Department of Physiology, School of Medical Sciences, University of Otago, Dunedin, New Zealand
Submitted 6 June 2003 ; accepted in final form 5 August 2004
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ABSTRACT |
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1-ethyl-2-benzimidazolone; vanadate; tyrphostin A23; cantharidic acid; phosphatase
Genistein has been reported to alter a number of ion transport proteins, such as the cystic fibrosis transmembrane conductance regulator (CFTR; the cAMP-dependent Cl channel) (13, 17, 24, 33, 34, 39, 41, 56), K+ channels (17, 34), and Na+-K+-2Cl cotransporter (43), in native epithelia and cultured epithelial cell lines. Recently, genistein was proposed as a possible pharmacotherapy for cystic fibrosis by modulation of Cl secretion (37, 48).
Channel activity of CFTR is reciprocally regulated by kinase-dependent phosphorylation and phosphatase-dependent dephosphorylation of nuclear-binding domains 1 and 2 (NBD1 and NBD2), respectively (22, 53). Therefore, CFTR is modulated by protein kinases (35), protein phosphatases (6, 35, 40, 54), and inhibitors of protein kinases and phosphatases (5, 34).
The mechanisms of action of genistein on CFTR include inhibition of tyrosine kinase (35, 52), modulation of CFTR by inhibition of protein phosphatases (34, 35, 46, 60), and direct activation of CFTR (3, 21, 45, 55, 57). Illek et al. (35) first demonstrated that genistein activated the CFTR via a tyrosine-dependent pathway by inhibiting a tyrosine kinase. In addition, Illek et al. (35) demonstrated that vanadate, an inhibitor of protein tyrosine phosphatase (25), prevented the genistein-induced activation of CFTR expressed in NIH/3T3 cells. Likewise, genistein activated Cl secretion in the rat colon; however, the action of genistein was suppressed by pretreatment of vanadate (17). Interestingly, Wang et al. (55) demonstrated that genistein activated CFTR in the presence of vanadate with excised patch-clamp experiments of CFTR expressed in Hi-5 insect cells. Tyrphostin 47, a tyrosine kinase inhibitor structurally unrelated to genistein, increased short-circuit current (Isc) of endogenous CFTR of human colonic epithelial monolayers (T84) (52); however, tyrphostin A23 and tyrphostin A51 did not stimulate transepithelial Cl secretion in T84 monolayers (34). Tyrphostin 47 failed to stimulate Cl current via CFTR, whereas genistein did stimulate Cl current for CFTR heterologously expressed in Xenopus oocytes (57). Thus the role of genistein in the tyrosine-dependent regulation of Cl secretion via CFTR is not well defined.
Non-tyrosine kinase effects of genistein on CFTR and the Cl secretory response have been reported. Genistein has been suggested to regulate CFTR by inhibiting protein phosphatases (35, 46, 60). Indeed, direct effects of genistein on CFTR have been suggested. French et al. (21) and Weinreich et al. (57), using patch-clamp studies of heterologously expressed CFTR, reported that genistein increased the activity of CFTR and speculated that genistein interacts directly with CFTR at NBD2. Randak et al. (45), using a fusion protein of NBD2, provided the first biochemical evidence that genistein interacts with the NBD2 of CFTR, thus inhibiting the NBD2 activity, and suggested that a similar mechanism could explain the genistein-dependent stimulation of Cl current via CFTR. Indeed, Al-Nakkash et al. (3) demonstrated that genistein stabilizes the open state of CFTR by inhibiting ATP hydrolysis at NBD2.
However, additional effects of genistein in cellular function include reduction of pp60c-src activity and reduction of nitrite levels and inducible nitric oxide synthase (iNOS) activity. Wijetunge et al. (59) reported that genistein reduced the channel current of L-type Ca2+ channels of smooth muscle cells of isolated rabbit ear arteries by reducing the pp60c-src activity. Chang et al. (11) suggested that induction of iNOS and nitrite accumulation in rat kidney mesangial cells occurs via a genistein-sensitive pathway. Similarly, Scuro et al. (51) showed that genistein reduced iNOS expression and nitric oxide production in mouse macrophages. Therefore, genistein has many effects on a wide number of cellular functions.
