Relaxation of Isolated Guinea Pig Trachea by Genistein
via Inhibition of Phosphodiesterase
Ching-Chi Lin1Junn-Lain Chen2 Wun-Chang Ko2
Affiliation
1Department of Internal Medicine, Macky Memorial Hospital, Taipei, Taiwan, R.O.C.
2Graduate Institute of Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C.
Correspondence Wun-Chang Ko ´ 250 Wu-Hsing St ´ Taipei 110 ´ Taiwan ´ Republic of China ´ Phone: +886-2-2736-1661 ext. 3197 ´ Fax: +886-2-2377-7639 ´ E-mail: [email protected] Received October 25, 2006 ´ Revised January 23, 2007 ´ Accepted February 5, 2007 Bibliography Planta Med 2007; 73: 323±329 Georg Thieme Verlag KG Stuttgart ´ New York DOI 10.1055/s-2007-967155 ´ Published online March 29, 2007 ISSN 0032-0943 Abstract
We investigated the mechanisms of the relaxant action of genis-tein, an isoflavone, phytoestrogen and non-specific protein tyro-sine kinase inhibitor. Changes in tension of guinea pig tracheal segments were isometrically recorded on a polygraph. Genistein concentration-dependently relaxed histamine (30mM)-, carba-chol (0.2mM)-, KCl (30 mM)- and leukotriene D4 (10 nM)-in-duced precontractions and inhibited cumulative histamine- and carbachol-induced contractions in a non-competitive manner. Genistein also concentration-dependently and non-competitive-ly inhibited the cumulative, Ca2+-induced contractions in the de-polarized (K+, 60 mM) trachealis. The remaining nifedipine (10 mM)-induced tension of the histamine (30 mM)-induced precon-traction was further relaxed by genistein, suggesting that regard-less of whether voltage-dependent calcium channels are blocked genistein may have other mechanisms of relaxant action. These other mechanisms of the relaxant effect of genistein appeared to be epithelium-independent and were not affected by the pres-ence of propranolol (1mM), 2¢,5¢-dideoxyadenosine (10 mM), me-thylene blue (25mM), glibenclamide (10 mM), Nw-nitro-L-arginine (20mM) or a-chymotrypsin (1 U/mL), suggesting that the mecha-nisms are unrelated to activation of theb-adrenoceptor, of ade-nylate cyclase, of guaade-nylate cyclase, of adenosine triphosphate-sensitive potassium channel opening, of nitric oxide formation or of neuropeptide release, respectively. However, genistein (17.5±35mM) produced parallel, leftward shifts in the concentra-tion-response curves of forskolin and nitroprusside and signifi-cantly increased the pD2values of these two agonists. Both
ge-nistein and 3-isobutyl-1-methylxanthine at various concentra-tions (10±300mM) concentration-dependently and significantly inhibited cAMP- and cGMP-phosphodiesterase (PDE) activities of the trachealis. The -log IC50values of genistein were estimated to be 4.28 and 4.17, respectively. The above results reveal that the mechanisms of the relaxant action of genistein may be due to its non-selective inhibition of both PDE activities.
Key words
Genistein ´ isoflavone ´ phosphodiesterase inhibitor ´ guinea pig tracheal relaxation ´ cyclic AMP-phosphodiesterase ´ cyclic GMP-phosphodiesterase
Abbreviations
IBMX: 3-ixobutyl-1-methylxanthine VDCCs: voltage-dependent calcium channels cAMP: adenosine 3¢,5¢-cyclic monophosphate cGMP: guanosine 3¢,5¢-cyclic monophosphate ATP: adenosine triphosphate
PDE: phosphodiesterase LTD4: leukotriene D4 L-NNA: Nw-nitro-L-arginine DMSO: dimethyl sulfoxide EGTA: N,N,N¢,N¢-tetraacetic acid ANOVA: analysis of variance
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Introduction
Flavonoids are naturally occurring polyphenolic compounds with a wide distribution in the plant kingdom. They possess an-tioxidant, antitumor, antiangiogenic, anti-inflammatory, antial-lergic, and antiviral properties [1], [2], [3]. Genistein, an isofla-vone and phytoestrogen found in high concentrations in soybean milk and tofu, has been reported to non-specifically inhibit pro-tein tyrosine kinases [4]. Genispro-tein, therefore, facilitates canine bronchial smooth muscle relaxation [5], [6] and attenuates anti-gen-induced guinea pig airway contractions [7]. In 1997, String-field and Morimoto reported that genistein can modulate adeno-sine 3¢,5¢-cyclic monophosphate (cAMP) levels in the HT4.7 neur-al cell line [8]. In the absence of phosphodiesterase (PDE) inhibi-tors, genistein causes increased intracellular cAMP levels. How-ever, when PDE inhibitors are included, cAMP levels decrease as a function of the concentration of genistein. This suggests that genistein inhibits both cAMP