amplitude of the contractile responses evoked by both a single stimulus and a bolus injection of exogenous ATP (Fig. 1). The facilitation of the purinergic contraction evoked by SOV was time-dependent. Thirty seconds after the introduction of SOV, the amplitude of the contractile response of the guinea pig vas deferens to a single impulse was tripled (data not shown). Full facilitation of the contractile response was maintained for at least 60 min (longer exposure to SOV has not been tested). The facilitation evoked by SOV was also reversible and reproducible. The control level of the amplitude of the purinergic contraction was restored within 30 min after washout of the drug. A new application of SOV produced virtually the same increase of the amplitude of contractions evoked by either EFS or ATP, and this cycle has been repeated up to five times without loss of effect.
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The effect of SOV on the purinergic contractile response was also concentration-dependent. SOV (0.1-1000 µM) had no effect on the resting tone of the guinea pig vas deferens but increased the response evoked by EFS (one impulse) from 0.62 ± 0.69 to 31.06 ± 1.1 g/s, which represents a 50.2 ± 3.2-fold increase. Facilitation was observed at approximately 3 µM SOV, and the half-maximal effect was achieved at 245 ± 23 µM (Fig. 2, A and B). However, when applied in concentrations greater than 1 mM, SOV produced spontaneous contractions of the guinea pig vas deferens. To avoid unspecific effects of SOV, concentrations higher than 1 mM were not used in this study.
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The contractile response of the guinea pig vas deferens evoked by exogenously applied ATP (100 µM) was increased from 4.3 ± 0.33 to 23.7 ± 1.2 g/s in the presence of SOV (0 to 1 mM) (Fig. 3). This change represents approximately 6-fold increase over the amplitude of the control contraction. The half-maximal facilitation, derived from nonlinear regression curves was achieved with 122 ± 42 µM SOV.
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We have tested the effect of SOV on the contractile response of the guinea pig vas deferens evoked by , mATP, a slowly degradable analog of ATP, which is also a more potent agonist of the P2X1 receptors. Accordingly, application of , mATP at a concentration of 3 µM produced smooth muscle contraction with an amplitude of 4.6 ± 0.49 g/s, which is similar to that produced by 100 µM ATP (Fig. 3). The contractile response evoked by , mATP in presence of SOV was also increased in a concentration-dependent manner to a maximum of 24.5 ± 1.3 g/s (a 6-fold increase). The half-maximal facilitation was achieved with 100 ± 39 µM SOV. The comparison of the effects of SOV on the contractions evoked by ATP and , mATP presented in Fig. 3 demonstrates that both agonists produce contractile responses that are similarly enhanced in the presence of increasing concentrations of SOV.
If an inhibition of a PTP was involved in the action of SOV, then pretreatment of the tissue preparations with an appropriate inhibitor of the tyrosine kinase responsible for the phosphorylation of the target protein(s) might prevent the SOV-evoked facilitation of the purinergic contractile response of the guinea pig vas deferens. To test this hypothesis, we have carried out experiments with a number of protein tyrosine kinase inhibitors. Two structurally unrelated antagonists of tyrosine kinase activity, genistein and 2,5-dihydroxycinnamic acid methyl ester, produced a dose-dependent inhibition of the SOV-evoked facilitation of the contractile responses to EFS (Fig. 2, C and D). The half-maximal inhibition was achieved with 2.47 ± 0.48 µM genistein and with 2.30 ± 0.38 µM DHCA (n = 4). When applied in concentration of 100 µM, genistein (Figs. 4 and 5) and 2,5-dihydroxycinnamic acid methyl ester (Fig. 5) inhibited the SOV-evoked facilitation of the contractile responses to EFS and exogenously applied ATP by more than 90%. In Fig. 4, we also show that daidzein, a structural analog of genistein that shares some of the effects of genistein but is not PTK inhibitor, does not inhibit the SOV-evoked facilitation of the purinergic contractions evoked by EFS or exogenously applied ATP. Although 2,5-dihydroxycinnamic acid methyl ester and genistein nearly abolished the effects of SOV (300 µM) on the contractile responses evoked by EFS and ATP, several other well known tyrosine kinase inhibitors, namely, butein, levandustin C, and thyrphostin 23 decreased but did not abolish the SOV-induced facilitation (Fig. 5, A and B). The inactive structural analog of thyrphostin 23, thyrphostin 1 had no effect (data not shown).