In the mammalian intestine, the small intestine (jejunum) is the primary site for secretion and absorption of fluid and electrolytes (10). Electrogenic Cl secretion is the primary transport mechanism responsible for fluid secretion within the crypt region (58). In this process, the combined action of the basolateral membrane Na+-K+-2Cl cotransporter, Na+/K+-ATPase, and K+ channel results in the level of Cl being raised above its electrochemical equilibrium within the cell. Then, upon the action of a secretagogue, Cl exits across the apical membrane via Cl channels (CFTR) (47). Coordinated activity of these transporters must occur for a sustained Cl secretory response to occur. Other than a recent report on the effects of genistein in the murine colon (24), very little is known about the action of genistein on the Cl secretory response of the mouse small intestine, and of the jejunum, in particular.
There were two aims of this study: first, to examine the action of genistein on the Cl secretory response of the mouse jejunum, and second, to begin to unravel the action of genistein on the Cl secretory response.
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MATERIALS AND METHODS |
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Electrophysiological measurements: Ussing chamber experiments. The Ussing chamber Isc experiments were performed as previously described (27, 28). Tissues were glued (Loctite 454; Loctite, Welwyn City, UK) to plastic annuli (0.7 cm2) and mounted in modified Ussing chambers. The epithelium was bathed on both mucosal and serosal sides with 10 ml of the NaCl Ringer's solution. Solutions were aerated (100% O2) and maintained at 37°C with the use of water-jacketed solution reservoirs.
Tissues were voltage clamped to 0 mV (Biodesign; South Campus Electronics, University of Otago) and Isc was continuously recorded with a MacLab data acquisition system (AD Instruments, Castle Hill, Australia) (8, 28). Data analysis was performed with MacLab Chart (version 3.6.3; AD Instruments). All tissues were pretreated with indomethacin (1 µM, mucosal and serosal sides) to reduce prostaglandin production (19) and with tetrodotoxin (0.6 µM, serosal side) to reduce enteric nerve activity (31) for 1 h (total time) to reduce transepithelial transport to basal conditions. The basal Isc was 116 ± 3 µA/cm2 (n = 66). The change in Isc (Isc) induced by a treatment was expressed as the difference between the former baseline value and the steady-state value. Dose-response curves were fit with the Hill equation. The presence of the circular and longitudinal muscle layers surrounding the jejunum did not impede access of chemicals to the epithelium as exhibited by very rapid responses of Isc with the addition of forskolin or bumetanide (see Figs. 1, 3, 4, 6, and 8).
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Statistical analysis. Statistical significance was assessed using Student's t-test (paired and unpaired). MacCurveFit (Kevin Raner Software, Victoria, Australia) was used for curve fitting and to determine the Km, Hill coefficients, and Vmax values of the dose-response experiments. Data are presented as means ± SE. In some figures, the SE bar is within the data point. A P value of 0.05 was considered significant. The number (n) of tissues used in each series of experiments is provided in the text.
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RESULTS |
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Others have demonstrated that phosphorylation of CFTR is required for participation in the stimulatory response of genistein (17, 21, 35, 46, 60). Clearly in the mouse jejunum, genistein alone activates Cl secretion (Fig. 1); however, we wanted to assess whether prior phosphorylation of CFTR would augment the dose response of genistein on the Isc of the mouse jejunum. Therefore, tissues were bathed in a NaCl Ringer's solution and pretreated with a low concentration of forskolin (0.25 µM, mucosal and serosal) before the addition of genistein. The pretreatment of forskolin increased Isc by 22 ± 8 µA/cm2. The dose-response curve of genistein in the presence of forskolin is shown in Fig. 2, B and C (circles, n = 4). The Km of the dose response of genistein in the presence of forskolin was shifted to 23 ± 6 µM but was not significantly different from that in the absence of forskolin (34 ± 3 µM). Similarly, Vmax (34 ± 6 and 34 ± 2 µA/cm2) and the Hill coefficients (2.7 ± 1.8 and 3.5 ± 0.6) were not altered in the presence and absence of forskolin, respectively.