synthesis and degradation. Cyclic AMP is mainly synthesized from adenosine triphosphate (ATP) via activation of adenylate cyclase and is degraded by PDE. Therefore, some investigators have focused on regulation of PDE by tyrosine phosphorylation either indirectly [9] or through di-rect interaction with a protein tyrosine kinase [10]. In 1999, Ni-chols and Morimoto reported that HT4.7 PDE activity can be regulated by genistein through a tyrosine kinase-independent mechanism [11]. It seems contradictory that genistein inhibits both cAMP synthesis and degradation. Therefore, we were inter-ested in the mechanism of tracheal relaxation by genistein. Materials and Methods
Reagents and drugs
Genistein (Fig.1, with a purity of > 98%), aminophylline, carba-chol, histamine, propranolol, 2¢,5¢-dideoxyadenosine, methylene blue, glibenclamide, Nw-nitro-L-arginine (L-NNA), a-chymotryp-sin, nifedipine, indomethacin, forskolin, sodium nitroprusside, ethylene glycol-bis(b-aminoethyl ether)-N,N,N¢,N¢-tetraacetic acid (EGTA), Trizma base, dl-dithiothreitol,b-mercaptoethanol, cyclic AMP, guanosine 3¢,5¢-cyclic monophosphate (cGMP), cal-modulin, leukotriene D4(LTD4), Dowex resin and Crotalus atrox snake venom were purchased from Sigma Chemical (St. Louis, MO, USA). [3H]cAMP and [3H]cGMP were purchased from Amer-sham Pharmacia Biotech (Uppsala, Sweden). 3-Isobutyl-1-me-thylxanthine (IBMX) was purchased from Aldrich Chemical (Mil-waukee, WI, USA). All other reagents, including KCl, were of ana-lytical grade. Glibenclamide was dissolved in dimethyl sulfoxide (DMSO). Genistein, IBMX, forskolin, indomethacin, and nifedi-pine were dissolved in ethyl alcohol. Other drugs were dissolved in distilled water. The final concentration of ethyl alcohol or DMSO was less than 0.1% and did not significantly affect the con-traction of the trachea.
Guinea pig trachea
Using a protocol approved by the Animal Care and Use Commit-tee of the Taipei Medical University, male Hartley guinea pigs (National Laboratory Animal Center, Taipei, Taiwan) weighing 250±450 g were sacrificed by cervical dislocation, and their tra-cheas were removed. Each trachea was cut into six segments. Each segment consisted of three cartilage rings. All segments were cut open opposite the trachealis. After the segments were randomized to minimize regional variability, a segment was tied at one end to a holder via silk sutures, placed in 5 mL of nor-mal or Ca2+-free Krebs solution containing indomethacin (3mM), gassed with a 95% O2/5% CO2mixture at 378C and attached by its other end to a force displacement transducer (Grass FT03, Grass; Quincy, MA, USA) for the isometric recording of tension changes on a polygraph (Gould RS3200, Gould; Valley View, OH, USA). The composition of the normal Krebs solution was (mM): NaCl 118, KCl 4.7, MgSO41.2, KH2PO41.2, CaCl22.5, NaHCO325, and dextrose 10.1. The isotonic high-K+, Ca2+-free Krebs solution con-sisted of the above composition without CaCl2, with the 60 mM NaCl being replaced by 60 mM KCl. The tissues were suspended in normal Krebs solution under an initial tension of 1.5 g and al-lowed to equilibrate for at least 1 h with washing at 15-min inter-vals. After the tissues were precontracted with histamine (30 mM), carbachol (0.2 mM), KCl (30 mM) or LTD4(10 nM), genistein (1±300mM) was cumulatively added to the organ bath, and its tracheal relaxant effects were allowed to reach a steady state at each concentration. At the end of the experiment without wash-out, 1 mM of aminophylline was added to standardize maximal tissue relaxation. The relaxant potencies of genistein were expressed as ±logIC50values. To determine the antagonistic ef-fects of genistein against contractile agonists, either histamine or carbachol was then cumulatively added to the normal Krebs solution, and the procedure was repeated until the contraction reached constancy after washout. Then, cumulative concentra-tion-response curves were constructed. The maximal contrac-tion of a trachea without incubacontrac-tion of drugs or their vehicles was set to 100 %. After the tissues were preincubated with genis-tein or its vehicle for 15 min, these two contractile agonists were also cumulatively added to the normal Krebs solution. The antag-onistic potencies of genistein were expressed as pD2¢ values when the antagonistic effect on these cumulative concentra-tion-response curves occurred in a non-competitive manner. In the case