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Regular Article |
a Department of Physiology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong
b The Population Council, Center for Biomedical Research, New York, New York 10021
c Department of Pharmacology, University La Sapienza, Rome, Italy
ABSTRACT
The cystic fibrosis transmembrane conductance regulator (CFTR) or the small conductance cAMP-activated chloride channel encoded by the CFTR gene has been shown to play an important role in the formation of the epididymal fluid microenvironment. Mutation of the gene has led to widespread effects on male reproduction. Like other ion channels, CFTR is amenable to pharmacological intervention. Blocking CFTR in the epididymis could in principle lead to disruption of the epididymal fluid environment. We report for the first time two indazole compounds: lonidamine and 1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid (AF2785) are potent blockers of CFTR in the epididymis. When added to the external solution under whole-cell patch clamp conditions, AF2785 and lonidamine inhibited the cAMP-activated chloride current in rat epididymal cells with apparent IC50 values of 170.6 and 631.5 µM, respectively; by comparison the IC50 value for diphenylamine-2-carboxylate, a well-known chloride channel blocker was 1294 µM. In cultured rat epididymal epithelia mounted in a Ussing chamber, AF2785 and lonidamine inhibited the cAMP-stimulated short-circuit current (a measure of chloride secretion) when added to the apical bathing solution with potency greater than any known chloride channel studied. It is proposed that in view of the important role CFTR plays in male reproduction, further study with these and other new indazole compounds for their CFTR blocking actions can provide a new avenue of research into the development of novel male contraceptives.
cAMP, epididymis, signal transduction
INTRODUCTION
It is known that a cAMP-activated chloride channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene plays an important role in male reproduction [1, 2]. In the genetic disease cystic fibrosis (CF) where mutation of the CFTR gene occurs, 97% of the men inflicted with the disease are infertile due to complete blockage or agenesis of the epididymis and vas deferens. Recently, evidence has been accumulating that the CFTR gene could have broader and more far-reaching effects on human reproduction. Genetic screening of healthy men with various diagnoses of poor sperm quality have revealed they have a high frequency of CFTR mutations [3, 4]. It transpires that the male reproductive system is exquisitely dependent on CFTR for its normal functions and consequently most vulnerable to modest loss of CFTR [2].
In epithelial tissues such as the airway, pancreas, gastrointestinal tract, sweat gland, etc., CFTR functions as a cAMP-activated chloride channel that effects transepithelial movement of electrolytes and fluid. In the epididymis, secretion of chloride through CFTR underlies formation of the epididymal fluid. Pharmacological intervention of CFTR activity could theoretically lead to alterations of the sperm microenvironment. As with other ion channels on the cell membrane, CFTR is amenable to regulation by pharmacological agents. For instance, genistein has been shown to increase chloride secretion in the epididymis by activating CFTR, an effect claimed to offer therapeutic benefit to CF men [5]. Conversely, blockers of CFTR could be used to disrupt the epididymal microenvironment by blocking fluid secretion. There are putative blockers of CFTR at present, but they are neither selective nor potent [6]. The present study is designed to explore some new blockers of epididymal CFTR with a view to developing them into potential antifertility agents for men. We found the antifertility agents lonidamine and its analogue AF2785 are potent blockers of CFTR and also chloride secretion in the epididymis.
MATERIALS AND METHODS
Culture of Rat Epididymal Epithelial Cells
Rat caudal epididymal cells were cultured as previously described [7, 8]. Briefly, immature male Sprague-Dawley rats weighing 150 g were used as a source of the epididymal tissue. Rats were killed by CO2 inhalation. The epididymis was dissected out and immersed in sterile Hanks balanced salt solution (HBSS). The tissue was cut into small pieces and treated successively with 0.25% trypsin (w:v) and 0.1% collagenase (w:v). The disaggregated cells were suspended in Eagles minimum essential medium (EMEM) 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 at a cell concentration of 105 cells/ml into a 35-mm dish for whole-cell patch-clamp study. In some experiments, cells were seeded onto the wells of millipore filter assemblies with a window area of 0.4 cm2 (plating density 5 x 104 cells/cm2 filter) for the measurement of short-circuit current as described previously [7, 8].
