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a Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, People's Republic of China
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cystic fibrosis (CF) is a common lethal genetic disease affecting mostly Caucasians. It is caused by mutation of the CFTR gene in various exocrine tissues. The resulting loss of cAMP-driven anion/fluid secretion may be responsible for the obstruction or agenesis of the vas deferens seen in men with CF [5]. Recently, genistein and flavonoids with similar structures have been reported to activate CFTR in the airway epithelium [6], colonic epithelium [7], and ventricular myocytes [8] without raising the cAMP level. Nevertheless, the mechanism of action of genistein remains controversial. As the epididymis is highly dependent on CFTR for its function, it is of interest to investigate the effect of genistein on anion secretion in cultured rat epididymal epithelia. The therapeutic potential of genistein in male infertility related to CF is discussed in this paper.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Minimum essential medium Eagle (MEM), fetal bovine serum, and nonessential amino acids were purchased from Gibco Laboratories (Grand Island, NY). Penicillin/streptomycin, Hanks' balanced salt solution (HBSS), sodium pyruvate, 5
-dihydrotestosterone, trypsin, collagenase I, genistein, frusemide, acetazolamide, nystatin, cAMP, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) were from Sigma (St. Louis, MO). Thapsigargin was from Research Biochemicals International (Natick, MA); and Calbiochem (San Diego, CA) was the source for the adenylate cyclase inhibitor MDL-12330, the protein kinase A inhibitor H-89, bisindolylmaleimide, phorbol 12-myristate 13-acetate (PMA), calyculin A, tyrphostin A47, and diphenylamine-2-carboxylate (DPC). The immunoassay kit for cAMP was purchased from R & B Systems (Minneapolis, MN).
Solutions
Krebs-Henseleit solution had the following composition: NaCl, 117 mM; KCl, 4.7 mM; KH2PO4, 1.2 mM; MgSO4·7H2O mM, 1.2; CaCl2·2H2O, 2.56 mM; NaHCO3, 24.8 mM; and glucose, 11.1 mM. This solution had a pH of 7.4 when gassed with 95% O2, 5% CO2. In Cl--free solution, NaCl, KCl, and CaCl2 were replaced by sodium gluconate, potassium gluconate, and calcium gluconate, respectively. When HCO3--free solution was used, NaHCO3 was replaced with NaCl, and the solution was buffered with 10 mM Hepes with a pH of 7.4 when gassed with 100% O2. In the Cl-- and HCO3--free solutions, NaCl and NaHCO3 were replaced by sodium gluconate, KCl by potassium gluconate, and CaCl2 by calcium gluconate. The solution was buffered with 10 mM Hepes with a pH of 7.4 when gassed with 100% O2. In all solutions, the osmolarity was adjusted to 290 mosmol/L with D-mannitol if necessary.
Tissue Culture Technique
The tissue culture procedures have been described previously [1,9]. Immature male Sprague-Dawley rats weighing 150 g were used as a source of cauda epididymidis. They were killed by CO2 inhalation. The epididymis was dissected out, finely chopped with scissors, and treated successively with 0.25% (w:v) trypsin and 0.1% (w:v) collagenase. The disaggregated cells were suspended in MEM containing nonessential amino acids (0.1 mM), sodium pyruvate (1 mM), glutamine (4 mM), 5-dihydrotestosterone (1 nM), 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 µg/ml), and seeded into the wells of Millipore (Bedford, MA) filter assemblies with a diameter of 0.4 cm2 (cell concentration 105 cells/ml, plating density 5 x 104 cells/cm2 filter) floating on 15 ml of culture medium. Cultures were incubated in 5% CO2 for 3 days at 32°C. Thereafter, the monolayers reached confluence and were ready for the measurement of short-circuit current (Isc). This culture method has been shown to yield epithelia with morphology resembling that of the intact rat epididymis and with the major cell type being principal cells [10].
Isc Measurement
Confluent epididymal monolayers were clamped between two halves of a Ussing chamber (World Precision Instruments, Inc., New Haven, CT) with a 0.6-cm2 window. The tissue was short-circuited with a voltage-clamp amplifier (DVC 1000; World Precision Instruments). The Isc was displayed on a pen recorder. Transepithelial resistance was obtained from Ohm's law by clamping the tissue intermittently at a voltage at 0.1–0.3 mV displaced from zero. Epithelia with transepithelial resistance of less than 300 
cm2 were discarded. Usually two channels of the amplifier were used simultaneously on parallel monolayers so that studies could be made under control and experimental conditions. In the majority of cases, the monolayers were bathed on both sides with Krebs-Henseleit solution, gassed with 95% O2/5% CO2, and warmed to 32°C.