of isotonic high-K+(60 mM)-depolarized tracheal pre-parations, the normal Krebs solution was replaced after equili-bration by a Ca2+-free Krebs solution without EGTA, and the seg-ments were washed with the Ca2+-free solution with 2 mM EGTA after the tracheal contraction reached constancy, followed by in-cubation for 5 min. After repeating the above procedure until no contraction was observed, Ca2+(0.01±10 mM) was cumulatively added, and contractions were elicited in the depolarized trachea-lis. The maximal contractile response elicited by Ca2+(10 mM) was taken as 100 %, and the cumulative concentration-response curve was constructed. The inhibitory effects of genistein on cu-mulative Ca2+-induced contractions in isotonic high-K+(60 mM)-depolarized tracheas were expressed as -logIC50values. The tra-cheal relaxant effects of cumulative genistein (10±100mM) on the histamine (30mM)-induced precontraction were allowed to reach a steady state at each concentration. All antagonists, in-cluding propranolol, glibenclamide, 2¢,5¢-dideoxadenosine, me-thylene blue, L-NNA,a-chymotrypsin and their respective vehi-Fig. 1 Chemical structure
of genistein (mol wt 270.23).
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324cles were individually incubated after the precontraction reached a steady state for 15 min prior to the first addition of genistein. Similarly, nifedipine (10mM) was added after the his-tamine (30mM)-induced precontraction reached a steady state, at 15 min prior to the addition of genistein (100mM) or its vehi-cle. At the end of the experiment without washout, 1 mM amino-phylline was added to standardize the maximal tissue relaxation (100%). To observe the effect of genistein on the relaxant re-sponse of forskolin or nitroprusside to the histamine (30 mM)-in-duced precontraction, genistein (17.5±35mM) was incubated for 15 min prior to the addition of histamine. Forskolin or nitroprus-side was cumulatively added to the organ bath after the sus-tained contraction had reached constancy. At the end of the ex-periment, aminophylline (1 mM) was also added to maximally relax the tissue. To investigate the effects of the epithelium on the relaxant response of genistein to the histamine (30 mM)-in-duced precontraction, some tracheal segments were denuded by rubbing with a moistened cotton-tipped applicator while the intact epithelium was retained in other segments. At the end of the experiment, aminophylline (1 mM) was also added to maxi-mally relax the tissue. The denuded and intact tissues were ex-amined using light microscopy after staining with hematoxylin and eosin (H&E) to determine the effectiveness of the epithelium removal procedure [12].
Phosphodiesterase activity
The isolated trachealis was homogenized with a glass/Teflon homogenizer (Glas-Col; Terre Haute, IN, USA) in 20 volumes of cold medium (pH 7.4) containing 100 mM Tris-HCl, 2 mM MgCl2 and 1 mM dithiothreitol. cAMP- and cGMP-PDE activities in the homogenate were measured by a modification of the method of Cook et al. [13]. The homogenate was centrifuged at 9500 rpm for 15 min, and the upper layer was decanted. Twenty-five micro-liters of the upper layer were taken for determination of enzyme activity in a final volume of 100mL containing 40 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, 3.75 mM mercaptoethanol, 0.1 unit cal-modulin (PDE activator), 10mM CaCl2and either 1mM cAMP with 0.2mCi [3H]cAMP or 1mM cGMP with 0.2 mCi [3H]cGMP. In tests of enzyme inhibition, the reaction mixture contained various con-centrations of genistein (10±300mM) or IBMX (10±300 mM) as the positive control. The reagents and homogenate were mixed on ice, and the reaction was initiated by transferring the mixture to a water bath at 378C. Following a 30-min incubation, the reac-tion was stopped by transferring the reacreac-tion vessel to a bath of boiling water for 3 min. After cooling on ice, 20mL of a 1 mg/mL solution of Crotalus atrox venom was added to the reaction mix-ture, and the mixture was incubated at 378C for 10 min. Unreac-ted [3H]cAMP or [3H]cGMP was removed by the addition of 500 mL of a 1-in-1 Tris-HCl (40 mM) buffer suspension of Dowex resin (18±200) with incubation on ice for 30 min. Each tube was then centrifuged for 2 min at 6000 rpm, and 150mL of the super-natant were removed for liquid scintillation counting. Less than 10% of the tritiated cyclic nucleotide was hydrolyzed in this as-say.