Whole-Cell Recordings
After 5 to 8 days in culture, subconfluent cell monolayers were incubated in Ca2+-free Hanks solution containing 1 mM EGTA for 20 min to separate the cells. Recordings were performed at room temperature using an Axopatch-1D amplifier and DigiData 1200 series Interface (Axon Instruments, Foster City, CA). Ionic current was recorded using conventional whole-cell patch-clamp technique [9]. The cell membrane potential was held at -70 mV. Signals were filtered at 1 kHz, then digitized with Digidata 1200 (Axon Instruments). Sampling rate was set at 500 µsec. The pClamp 8 program was used for data recording and analysis. Patch pipettes (2–5 M
) were pulled from 1.0-mm outer diameter, 0.5-mm inner diameter borosilicate glass pipettes (Sutter Instrument Co., Novato, CA) using a horizontal puller (Sutter Instrument Co.). They were polished before use. Normally, the pipettes were filled with a solution containing 120 mM CsCl, 20 mM tetraethyl ammonium chloride (TEA)-Cl, and 10 µM cAMP, pH adjusted to 7.4 with CsOH. The bath solution was Krebs-Henseleit solution (K-H solution) that contained 117 mM NaCl, 4.7 mM KCl, 2.56 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 24.8 mM NaHCO3, 11.1 mM glucose, pH adjusted to 7.4 with 0.3 N NaOH. 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. In Cl-- and HCO3--free solution, NaCl, and NaHCO3 were replaced with sodium gluconate, KCl with potassium gluconate, and CaCl2 with calcium gluconate. All solutions were buffered with 10 mM Hepes at pH 7.4, and the osmolality was adjusted to 290 mosmol kg-1 with D-mannitol if necessary. The liquid junction potential was corrected when Cl-- and HCO3--free solutions were used.
Lonidamine, its analogue, and other chloride channel blockers were applied by the use of a linear array of eight tubings connected to solution reservoirs with appropriate mixtures of K-H solution and antagonists. Solution flow through each pipe was individually controlled by a perfusion system (Scientific Instruments, New York) and the pClampex 8 manually. The solution flow rate was 0.5 ml per min. Once whole-cell recording was achieved, the epididymal cells under study were continuously superfused by a stream of solution from flow pipe; only one barrel was used for perfusion at a given time. To achieve fast solution changes, a barrel with a small dead volume (10 µl) was used.
Short-Circuit Current Measurement
Short-circuit current (Isc) was measured by a method previously described [7, 8, 10]. Confluent epididymal monolayers were clamped between two halves of Ussing chambers with a 0.6-cm2 window. The tissue was short-circuited with a voltage-clamp amplifier (DVC 1000; World Precision Instrument, New Haven, CT). The Isc was displayed on a pen recorder. Transepithelial resistance was obtained from ohms' law by clamping the tissue intermittently at a voltage of 0.1 to 0.3 mV displaced from zero. Epithelia with transepithelial resistance of less than 300 /cm2 were discarded. In the majority of cases, the monolayers were bathed on both sides with K-H solution, gassed with 95% O2/5% CO2, and warmed to 32°C.
Data Analysis
Whole cell current analysis was performed using pClamp8 software (Axon Instruments). For both whole-cell current and Isc measurement, the concentration-inhibition data were fit with equation:
where I is the measured current, Icontrol is the current in the absence of antagonist, IC50 is the concentration of drug that causes 50% inhibition of the current, and n is the slope factor of the inhibition curve. Results are expressed as mean + SEM.
Chemicals
The EMEM, fetal bovine serum, and nonessential amino acids were purchased from Gibco Laboratories (New York). Penicillin/streptomycin, HBSS, sodium pyruvate, 5-dihydrotestosterone, trypsin, collagenase I, cAMP, and DIDS were from Sigma (St. Louis, MO). Lonidamine and AF2785 [1-(2,4-dichlorobenzyl)-indazole-3-acrylic acid] were synthesized by procedures as detailed elsewhere [11, 12]. Diphenylamine-2-carboxylate (DPC) was obtained from Riedel-de-Haën, Seelze, Germany. Glibenclamide was purchased from Sigma and indanyloxyacetic acid, IAA-94, from Research Biochemicals International (Natick, MA). Lonidamine, AF2785, DPC, and glibenclamide were dissolved in dimethyl sulfoxide and IAA-94 in 95% ethanol. These solvents alone were found not to affect the whole-cell or short-circuit current. The chemical structures of the chloride channel blockers used in the study are shown in Figure 1.