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The level of tyrosine phosphorylation of proteins from guinea pig vas deferens treated with ATP alone or in the presence of SOV or SOV and genistein was studied in Western blot experiments. The results showed that SOV (Fig. 6A, lane 1) promoted a prominent increase of the anti-phosphotyrosine immunoreactivity in at least five bands containing proteins with estimated molecular masses of 180, 123, 97, 77, and 42 kDa (compare lane 1 with lane 3). Pretreatment with genistein inhibited the increase in tyrosine phosphorylation evoked by SOV in all those bands (Fig. 6A, lane 2). The level of tyrosine phosphorylation of proteins with molecular mass of 180 and 123 kDa observed under control conditions (lane 3) was also significantly decreased by genistein (Fig. 6B).
Evidence in the literature demonstrates that the vasoconstriction evoked by NE or selective 2-adrenoceptor agonists is inhibited by genistein (Abebe and Agrawal, 1995; Jinsi et al., 1996). These authors have suggested that tyrosine kinases may play an important role in the adrenergic contraction of vascular smooth muscle mediated via 2-adrenoceptors. We have also tested the effects of genistein on the adrenergic contraction, which in the guinea pig vas deferens is mediated via 1-adrenoceptors. Prolonged neurogenic electrical stimulation (20 s at 8 Hz) evokes a biphasic contractile response of the guinea pig vas deferens (Fig. 7). The first twitch contraction is mediated predominantly by ATP, whereas the second, tonic contraction is mediated exclusively by NE. Consistent with the evidence presented above, the purinergic twitch contraction was significantly increased in the presence of SOV (300 µM). Pretreatment with genistein (100 µM) prevented this facilitation and further reduced the purinergic twitch. The adrenergic tonic component of the contractile response, however, was affected to a much lesser extent (Fig. 7). Similar results were obtained in experiments with exogenously applied NE. There was approximately 50% increase of the amplitude of the NE-evoked contraction in the presence of SOV. The amplitude of this effect seems to be relatively modest in comparison with the 8-fold increase of the contractile response evoked by exogenously applied ATP. A moderate decrease of contractions evoked by exogenously applied NE was also observed in the presence of genistein (data not shown).
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Although widely used as an antagonist of PTP, when applied in higher concentrations SOV can also inhibit other enzymes, including the Na+/K+-ATPase. To test whether effects other than inhibition of PTP might be involved in the observed facilitation of the purinergic contractile responses by SOV, we have compared the effects of SOV with that of ouabain, a selective inhibitor of the Na+/K+-ATPase. The results presented in Fig. 8 show that there are significant differences between the modes of action of the two drugs. Application of ouabain (300 µM) produced an increase of the amplitude of the neurogenic contractile response of the guinea pig vas deferens evoked by a single impulse of EFS (Fig. 8A). Approximately 15 min after the exposure to ouabain a spontaneous contraction developed, and the tissue preparations stopped responding to nerve stimulation. Application of SOV (300 µM) produced a greater increase of the amplitude of the neurogenic contractions than that produced by ouabain (Fig. 8, compare A with B). Unlike ouabain, SOV did not produce a spontaneous contraction, and its facilitatory effect continued after the tissues treated with ouabain stopped responding to neurogenic stimulation (Fig. 8, A and B). Addition of genistein (100 µM) to the superfusing solution already containing SOV quickly inhibited the facilitation of the neurogenic contractile responses evoked by SOV (Fig. 8C).