Measurement of cAMP
Rat epididymal cell monolayers were grown on 24-well plates (Costar, Cambridge, MA). After reaching confluence, they were washed twice with Krebs-Henseleit solution and then incubated in 0.5 ml of the same buffer containing isobutylmethylxanthine (IBMX, 1 mM) for 10 min at 32°C. Genistein was added to the wells and incubated for an additional 10 min. The reaction was terminated by adding 10 µl (60% w:v) perchloric acid to each well. The content of each well was mixed thoroughly and transferred to a 1.5-ml microcentrifuge tube and was then centrifuged at 10 000 x g for 5 sec. Supernatant (300 µl) was neutralized by KOH (1 M). The mixture (100 µl) was assayed for cAMP by an immunoassay kit.
Statistical Analysis
Results are expressed as means ± SEM. Comparisons between groups of data were analyzed by one-way ANOVA followed by Duncan's multiple-range procedure (for comparison of cAMP levels) or Student's unpaired t-test. A P value of less than 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When bathed in normal Krebs-Henseleit solution, the cultured epididymal epithelia exhibited a transepithelial potential difference of 2.3 ± 0.1 mV (n = 118 monolayers from 10 different cell preparations), a basal Isc of 5.4 ± 0.2 µA/cm2 (n = 118 monolayers from 10 different cell preparations), and a transepithelial resistance of about 500 cm2. When added to the apical side, genistein elicited a dose-dependent increase in Isc (Fig. 1, B and C). However, no change in Isc was observed when genistein was added to the basolateral side (Fig. 1A). The maximal Isc response to genistein was 6.25 ± 0.32 µA/cm2, and the median effective concentration was 10 µM. In all subsequent experiments, genistein was added to the apical side of the tissue.
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Effect of Ion Substitution
Figure 2 shows the concentration-response curves of genistein when anions in the bathing media were omitted. In Cl--free solution (HCO3--solution), the threshold dose and the maximum Isc response to genistein were 10 µM and 1.53 µA/cm2 (76% inhibition of the response in normal Krebs-Henseleit solution), respectively. In HCO3--free solution (Cl--solution), the threshold dose and the maximum response were 1 µM and 3.41 µA/cm2 (45% inhibition), respectively. The Isc response to genistein when both anions were omitted from the bathing solution was negligible.
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Involvement of Apical CFTR
To investigate whether the genistein-induced Isc response was mediated by activation of the apical Cl--channel or the basolaterally placed transporters or Na+-K+ ATPase, the pore-forming antibiotic, nystatin (500 µM), was used to selectively permeabilize the basolateral membrane. Solutions were used to achieve a Cl- gradient of 40:120 mM (apical to basolateral) across the epithelium after the active transport mechanism was disabled by permeabilization of the basolateral membrane. As shown in Figure 3, an Isc response to genistein (5.00 ± 0.72 µA/cm2) was observed after pretreatment with nystatin for 45–60 min. At the plateau of the response, basolateral addition of frusemide (100 µM), an inhibitor of the Na+/K+/2Cl- symport, did not inhibit the Isc, confirming that the basolateral membrane had been successfully permeabilized by nystatin. Apical application of DPC (1 mM) completely blocked the Isc response to genistein, indicating the involvement of the apical Cl- channel.
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The effects of different Cl- channel blockers were examined to investigate the types of Cl- channel activated by genistein. At the plateau phase of the Isc response to genistein, DPC (1 mM) or DIDS (100 µM) was added apically to determine whether the Isc response to genistein was due to activation of the cAMP-activated channel (CFTR) or the Ca2+ activated channel. As shown in Figure 4, DPC completely blocked the Isc response to genistein whereas DIDS, at a concentration that should have inhibited the Ca2+-activated Cl- channel in the epididymis [11], did not exert any significant inhibitory effect.
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Effect of Thapsigargin on the Genistein-Induced Isc
Figure 5B shows that pretreatment with thapsigargin (1 µM), a microsomal Ca2+-ATPase inhibitor, elicited an Isc response due to the release of Ca2+ from internal calcium store. When the current had returned to the basal level, addition of genistein produced an Isc response that was not significantly different from that produced by the same concentration of genistein without prior treatment of thapsigargin (Fig. 5A).