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RESULTS
Activation of Cl- Current by cAMP in the Rat Epididymal Cells
Figure 2 shows the time course of whole-cell current activation in cultured epididymal cells by addition of cAMP in the pipette solution. The internal solution containing 120 mM CsCl and 20 mM TEA-Cl was used to block potassium channels. No currents were detected when the internal solution contained only 120 mM CsCl and 20 mM TEA-Cl (Fig. 2). Addition of 1 µM cAMP into the internal solution resulted in a slow activation of the channel, which demonstrated an inward current. The current reached a maximal value after about 5 min. The current peaked more rapidly if more than 10 µM cAMP was applied in the internal solutions. The currents reached a peak level after 1.5 min and 2.5 min for 100 µM and 10 µM cAMP, respectively (Fig. 2).
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Effects of Cl- and HCO3- Concentrations on the cAMP-Activated Current
To test whether the inward current is caused by anion secretion (Cl-, HCO3-), we compared the effects of different concentrations of Cl-, HCO3- on the whole-cell current activated by cAMP. Four different perfusion solutions were applied as the external solution. Figure 3 shows current responses under different perfusion solutions. The K-H solution contained 124.26 mM Cl- and 24.8 mM HCO3-. The HCO3--free solution, Cl--free solution, and HCO3-- and Cl--free solutions contained 149 mM Cl-, 0 mM HCO3-; 0 mM Cl-, 24.8 mM HCO3-; and 0 mM Cl-, 0 mM HCO3-, respectively. It is apparent that the current was affected by the extracellular Cl- concentration (Fig. 3). Higher external Cl- concentration (obtained with HCO3--free solution containing 149 mM Cl-) decreased the inward current. The epididymal cell current was greatly increased if the perfusion solution (K-H solution) was changed to Cl--free, or Cl-- and HCO3--free solutions. No significant difference was observed between Cl--free and Cl-- and HCO3--free solutions, indicating little contribution of HCO3- to the cAMP-activated whole-cell current.
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Blockage of Cl- Channel by Lonidamine and Its Analogue AF2785
Figure 4 shows a typical time course experiment in which three different concentrations of AF2785 (10, 100, and 500 µM) were added to the external K-H solution superfusing the cell. The AF2785 induced a fast and concentration-dependent block of the current. This effect was rapidly reversible upon washout of the drug. Similar results were obtained with lonidamine (Fig. 4). However, inhibition by lonidamine was not as great as that by AF2785.
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Comparison of the Effects of Lonidamine, AF2785, and DPC on the cAMP-Activated Current
Figure 5 displays whole-cell current recorded from an epididymal cell exposed to an equimolar concentration of AF2785, lonidamine, and DPC. Addition of 100 µM of AF2785, lonidamine, or DPC caused a reduction in the current albeit with different magnitudes and time course. The AF2785 produced the largest inhibition and the time course of inhibition and recovery was most rapid. The inhibition elicited by DPC was small. Lonidmaine produced an inhibition with magnitude only slightly less than that produced by AF2785; however, the time course of inhibition and recovery appeared to be slower than the other two drugs.
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Concentration-Inhibition Curves for Different Chloride Channel Blockers
Using the experimental protocols shown in Figures 4 and 5, we have studied the concentration-inhibition relationship for five different chloride channel blockers on the cAMP-activated Cl- current in rat epididymal cells under whole-cell patch-clamp conditions. Figure 6 summarizes the results from different chloride channel blockers. The data were fit according to a logistic equation (see Materials and Methods), and the concentration at which 50% of the cAMP-evoked response was blocked (IC50) was calculated from the equation. The IC50 values derived from the fits were found to be 170.6, 631.5, and 1294 µM, for AF2785, lonidamine, and DPC, respectively; and the slope factors (n value) were -0.71, -0.73, and -0.84 for AF2785, lonidamine, and DPC, respectively.
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Effects of AF2785, Lonidamine, and Other Putative Chloride Channel Blockers on cAMP-Activated Short-Circuit Current (Isc) in Epididymal Epithelia
Figure 7 shows the effects of AF2785 and lonidamine on the cAMP-induced short-circuit current (Isc). Both drugs caused a dose-dependent inhibition of the current when added to the apical bathing solution. Figure 8 summarizes the concentration-inhibition curves for AF2785, lonidamine, and other putative chloride channel blockers. The IC50 values computed from the data were 106.5, 168.8, 729.5, and 1290 µM for AF2785, lonidamine, glibenclamide, and DPC, respectively.