Statistical analysis
The antagonistic effects of genistein on these cumulative con-centration-response curves are expressed as pD2¢ values, and the relaxing effects of forskolin and nitroprusside against hista-mine (30mM)-induced precontractions are expressed as pD2
val-ues, according to the method described by Arins and van Ros-sum [14]. The pD2values are the negative logarithm of the molar concentrations of forskolin and nitroprusside at which the half-relaxing effects on histamine (30mM)-induced precontractions were observed. pD2¢ = pDx¢ + log (x - 1), where pDx¢ is the nega-tive logarithm of the molar concentration of genistein and x is the ratio between the maximal effect of the agonist in the ab-sence and preab-sence of genistein. The -log IC50value was consid-ered to be equal to the negative logarithm of the molar concen-trations of genistein at which a half-inhibitory effect on agonist-induced precontractions, the Ca2+(10 mM)-induced contraction or cyclic nucleotide PDE activity was observed. The IC50value was calculated by linear regression. All values are shown as the mean SEM. Differences among these values were statistically calculat-ed by one-way analysis of variance (ANOVA), then determincalculat-ed by Dunnett's test. The difference between the two values, however, was determined using Student's unpaired t-test. Differences were considered statistically significant if the p value was < 0.05. Results
Genistein concentration-dependently and almost completely re-laxed the histamine (30mM), carbachol (0.2 mM), KCl (30 mM) and LTD4(10 nM)-induced precontractions (Fig. 2). Their -log IC50 values were 4.55 0.06 (n = 7), 4.41 0.04 (n = 7), 4.40 0.05 (n = 6) and 4.57 0.07 (n = 6), respectively. They are not statis-tically different. Genistein (35±100 mM) concentration-depen-dently inhibited the log concentration-response curves of cumu-lative histamine in a non-competitive manner (Fig. 3A). The same was true for genistein (45±200mM) in the curves for carba-chol (Fig. 3B). The pD2¢ values were 4.04 0.15 (n = 5) and 3.61 0.01 (n = 6), respectively, which significantly differ from each other. This suggests that the antispasmodic effects of genistein against histamine are more potent than those against carbachol. In addition, these pD2¢ values were significantly less than the -lo-gIC50values of genistein against histamine- and
carbachol-in-Fig. 2 The relaxant effects of genistein on histamine-, carbachol-, KCl-, and leukotriene D4-induced precontractions in guinea pig trachealis. The relaxant effects do not include those of the respective vehicle. Each point represents the mean SEM of 6 or 7 experiments. AP = aminophylline.
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duced precontractions, respectively. In isotonic Ca2+-free high-K+ (60 mM)-depolarized tracheas, genistein (25±100mM) also con-centration-dependently inhibited the log concentration-re-sponse curves of cumulative Ca2+(0.01±10 mM) in a non-com-petitive manner (Fig. 4). The -logIC50 value was 4.47 0.06 (n = 4), which did not significantly differ from that against the KCl-induced precontraction. Nifedipine (1mM), a selective vol-tage-dependent calcium channel (VDCC) blocker [15], has been reported to completely inhibit calcium-induced contractions in the deporalized trachealis [16]. In this present experiment, nife-dipine (10mM), however, only relaxed 10.4% of the histamine (30 mM)-induced precontraction in the trachealis. The remaining ni-fedipine (10mM)-induced tension of the trachealis was further relaxed by genistein (100mM) to approximately 90% and then completely relaxed by the addition of 1 mM aminophylline (Fig. 5). This suggests that regardless of whether genistein blocks the VDCCs, it may have other mechanism(s) of relaxant action. However, neither removal of the epithelium nor the presence of an antagonist, such as propranolol (1mM), 2¢,5¢-dideoxyadeno-sine (10mM), methylene blue (25 mM), glibenclamide (10 mM), L-NNA (20mM) or a-chymotrypsin (1 U/mL), affected the log
con-centration-relaxing response curves of cumulative genistein on the histamine (30mM)-induced precontraction in normal Krebs solution (data not shown).