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Cyclic AMP Level After Genistein
Immunoassays were performed to study the effects of genistein on intracellular cAMP level in the epididymal epithelia. The results are shown as Figure 6. The intracellular cAMP content under basal conditions and after incubation with genistein (20 µM) were 47.6 ± 9.6 pmol/well (n = 6) and 51.0 ± 15.0 pmol/well (n = 5), respectively. The difference was not statistically significant. However, forskolin, serving as a positive control, stimulated a rise in cAMP level to 130.2 ± 14.4 pmol/well (n = 5; P < 0.01).
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Effects of Inhibitors of PKA, PKC, and Adenylate Cyclase
To further elucidate the involvement of cAMP/PKA pathway in the response to genistein, the effect of the PKA inhibitor, H-89, was examined. As shown in Figure 7, the Isc response to genistein could be completely abolished upon pretreatment with H-89.
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Another set of experiments was performed to examine the role of phosphorylation of CFTR by PKA in the action of genistein. After permeabilization of the basolateral membrane by nystatin, the Isc response to genistein could be blocked by pretreatment with MDL-12330A (50 µM), a specific adenylate cyclase inhibitor that would have depleted intracellular cAMP (Fig. 8B). Nevertheless, subsequent addition of cAMP (5 µM), which had very little effect on Isc at this concentration, fully restored the Isc response to genistein (Fig. 8, A and C).
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To study the involvement of PKC in the Isc response to genistein, the effect of the PKC inhibitor bisindolylmaleimide (BIS) was examined. As shown in Figure 9, pretreatment with BIS (1 µM) did not affect the Isc response to genistein but reduced most of the Isc response to the PKC activator, PMA (1 µM).
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Effects of Tyrphostin A47 and Calyculin A
As shown in Figure 10, tyrphostin A47 (20 or 200 µM), at concentrations known to inhibit tyrosine kinase, and calyculin A (20 or 200 nM), at concentrations known to inhibit protein phosphatase (PPase), did not affect the basal Isc in rat epididymal epithelium, nor did they affect the Isc response to genistein.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There has been much debate on the mechanism underlying the activation of CFTR by genistein. Since genistein is a well-characterized tyrosine kinase inhibitor in multiple cell types, it was initially thought to activate CFTR by inhibition of protein tyrosine kinase [7,13]. However, the present study demonstrated that tyrphostin A47, at a dose found to inhibit tyrosine kinase in colonic cells [7], was unable to mimic the action of genistein (Fig. 10). Similar observations in other epithelia [14–16] strengthen the idea that genistein activates CFTR via a tyrosine kinase-independent pathway.
Genistein stimulated anion secretion without raising intracellular cAMP (Fig. 7). This finding ruled out the possibility that genistein activated adenylate cyclase. Instead, constitutive phosphorylation of CFTR by PKA might be required for the action of genistein as inhibition of PKA by H-89 inhibited the genistein-induced Isc response (Fig. 8). Moreover, in nystatin-permeabilized monolayers, genistein did not stimulate Isc when the intracellular cAMP level had been previously suppressed by the adenylate cyclase inhibitor MDL-12330A. Replenishment of a low level of cAMP (5 µM), however, restored the normal response to genistein (Fig. 9).
It is generally held that the CFTR activity stimulated by PKA can be reversed by dephosphorylation with PPase. Two distinct PPases have been proposed to dephosphorylate different phosphorylation sites on CFTR [17,18]. The activation site is dephosphorylated by PPase 2A (calyculin A-sensitive), whereas the modulation site is dephosphorylated by PPase 2C (calyculin A-insensitive). Illek et al. [16] suggested that inhibition of PPase by genistein might prevent dephosphorylation and enhance CFTR activity. However, our data demonstrated that calyculin A, at concentrations found to inhibit PPase 2A [17], did not affect the response to genistein (Fig. 10), casting doubt on the involvement of PPase 2A in the regulation of CFTR activity in epididymal epithelium. Genistein may act via inhibition of PPase 2C [17–19]. However, this possibility awaits further investigation when specific PPase 2C inhibitors become available in the future. Although it is generally believed that phosphorylation by PKA is the principle pathway regulating the gating of CFTR, CFTR can also be phosphorylated by PKC [20]. Constitutive PKC phosphorylation has been found to be a prerequisite for the subsequent activation by PKA [21]. Previous work has provided evidence that PKC plays an important role in electrolyte secretion by the epididymis [22]. In our present study, pretreatment with BIS, a specific PKC inhibitor, did not reduce the Isc response to genistein (Fig. 9). Therefore, the stimulatory effect of genistein is unlikely to be mediated by PKC.