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It can be argued that if SOV can inhibit enzymes such as phosphatases and the Na+/K+-ATPase, it might also inhibit the breakdown of extracellular ATP and thus enhance its contractile action. To test this hypothesis, we studied the breakdown of extracellular ATP by the ecto-enzymes expressed in the guinea pig vas deferens. The amount of e-ATP present in the sample taken after 10 min of incubation with the tissue preparations was subtracted from the amount of e-ATP present in the buffer that has not been in contact with a tissue. The difference, measured in picomoles, represents the amount of e-ATP that has been metabolized by ectoenzymes and reflects the sum total of the ecto-ATP-metabolizing activity of the guinea pig vas deferens. The results presented in Fig. 9 show that the plot of substrate (e-ATP) concentrations versus the ATP-metabolizing activity had a hyperbolic fit with a KD value of 414 ± 68 µM. In presence of the phosphatase inhibitor cocktail 2, which contains sodium orthovanadate, sodium molybdate, sodium tartrate, and imidazole, the observed KD value was increased to 673 ± 170 µM, whereas the Vmax value was increased from 1768 ± 103 to 2075 ± 236 pmol/10 min. A comparison of the best fit values by t test (GraphPad Prism) revealed that there were no significant differences between the ATP-metabolizing activity of the vas deferens tissue preparations under control conditions and in the presence of the phosphatase inhibitor cocktail 2 (P < 0.05). The results demonstrate that ATP is not significantly metabolized by the guinea pig vas deferens tissue preparations. From the initially applied 100 µM e-ATP, only 350 pM was metabolized within 10 min. The evidence also shows that neither SOV nor the other phosphatase inhibitors have any significant effect on the metabolism of extracellular ATP.
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Discussion |
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) as well as soluble nucleotidases that are released during nerve activation (Todorov et al., 1997). It has been shown that SOV has no effect on the activity of either group of enzymes (Mihaylova-Todorova et al., 2001, 2002). However, the possibility that the breakdown of extracellular ATP by the ecto-enzymes expressed in the guinea pig vas deferens tissue preparations may involve not only ecto-ATPases but also ecto-phosphatases has not been addressed. Our results demonstrate that not only SOV but also other phosphatase inhibitors such as sodium molybdate, sodium tartrate, and imidazole, all of which are included in the phosphatase inhibitor cocktail 2 (Sigma-Aldrich) failed to affect the breakdown of ATP by the guinea pig vas deferens tissue preparations. The changes of both KD and Vmax of the enzymatic breakdown of ATP after pretreatment of the tissue preparations with the phosphatase inhibitor cocktail 2 were not statistically significant (Fig. 9). The results from this study have also demonstrated that only a minimal amount of the exogenously applied ATP has been metabolized by the guinea pig vas deferens tissue preparations even though the reaction was let to continue for 10 min. Moreover, the results shown in Fig. 3 show that SOV increases the contractile effects not only of ATP but also of its metabolically stable analog , mATP. Together, this evidence argues against the possibility that SOV facilitates the ATP-evoked contractile response of the vas deferens by preventing its clearance.
Another side effect of SOV might be related to its ability to block the Na+/K+-ATPase, i.e., it may produce an "ouabain-like" effect. Indeed, an inhibition of the electrogenic Na+/K+ pump could cause cell membrane depolarization and thereby an increase of the amplitude of the contractile response to ATP (Urquilla at al., 1978). However, an inhibition of the Na+/K+ pump should also produce a spontaneous contractile activity within a short period of time, after which the tissue preparation should stop responding to stimulation due to equilibration of the concentration gradients of Na+ and K+. The results presented in Fig. 8A demonstrate that ouabain produces all these expected effects. However, the evidence from our studies argues against the possibility that an ouabain-like or another side effect such as inhibition of Ca2+ ATPases is involved in the facilitation evoked by SOV. First, the dose-response curve presented in Fig. 2 demonstrate that SOV facilitates the purinergic contractile response of the guinea pig vas deferens with threshold concentration in the low micromolar range and with EC50 value of approximately 100 µM. SOV acts as a selective antagonist of protein tyrosine phosphatases when used in this range of concentrations (Taylor, 2003). Second, even when applied at the concentration of 300 µM, which produces near maximal facilitation effect, SOV does not trigger spontaneous contractile activity (Fig. 8B). A contractile effect should accompany any significant increase of intracellular Ca2+ due to increased influx of Ca2+ (i.e., ouabain-like effect) or inhibition of Ca2+ extrusion due to an inhibition of the Ca2+ pump. Third, longer exposure to SOV (up to 60 min) did not cause a spontaneous contraction and did not abolish the response of the tissue to neurogenic stimulation or application of exogenous ATP. Fourth, genistein and 2,5-dihydroxycinnamic acid methyl ester, two structurally unrelated inhibitors of tyrosine kinase activity, abolished the facilitation of the purinergic contractions evoked by SOV. Fifth, the changes in the level of protein tyrosine phosphorylation evoked by SOV and genistein are consistent with their functional effects.