In contrast, genistein (17.5±35 mM) shifted the log concentra-tion-response curves of forskolin (Fig. 6A) and nitroprusside (Fig. 6B) to histamine (30mM)-induced precontractions of the trachealis to the left in a parallel manner and significantly in-creased the pD2values of forskolin and nitroprusside (Table 1). This reveals that the relaxant effect of genistein may occur via the inhibition of cAMP- and cGMP-PDE and subsequent increas-es in thincreas-ese two cyclic nucleotidincreas-es. Indeed, in the princreas-esent study, genistein at various concentrations (10±300 mM), concentra-tion-dependently and significantly inhibited cAMP- and cGMP-PDE activities. The -logIC50values of genistein were estimated
Fig. 3 Inhibitory effects of genistein on cumulative histamine (A)- and carbachol (B)-induced contractions in guinea pig trachealis in normal Krebs solution. Each point represents the mean SEM of 5 or 6 experi-ments. * P < 0.05, ** P < 0.01, *** P < 0.001 when compared with the corresponding value of the vehicle.
Fig. 4 Inhibitory effects of genistein on cumulative calcium-induced contractions in guinea pig trachealis depolarized by 60 mM KCl in Ca2+-free medium. Each point represents the mean SEM of 4 experi-ments. ** P <0.01, *** P < 0.001 when compared with the correspond-ing value of the vehicle.
Fig. 5 Tracing of the relaxant effect of genistein on the histamine (30 mM)-induced precontraction in guinea pig trachealis in normal Krebs solution. Genistein (100mM), compared to its vehicle (A), further re-laxed the remaining nifedipine (Nif, 10mM)-induced tension (B). At the end of the experiment, aminophylline (AP, 1 mM) was added to completely relax the trachealis.
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to be 4.28 0.06 (n = 8) and 4.17 0.07 (n = 4), respectively, which do not significantly differ from each other. Therefore, gen-istein appeared to have non-selective inhibitory effects on both PDE activities, although the inhibitory effect of genistein at 100 mM on cAMP-PDE activity was statistically more potent (p < 0.01) than that on cGMP-PDE activity (Fig. 7). The -log IC50values of IBMX, the positive control, were estimated to be 5.61 0.36 (n = 4) and 4.84 0.34 (n = 4), respectively, which also did not significantly differ from each other. There was also no selectivity for either PDE activity observed when IBMX was used (Fig. 7).
Discussion
Removal of the epithelium did not affect the log concentration-relaxing response curve of cumulative genistein for histamine (30mM)-induced precontraction suggesting that the relaxant ef-fect of genistein is epithelium-independent. The log concentra-tion-relaxing response curve of cumulative genistein to the his-tamine (30mM)-induced precontraction was not affected by pro-pranolol (1 mM), a non-selective b-adrenoceptor blocker, sug-gesting that its relaxant effect is not via activation of the b-adre-noceptor. Neither 2¢,5¢-dideoxyadenosine, an adenylate cyclase inhibitor [17], nor methylene blue, a soluble guanylate cyclase inhibitor [18], affected the log concentration-response curve of genistein, although baseline tensions were mildly relaxed during incubation with these two inhibitors (data not shown). However, relaxation of the baseline tension was unrelated to inhibition of adenylate cyclase or soluble guanylate cyclase by these two inhi-bitors. Therefore, this reveals that the relaxant effect of genistein occurs via activation of neither adenylate cyclase nor guanylate cyclase. Glibenclamide, an ATP-sensitive potassium channel blocker [19], also did not affect the log concentration-response curve of genistein, suggesting that its relaxant effect is not via the opening of ATP-sensitive potassium channels. L-NNA (20 mM), a nitric oxide (NO) synthase inhibitor [20], did not affect the log concentration-response curve of genistein, suggesting that its relaxant effect is unrelated to NO formation. a-Chymo-trypsin (1 U/mL), a peptidase, also did not affect the log concen-tration-response curve of genistein, suggesting that its relaxant effect is unrelated to the neuropeptides.
Genistein (25±100 mM) concentration-dependently and non-competitively inhibited cumulative Ca2+-induced contractions in the depolarized (K+, 60 mM) trachealis. Therefore, it may in-hibit Ca2+influx via VDCCs opened by 60 mM KCl. For example, nifedipine, a selective VDCC blocker, at concentrations below 1mM, also inhibited those contractions in a non-competitive Fig. 6 Potentiating effects of genistein on the relaxant responses of
cumulative forskolin (A) and nitroprusside (B) to the histamine (30 mM)-induced precontraction in guinea pig trachealis. Each point repre-sents the mean SEM of 5 or 6 experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 when the compared with corresponding value of the ve-hicle. AP = aminophylline.