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DISCUSSION
The present study confirms previous findings that intracellular cAMP activated an inward current in mammalian epididymal cells [13, 14]. The current was transient and the magnitude and the time course of the current response depended on the concentration of cAMP used. The magnitude of the current response reached a maximum at 10 µM cAMP. However, the time course of the response produced by 100 µM cAMP was faster than that produced by 10 µM cAMP (Fig. 2). Several features of the current suggest that it was a chloride current. Firstly, the current was sensitive to changes in chloride concentration in the external solution but not to bicarbonate (Fig. 3), sodium (results not shown), or potassium (K+ channels blocked by Cs and TEA in the pipette). Secondly, the current reversed at about +5 mV (results not shown), which is the expected equilibrium potential for chloride under the prevailing condition. Thirdly, the current was time and voltage independent and exhibited a linear current-voltage relationship (results not shown). These features are characteristic of a cAMP-activated chloride current [14] flowing through the CFTR or cAMP-activated chloride channel [15, 16]. Lastly, the current was sensitive to agents that are known to block CFTR. The present study showed for the first time that the antispermatogenic indazole compounds lonidamine and AF2785 inhibited the cAMP-activated chloride current, probably by blocking CFTR. Judging by the IC50 values, these two compounds are more potent than the conventional chloride channel blockers in blocking chloride conductance (Fig. 6). The indazoles (Fig. 1) may represent a completely new class of compounds with chloride channel blocking activities.
The chloride channel activated by cAMP plays an integral role in the secretion of electrolytes and water by the epididymis [1, 2]. The inhibition by lonidamine and AF2785 of the channel should lead to inhibition of transepithelial secretion of chloride (and secondarily fluid). Cultured rat epididymal epithelia have been used as a model to study transepithelial secretion of electrolytes in the epididymis [10]. When stimulated with exogenous cAMP or cAMP-elevating agonists, these epithelia respond by an increase in electrogenic chloride secretion (measured as short-circuit current, Isc) [17, 18] that can be blocked by putative chloride channel blockers, such as the arylaminobenzoates and sulfonylureas [19]. In this study, apically applied lonidamine and AF2785 were found to inhibit the cAMP-induced short-circuit current (Fig. 7), in keeping with their effects on chloride conductance. As with the whole-cell patch-clamp study, they were more potent than the conventional chloride channel blockers as evaluated by the IC50 values. The order of potency in inhibiting the cAMP-activated Isc was AF2785 > lonidamine > glibenclamide > DPC, in agreement with the order obtained from the inhibition of chloride conductance in rat epididymal cells. Although direct comparison between the whole-cell patch clamp (measurement made on isolated cells) and the short-circuit current (measurement made on intact epithelia) techniques is difficult, the IC50 values of the drugs obtained from the two studies were not dissimilar. In both cases, AF2785 was about seven times more potent than the putative chloride channel blocker DPC. These results are promising as they point to the potential use of these compounds in blocking fluid secretion by the epididymis. By virtue of their inhibition of the formation of the epididymal microenvironment, they can evolve into novel male contraceptives. Systematic screening of indazole compounds based on lonidamine and AF2785 (Fig. 1) for their ability to block epididymal CFTR will prove fruitful.
Lonidamine [11, 20, 21], AF2785 ([12], unpublished results), and analogues have been shown to have antispermatogenic activity. When given to rats they caused a premature shedding of spermatozoa from the testis (unpublished results). Although these effects have been attributed to a disruption of germ cell-Sertoli cell junctional complex, their action on germ cell CFTR cannot be excluded. Using in situ hybridization, Buchwald and colleagues [22] have detected CFTR mRNA in round spermatids (spermatogenic stages V–X) in rat testis. While the role of the germ cell CFTR in spermatogenesis remains unknown, it is conceivable that the antifertility effects of the indazole compounds may in part be due to an inhibition of germ cell CFTR. Given the widespread role of this membrane protein in male reproduction [2], screening of testis-specific CFTR inhibitors can provide a new avenue of research into the development of novel male contraceptives.
FOOTNOTES
First decision: 17 March 2000.
1 This work was supported by a grant from the Rockefeller Foundation/Ernst Schering Research Foundation to P.Y.D.W. 
2 Correspondence. FAX: 852 2603 5022; patrickwong{at}cuhk.edu.hk
Accepted: April 20, 2000.
Received: February 10, 2000.
REFERENCES
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