Genistein inhibits the activity of tyrosine kinases by interacting with their ATP-binding sites (Akiyama et al., 1987). In addition, it has been shown that genistein may also inhibit the function of L-VDCCs. The ATP-induced smooth muscle contraction is mediated via P2X1 receptors, which when activated lead to the opening of L-VDCCs. The resulting influx of extracellular Ca2+ triggers action potentials and the twitch-like purinergic contraction (Sneddon and Westfall, 1984). It seems possible, therefore, that genistein could inhibit the purinergic contractile responses of the guinea pig vas deferens by blocking the P2X receptors and/or the L-VDCC directly. Several lines of evidence from studies of cardiac and vascular smooth muscle suggest that tyrosine phosphorylation may be involved in the regulation of the activity of L-VDCC (Hughes and Wijetunge 1998; Davis et al., 2001). Our findings that genistein not only prevents the facilitation of the purinergic contraction evoked in the presence of SOV but also reduces the purinergic contraction itself is consistent with the possibility that protein tyrosine phosphorylation may be involved in the regulation of the activity of L-VDCC in the smooth muscle of the guinea pig vas deferens as well.
However, there are publications that argue that L-VDCCs are not regulated by PTK and the inhibitory effect of genistein might be a result from a direct noncatalytic blockade of these channels. The proponents of this view have also reported that daidzein mimics the inhibitory action of genistein at the L-VDCC (Hool et al., 1998). The evidence from our study favors the view that the inhibition of the SOV-induced facilitation of the purinergic contraction by genistein is indeed due to an inhibition of protein tyrosine phosphorylation rather than direct inhibition of influx of Ca2+. For example, we found that daidzein, which like its analog genistein, blocks Ca2+ influx but is inactive as PTK inhibitor had no effect on the amplitude of the purinergic contraction or its facilitation by SOV. In addition, 2,5-dihydroxycinnamic acid methyl ester, a PTK inhibitor, which is structurally unrelated to genistein prevented the SOV-evoked facilitation of the purinergic contraction.
Our results have demonstrated certain selectivity of the drugs used to modulate the activity of protein tyrosine kinases. From the wide spectrum of PTK inhibitors used, two, genistein and 2,5-dihydroxycinnamic acid methyl ester, were the most effective inhibitors of the SOV-evoked facilitation of the purinergic contractile response of the guinea pig vas deferens. The other three PTK antagonists, butein, levandustin C, and thyrphostin 23, were less effective in reducing the effects of SOV. This pharmacological profile of the PTK antagonists may prove valuable for the ultimate identification of the protein(s) involved in modulation the smooth muscle responsiveness to purinergic stimulation.
Interestingly, we have shown that both PTK and PTP inhibitors affected to a much greater extent the facilitation of the contractile responses produced by ATP than the contractile responses produced by the cotransmitter NE. This finding suggests that the second messenger system that mediates the smooth muscle contractile response to adrenergic stimulation may be less dependent on protein tyrosine phosphorylation than is the response to purinergic stimulation. No further effort to clarify the role of protein tyrosine phosphorylation in the adrenergic contractile response of the guinea pig vas deferens has been made in this study.
Although not entirely selective at higher concentrations, the drugs used in this study seem to have only one common mechanism of action. They increase (SOV) or decrease (genistein and 2,5-dihydroxycinnamic acid methyl ester) the level of protein tyrosine phosphorylation. Therefore, the pharmacological evidence from our functional experiments, together with the evidence from our Western blot experiments seem to suggest that tyrosine phosphorylation of a protein(s), whose identity and signaling pathway are not known at this time, is involved in the regulation of the amplitude of the purinergic smooth muscle contraction. This novel element of the purinergic signaling in the smooth muscle may prove to be an appropriate pharmacological target for effective management of smooth muscle contractility.
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Footnotes |
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ABBREVIATIONS: NE, norepinephrine; L-VDCC, L-type voltage-dependent calcium channel; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase(s); EFS, electrical field stimulation; , mATP, , methylene adenosine 5' triphosphate; SOV, sodium orthovanadate; e-ATP, 1,N6-etheno-ATP; HPLC, high-performance liquid chromatography; ERK, extracellular signal-regulated kinase.
Address correspondence to: Dr. Latchezar D. Todorov, Department of Pharmacology, University of Nevada School of Medicine, 1664 N Virginia St., Howard Medical Sciences Bldg. MS 318, Reno, NV 89557-0270. E-mail: todorov{at}med.unr.edu
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