Fig. 7 Inhibitory effects of genistein and IBMX, a positive control, on cAMP- and cGMP-PDE activities. The inhibitory effects do not include those of the respective vehicle. Each column represents the mean SEM of 4±8 experiments. ** P < 0.01 when compared with the cor-responding column of cGMP-PDE activity.
Table 1 pD2values of forskolin and nitroprusside against histamine (30mM)-induced precontractions in the absence and pres-ence of genistein Forskolin Nitroprusside Genistein Vehicle 6.81 0.06 (6) 5.67 0.10 (6) 17.5mM 7.06 0.06 (5)* 6.16 0.07 (5)* 35mM 7.45 0.09 (5)* 6.65 0.14 (5)*
Values are presented as the mean SEM (n); n is the number of experiments. * P < 0.05, when compared to the corresponding value of the vehicle.
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manner. Nifedipine at 1mM can completely inhibit such contrac-tions [16]. In the present study, nifedipine (10mM) only partially (10.4%) relaxed the histamine-induced precontraction in normal Krebs solution. The remaining nifedipine-induced tension was further (90 %) relaxed by 100mM genistein, suggesting that re-gardless of whether it blocks the VDCCs or not, it may have other mechanisms of relaxant action. Genistein concentration-depen-dently relaxed histamine (30mM), carbachol (0.2 mM), KCl (30 mM) and LTD4 (10 nM)-induced precontractions. The -logIC50 values against these four agonists did not significantly differ from each other. However, the -logIC50 values of genistein against histamine- and carbachol-induced precontractions were significantly greater than the pD2¢ values of genistein against cu-mulative histamine- and carbachol-induced contractions, respectively. It has been reported that the phasic response to agonists involves the release of stored Ca2+, and the tonic re-sponse is due to an increased influx of Ca2+across the membrane [21], [22]. This suggests that genistein more selectively inhibits calcium influx than calcium release from calcium stores. In addi-tion, the pD2¢ value of genistein against cumulative histamine-induced contractions was significantly greater than that against carbachol. This suggests that the antispasmodic effects of genis-tein against histamine are more potent than those against carba-chol. Although the exact reason is not clear, it has been estab-lished that carbachol may activate muscarinic M2 receptors, a major (80%) receptor population, via a pertussis-toxin-sensitive G protein, Gi, which inhibits adenylate cyclase activity [23] and causes an indirect contraction thus attenuating the relaxant ef-fects of genistein. Although the highest concentrations (100 and 200mM) of genistein used with histamine and carbachol, respec-tively, are impossible to reach in the blood due to their cytotoxi-city in in vivo studies, the above results of the in vitro study clear-ly suggest that genistein is a non-specific antispasmodic [24]. Genistein (17.5±35mM) shifted both the log concentration-re-sponse curves of forskolin, an activator of adenylate cyclase [25], and that of nitroprusside, an activator of guanylate cyclase [26], to histamine (30mM)-induced precontractions of the tra-chealis to the left in a parallel manner and significantly increased the pD2values of forskolin and nitroprusside (Table 1). This re-veals that the relaxant effect of genistein may occur via inhibi-tion of cAMP- and cGMP-PDE, and the subsequent increase in these two cyclic nucleotides. The increased cAMP or cGMP level subsequently activates cAMP- or cGMP-dependent protein ki-nase which may phosphorylate and inhibit myosin light-chain kinase, thus inhibiting contractions [27]. The precise mechanism by which relaxation is produced by this second-messenger path-way is not known, but it may result from decreased intracellular Ca2+([Ca2+]
i). The decrease in [Ca2+]imay be due to a reduced in-flux of Ca2+, enhanced Ca2+uptake into the sarcoplasmic reticula or enhanced Ca2+extrusion through the cell membrane [27]. In the present study, genistein or IBMX, a positive control, at var-ious concentrations (10~300 mM), significantly inhibited cAMP-and cGMP-PDE activities. Therefore, we can not exclude the pos-sibility that the relaxant effects of genistein may be due to its in-hibitory effect on both enzyme activities and its subsequent re-ducing effect on [Ca2+]
iof the trachealis.
Acknowledgements
The support for this work by a grant (93MMH-TMU-11) from Macky Memorial Hospital is gratefully acknowledged.
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