These results confirm that genistein activates a sustained, bumetanide-sensitive Cl secretory response by the mouse jejunum. In addition, prior phosphorylation of CFTR did not augment the genistein-stimulated Cl secretory response.
CFTR participates in the genistein-stimulated Isc response. Many studies have demonstrated that genistein stimulates Cl secretion with the participation of CFTR (13, 17, 24, 39, 52). Indeed, genistein has been shown to directly activate CFTR in patch-clamp experiments (21, 32, 35, 55, 57). We predicted that if CFTR participates in the genistein response of the mouse jejunum, then genistein would not alter Isc in the presence of a maximum concentration of forskolin, an activator of CFTR via cAMP. To test this hypothesis, tissues were bathed in NaCl Ringer's solution and either pretreated with a maximum concentration of forskolin (10 µM, mucosal and serosal; unpublished data) followed by the addition of genistein (50 µM, mucosal and serosal) or treated with DMSO before the addition of genistein. Pretreatment of tissues with forskolin increased Isc by 106 ± 8 µA/cm2 (Fig. 3B, n = 10). As noted above, genistein stimulated a sustained increase in Isc in the absence of forskolin, as shown in Fig. 3A (n = 5). However, in the presence of a maximally forskolin-stimulated Isc response, genistein failed to further increase Isc (Fig. 3B, n = 10). These experiments are summarized in Fig. 3C.
Working from these data, we predicted that if CFTR participates in the genistein-activated Isc response, then pretreating tissues with genistein would reduce the forskolin-stimulated Isc response of the mouse jejunum. To test this hypothesis, tissues were bathed in NaCl Ringer's solution and subjected to genistein (50 µM, mucosal and serosal) followed by forskolin (10 µM, mucosal and serosal) or to DMSO followed by forskolin (Fig. 4, A and B). Indeed, forskolin alone increased Isc by 129 ± 28 µA/cm2 (n = 3), a response that was significantly reduced by 35% when forskolin was added in the presence of genistein (84 ± 4 µA/cm2; Fig. 4C, n = 7).
Furthermore, glibenclamide, a known blocker of CFTR (50, 53), was used to confirm the role of CFTR in the genistein-stimulated Isc response. Recently, we reported that mucosal rather than serosal application of glibenclamide reduced methoxsalen-stimulated Cl secretion of the mouse jejunum (27), suggesting that glibenclamide did not have access to the opposite side of the epithelium. In this series of experiments, all tissues were bathed with NaCl Ringer's solution. Experimental tissues were pretreated (30 min) with glibenclamide (100 µM, mucosal) whereas control tissues received DMSO, after which genistein (50 µM, mucosal and serosal) was added to all tissues. Genistein activated Isc as previously demonstrated (n = 8); however, genistein failed to stimulate Isc in the presence of glibenclamide (Fig. 5A, n = 7). Because glibenclamide prevented genistein's activation of Isc, we wanted to confirm that glibenclamide could reduce forskolin-stimulated Isc, as well. In fact, in experiments similar to those described above, pretreatment of tissues with mucosal glibenclamide (100 µM) reduced the forskolin (10 µM, mucosal and serosal)-stimulated Isc by 60% (Fig. 5B, n = 3).