CFTR belongs to the ATP-binding cassette superfamily of transporters. CFTR is activated by phosphorylation of the R domain, and its channels are gated by the hydrolysis of ATP in two nucleotide binding domains (NBDs). NBD1 controls the opening whereas NBD2 controls the closing [23]. It was proposed that genistein may bind directly to the ATP-binding site in NBD2 to prevent closure of CFTR channels [24–26]. It is known that the binding and hydrolysis of ATP in NBDs require phosphorylation of the R domain [27–30]. Our finding that CFTR must be in a phosphorylated form before genistein can exert its effect is consistent with a direct interaction of genistein with CFTR in the epididymis.
CF results in defective anion and fluid secretion in various epithelia such as the airway, the pancreatic duct, and the gastrointestinal tract. In the epididymis, defective fluid transport leads to male infertility [5]. At the present time, there are two major approaches in the treatment of CF. Gene therapy can be applied to transfer the normal CFTR gene into the affected cells [31]. Another strategy involves the use of pharmacological agents to modulate ion channels other than CFTR. For instance, amiloride and uridine-5'-triphosphate have been used to reduce Na+ (water) reabsorption and increase Ca2+-driven anion (water) secretion, respectively [32]. Although much of the mutated CFTR is degraded during processing (e.g., 
F508) [33], recent studies showed that a low level of mutated CFTR protein can still reach the apical membrane [34], in which they are amenable to pharmacological intervention. In heterologous cell expression systems, genistein has been shown to potentiate the cAMP-dependent activation of G551D-CFTR [35] and F508-CFTR [19,36], the most common mutations in CF. Although the development of pharmacological agents targeted at the CFTR is still at an early stage, our present demonstration that genistein stimulates anion secretion in the epididymis (probably by direct interaction with CFTR) has led to the premise that genistein may be of therapeutic benefit in the reversal of epididymal anomalies in CF men. This may offer new hope for men with CF to father children. However, before such measures are taken, genetic screening and counseling for the men and their partners are mandatory to safeguard the offspring from the risk of clinical CF [5].
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FOOTNOTES |
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1 This work was supported by the Research Grants Council of Hong Kong and the International Consortium on Male Contraception, Population Council, New York. 
2 Correspondence. FAX: 852 2603 5022; patrickwong{at}cuhk.edu.hk
Accepted: August 28, 1999.
Received: June 24, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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F508-CFTR channel activity. Am J Physiol 1997; 273:C988-C998.This article has been cited by other articles:
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  C. Isnard-Bagnis, N. Da Silva, V. Beaulieu, A. S. L. Yu, D. Brown, and S. Breton Detection of ClC-3 and ClC-5 in epididymal epithelium: immunofluorescence and RT-PCR after LCM Am J Physiol Cell Physiol, January 1, 2003; 284(1): C220 - C232. [Abstract] [Full Text] [PDF]
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 X.D. Gong, P. Linsdell, K.H. Cheung, G.P.H. Leung, and P.Y.D. Wong Indazole Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Cl- Channels in Rat Epididymal Epithelial Cells Biol Reprod, December 1, 2002; 67(6): 1888 - 1896. [Abstract] [Full Text] [PDF]
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 R. Derand, L. Bulteau-Pignoux, and F. Becq The Cystic Fibrosis Mutation G551D Alters the Non-Michaelis-Menten Behavior of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel and Abolishes the Inhibitory Genistein Binding Site J. Biol. Chem., September 20, 2002; 277(39): 35999 - 36004. [Abstract] [Full Text] [PDF]
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 M. Kulka, M. Gilchrist, M. Duszyk, and A. D. Befus Expression and functional characterization of CFTR in mast cells J. Leukoc. Biol., January 1, 2002; 71(1): 54 - 64. [Abstract] [Full Text] [PDF]
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J. E. Smitham and K. E. Barrett Differential effects of apical and basolateral uridine triphosphate on intestinal epithelial chloride secretion Am J Physiol Cell Physiol, June 1, 2001; 280(6): C1431 - C1439. [Abstract] [Full Text] [PDF]
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X.D. Gong, Y.L. Wong, G.P.H. Leung, C.Y. Cheng, B. Silvestrini, and P.Y.D. Wong Lonidamine and Analogue AF2785 Block the Cyclic Adenosine 3',5'-Monophosphate-Activated Chloride Current and Chloride Secretion in the Rat Epididymis Biol Reprod, September 1, 2000; 63(3): 833 - 838. [Abstract] [Full Text]
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