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Effect of genistein on a maximally 1-EBIO-activated Isc. 1-EBIO is a known activator of K+ channels, particularly the intermediate-conductance Ca2+-dependent K+ channel (IKCa) (15, 28). Also, 1-EBIO has been reported to stimulate intracellular levels of cAMP in native tissue (12) and to increase the activity of CFTR in cultured cells (14). Investigators (Carter DJ, Hamilton KL, and Butt AG, unpublished observations) have demonstrated that 1 mM 1-EBIO stimulates a maximal Isc response of the mouse jejunum bathed in HEPES-buffered Ringer's solution. The IKCa is present in the basolateral membrane of isolated mouse jejunal crypts and is activated by 1-EBIO (9, 28). Therefore, it was of interest to examine the effects of genistein in the presence of a maximum concentration of 1-EBIO. To examine these effects, tissues were bathed in NaCl Ringer's solution and either pretreated with 1-EBIO (1 mM, serosal, 20 min) followed by the addition of genistein (50 µM, mucosal and serosal) or treated with genistein alone. Genistein stimulated a sustained increase in Isc in the absence of 1-EBIO, as shown in Fig. 6A (n = 7). Interestingly, in the presence of a maximally 1-EBIO-stimulated response, genistein further increased Isc (Fig. 6B, n = 15). However, the genistein-stimulated increase in Isc in the presence of 1-EBIO was significantly less than in the absence of 1-EBIO (Fig. 6C). These data suggest that genistein may be increasing the activity of CFTR and/or altering the basolateral K+ conductance.
IKCa is not involved in genistein-stimulated Isc. The above data suggest that genistein increases Isc of the mouse jejunum with the cooperation of CFTR. However, to establish a sustained Isc secretory response, there must be an increase in the basolateral K+ conductance coupled with the increase in apical Cl conductance (i.e., CFTR activity) or, alternatively, a basal K+ conductance must be large enough to maintain a driving force for Cl to exit across the apical membrane (13, 47). IKCa is one of the predominant K+ channels of the basolateral membrane of the mouse jejunum (9, 28). In fact, we recently reported that methoxsalen, a psoralen, stimulated a sustained increase Cl secretory response of the mouse jejunum with the participation of CFTR and activation of the basolateral IKCa (27). To determine whether IKCa is involved in the genistein-stimulated Isc response, we examined the effects of clotrimazole, a known blocker of IKCa (16), on the action of genistein on the Isc of the mouse jejunum. In this series of experiments, all tissues were bathed with NaCl Ringer's solution. Experimental tissues were pretreated (30 min) with clotrimazole (10 µM, serosal), whereas control tissues received DMSO, after which genistein (50 µM, mucosal and serosal) was added to all tissues. Clotrimazole had no effect on the action of genistein in that genistein had a similar effect on Isc whether clotrimazole was present or not (19 ± 3 and 17 ± 6 µA/cm2 in the absence and presence of clotrimazole, respectively; n = 56) (data not shown). These data suggest that the basolateral IKCa does not participate in the genistein-stimulated response.
Influence of genistein on phosphorylation of CFTR? The overall channel activity of CFTR, and thus the rate of Cl secretion, is reciprocally regulated by kinase-dependent phosphorylation and phosphatase-dependent dephosphorylation of nuclear-binding domains (22, 53). Therefore, we examined the effects of cantharidic acid, an inhibitor of the serine/threonine protein phosphatase type 2A (PP2A) (29), sodium orthovanadate (vanadate), an inhibitor of protein tyrosine phosphatases (25), and tyrphostin A23 (23), a tyrosine kinase inhibitor, on the stimulatory action of genistein.
To test the involvement of serine protein phosphatases in the genistein-stimulated Isc response, tissues were pretreated (30 min) with either cantharidic acid (5 µM, serosal) or with DMSO followed by the addition of genistein (50 µM, mucosal and serosal). Genistein alone activated a sustained increase in Isc (Fig. 7A). Cantharidic acid had no effect on the basal current (Fig. 7B); however, the genistein-stimulated Isc was greater in the presence than in the absence of cantharidic acid (Fig. 7, B and C, n = 11). The time dependence of the activation of Isc by genistein was not different in the absence (670 ± 115 s) or presence (641 ± 90 s) of cantharidic acid (n = 11). These results suggest that genistein does not increase Isc by inhibition of PP2A.
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Tyrphostin A23, an inhibitor of protein tyrosine kinase, was used to test whether genistein was activating Isc by inhibiting a protein tyrosine kinase that had a negative influence on CFTR (35). Initially, tissues were bathed in NaCl Ringer's solution pretreated with either tyrphostin A23 (100 µM, mucosal and serosal) or DMSO, after which genistein (50 µM, mucosal and serosal) was added to the tissues. Tyrphostin A23 reduced the basal Isc by 30 ± 3 µA/cm2 (n = 5; data not shown). Genistein failed to increase Isc in the presence of tyrphostin A23 (2 ± 1 µA/cm2, n = 3) but stimulated a robust Isc in control tissues (27 ± 5 µA/cm2, n = 3; data not shown). To test the viability of the tissues, ouabain (2 mM, serosal) reduced the Isc of all tissues. Lower concentrations of tyrphostin A23 (20 and 50 µM) produced inconsistent results (data not shown).
These data suggest that the action of genistein on Cl secretion of the mouse jejunum is likely via a tyrosine phosphorylation-dependent pathway.
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DISCUSSION |
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Others have reported that genistein can activate CFTR in heterologously expressed CFTR in expression systems, cultured cell lines, and a few native tissues (13, 17, 21, 24, 3235, 39, 46, 52, 55, 60). The mechanism of action of genistein on the Cl secretory response is still a matter of contention. In some studies, genistein alone elicits a sustained secretory response; however, investigators in other studies report that CFTR must be phosphorylated before the addition of genistein to initiate a stimulatory effect (35, 46, 60). Clearly, genistein can activate CFTR directly, as has been demonstrated with cell-attached and excised patches of various cell types (21, 32, 35, 55, 57). It has been demonstrated that a basal level of activity of protein kinase A, and thus phosphorylation of CFTR via cAMP, is clearly necessary for stimulation of CFTR by genistein in other studies (46, 60). However, elevated levels of cAMP are not mandatory for a stimulatory action of genistein, because many have reported genistein-stimulated Cl secretion without changes of intracellular cAMP (3335, 39, 52).
In the current study, genistein alone stimulated a robust sustained Cl secretory response in the mouse jejunum (Fig. 1) as has been reported by others for a native tissue (24) and cultured cells (13, 24, 52). However, we report that prior phosphorylation of CFTR did not alter the efficacy of genistein, as shown in the dose-response experiments (Fig. 2). In the presence of phosphorylated CFTR, concentrations >100 µM genistein caused a reduction in Isc. This may be due to inhibition of K+ channels by genistein (17, 34, 41); however, recent evidence from our laboratory showed that 125 and 200 µM genistein increased Isc of the mouse jejunum by 37 ± 6 and 35 ± 5 µA/cm2, respectively (Chao PCT and Hamilton KL, unpublished data). This finding is in agreement with the dose-response curve in the current study (Fig. 2, squares). Nonetheless, Deiner and Hug (17) clearly demonstrated that the genistein-stimulated Isc response of the rat colon was quite small but was greatly enhanced in the presence of a low concentration of forskolin (0.2 µM). Similarly, heterologously expressed CFTR in a variety of expression systems (NIH/3T3 fibroblasts and Hi-5 insect cells) required a basal level of protein kinase A activity to ensure a stimulatory effect of genistein (34, 46, 60).
Sustained genistein response on Isc. The nature of the sustained Cl secretory response stimulated by genistein in the present study suggests that various components of the Cl secretory pathway (e.g., CFTR, K+ channels) may be upregulated (47). It is not surprising to conclude that the genistein response involves the participation of CFTR (55, 57).
Several lines of evidence suggest that CFTR is the Cl channel participating in the genistein response of the mouse jejunum. First, CFTR is expressed in the mammalian intestine and has been identified in the mouse jejunum in particular (4). Second, pretreatment of the jejunum with a maximum concentration of forskolin to maximally activate (phosphorylate) CFTR prevented a further increase in Isc by genistein (Fig. 3). Third, pretreatment of the jejunum with genistein reduced the forskolin-stimulated Isc (Fig. 4), suggesting that genistein and forskolin may use a common pathway for increasing Cl secretion by the mouse jejunum. Finally, glibenclamide, a known inhibitor of CFTR (50, 53), prevented an increase in Isc by genistein and reduced the Isc activated by forskolin (Fig. 5A).
Genistein's effect on basolateral K+ conductance. Genistein has varying effects on K+ channels of epithelial and nonepithelial tissues. Genistein has inhibitory effects on basolateral Ca2+-activated K+ channels as previously reported for the human rectum, rat colon, and CFTR-expressing colonic cell lines (17, 34, 41). Alternatively, genistein has been reported to stimulate a small-conductance, Ca2+-activated K+ channel of rat cortical collecting ducts (56) and K+ channels of human lung adenocarcinoma and fibroblast cultured cells (49). Therefore, it is possible that genistein may increase Cl secretion of the mouse jejunum by activating basolateral K+ channels. An increased K+ conductance (activation of K+ channels) would hyperpolarize the jejunal cells, resulting in an enhanced driving force for Cl secretion. The IKCa is one of the more prevalent K+ channels of the basolateral membrane of the mouse jejunum (9, 28). Thus we examined the effect of clotrimazole, a specific blocker of IKCa (16), on the ability of genistein to stimulate a Cl secretory response. However, in the presence of clotrimazole, genistein still activated a sustained Cl secretory response, suggesting that IKCa does not participate in the genistein response. This finding does not preclude the possibility that another type of K+ channel may be activated by genistein. However, it is possible that the basal K+ conductance of the jejunal epithelium may be adequate to maintain the driving force for Cl secretion as has been suggested for other secretagogues (13).
Additional evidence suggesting that genistein may not be activating IKCa comes from the 1-EBIO/genistein experiments (Fig. 6). In the presence of a maximal 1-EBIO stimulation of Isc [presumably a maximal activation of IKCa (28)], genistein clearly stimulated Isc, lending further support to the possibility that genistein is not activating IKCa and, thus, may be stimulating Cl secretion via CFTR as has been reported by others (21, 35, 55).
It is possible that genistein is activating another type of K+ channel. If this were so, then we would have expected genistein to increase Isc in the presence of a maximum concentration of forskolin, which did not occur. It is conceivable that forskolin leads to the activation of multiple types of K+ channels; therefore, genistein might not have the opportunity to exhibit any further effect on Isc. Indeed, the reduced effect of forskolin on Isc in the presence of genistein (Fig. 4) suggests that if genistein is activating a basolateral K+ channel, then that channel may also be involved in the forskolin-stimulated Isc response of the mouse jejunum. In support of this hypothesis, we have demonstrated that both the IKCa and cAMP-dependent K+ channels participate in the forskolin-stimulated Isc of the mouse jejunum (Carter DJ, Hamilton KL, and Butt AG, unpublished data).
Tyrosine phosphorylation, CFTR, and genistein. Gating of CFTR can be regulated by phosphorylation and ATP hydrolysis and is modulated by the action of protein phosphatases at the nuclear-binding domains of CFTR (6, 60). Studies have reported that genistein modulates Cl secretion by altering the overall phosphorylated state of CFTR by inhibition of either protein kinases (35, 52) or protein phosphatases (34, 35, 46, 60). Therefore, if the observed genistein-stimulated secretory response (i.e., Isc) of the mouse jejunum occurs via inhibition of a serine phosphatase, for instance, then prior treatment with a serine phosphatase inhibitor should reduce the efficacy of genistein. Alternatively, if tyrosine kinase phosphorylation is involved in the genistein-activated Cl secretory response, then we would expect an associated tyrosine phosphatase to be involved (57), and thus the inhibition of that phosphatase should alter the action of genistein.
PP2A has been demonstrated to dephosphorylate CFTR (6, 60) and reduce the activity of CFTR (40). Cantharidic acid, a known inhibitor of PP2A (29), failed to prevent the action of genistein in the present study. Indeed, tissues pretreated with cantharidic acid exhibited an increased Isc, stimulated by genistein. The enhanced genistein response in the presence of cantharidic acid may result by cantharidic acid preinhibiting specific phosphatases, thus permitting an action of genistein to proceed without a time lag imposed by dephosphorylation events, or else another phosphatase(s) may be inhibited by genistein. Nonetheless, genistein's action on the Cl secretory response of the mouse jejunum was clearly independent of PP2A. These results are consistent with others showing that genistein exhibited a further effect on 125I efflux of CFTR-expressing NIH/3T3 fibroblasts in the presence of calyculin A, an inhibitor of PP2A (46). Similarly, Wang et al. (55) demonstrated that genistein increased CFTR channel current in the presence of saturating concentrations of forskolin and calyculin A in cell-attached patches of CFTR-expressing NIH/3T3 fibroblasts and Hi5 insect cells.
Regulation of tyrosine phosphorylation has been proposed to modulate CFTR, and thus Cl secretion, in native tissue (39) and cultured cells (35, 52). In the present study, vanadate, an inhibitor of tyrosine protein phosphatase (25), greatly reduced the genistein-stimulated Isc (Fig. 8). One explanation for the reduced genistein response in the presence of vanadate is that the protein tyrosine kinase-induced phosphorylation exerts a negative effect on CFTR channel activity and, hence, reduced Cl secretion (35). Therefore, pretreating tissues with vanadate thus yields increased tyrosine phosphorylation, resulting in blocking of the stimulatory effect of genistein (35). Illek et al. (35) demonstrated that vanadate reversibly inhibited genistein-stimulated CFTR currents in cell-attached patches of CFTR-transfected NIH/3T3 fibroblasts as well as genistein-stimulated 125I efflux via CFTR. In the current study, it is possible that vanadate inhibited the Na+-K+-ATPase; however, we observed bumetanide-sensitive Isc in the presence of vanadate (Fig. 8B), suggesting that the Na+-K+-2Cl-cotransporter as well as the Na+-K+-ATPase was still functional. Nonetheless, our data provide additional support for the contention of Illek et al. (35) that genistein regulates CFTR, and thus Cl secretion, via a tyrosine kinase-dependent pathway.
A perplexing result was that tyrphostin A23 reduced basal Isc and prevented any stimulatory effect by genistein in the mouse jejunum. Tyrphostins are a class of tyrosine kinase inhibitors that are structurally different from genistein. Genistein contains an isoflavone structure (2). It has been suggested that the action of particular tyrosine kinase inhibitors on transport function is dependent on the structural nature of the inhibitor (43). Indeed, Niisato et al. (43) demonstrated that tyrphostin A23 (100 µM) reduced Isc and Cl conductance in renal epithelial A6 cells, whereas genistein increased Isc. Similarly, Illek et al. (34) reported that tyrphostin A23 (50 µM) and tyrphostin 51 (40 µM) failed to stimulate Cl secretion in the human colonic T84 cell line even though genistein clearly activated Cl secretion. In contrast, Yang et al. (60) demonstrated that tyrphostin 51 enhanced forskolin-activated CFTR channel activity in CFTR-expressing Hi-5 insects cells. In the present study, the lack of response of genistein in the presence of tyrphostin A23 may be the result of inhibition of the Na+-K+-2Cl cotransporter by tyrphostin A23 as recently reported (44). However, others are not convinced that there are kinase inhibitors that totally inhibit the Na+-K+-2Cl cotransporter (20). Future work is required to further examine the complex role of genistein in the Cl secretory response of the mouse jejunum.
In summary, we have used the Ussing chamber short-circuit current technique to demonstrate that genistein activates a Cl secretory response of the mouse jejunum. Activation of Cl secretion by genistein involves the participation of CFTR, resulting in increased fluid secretion. Certainly there is no definitive action of various tyrosine kinase inhibitors on CFTR and Cl secretion. However, genistein appears to participate in the tyrosine-dependent phosphorylation pathway. Furthermore, these results demonstrate in a native epithelium that genistein targets a component(s) of the Cl secretory response.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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