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Male Reproductive Tract |
a Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada
b Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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epididymis, male reproductive tract
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have studied the role of CFTR in anion and fluid secretion in our laboratory and have found that a variety of neurohumoral agents [4–7] regulate anion secretion via CFTR activation. These effects may have an important influence on the formation of epididymal fluid. It transpires that pharmacological intervention of CFTR function may have far-reaching effects on epididymal functions and male fertility. We previously reported that genistein increases Cl- secretion in the epididymis by activating CFTR, an effect that likely provides therapeutic benefit to men with cystic fibrosis [8]. Conversely, inhibition of CFTR is expected to disrupt formation of the epididymal environment, which could lead to induction of sterility. Recently, two indazole compounds with antifertility activity, lonidamine and its analogue AF2785, were studied to assess their effects on CFTR in rat epididymal epithelial cells. The results show that these drugs inhibit epididymal secretion of Cl- by blocking CFTR [9], and they do so with greater efficacy than conventional CFTR-Cl- channel blockers [9]. Characterization of the block using a whole-cell patch-clamp method revealed that the block is voltage-dependent [10]. The degree of the block increases when extracellular pH decreases [10]. To understand how these two compounds inhibit CFTR we used a spectral analysis protocol to study the whole-cell current variance. We also studied single CFTR-Cl- channel activity in excised inside-out membrane patches from rat epididymal epithelial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All experiments on animals were performed in accordance with guidelines on the use of laboratory animals established by the Animal Ethics Committee of the Chinese University of Hong Kong. The tissue culture procedures were described previously [5]. In brief, immature male Sprague-Dawley rats weighing 150–200 g were killed by CO2 inhalation. Cauda epididymides were dissected out, finely minced with scissors, and treated successively with 0.25% (w/v) trypsin and 0.1% (w/v) collagenase. The disaggregated cells were suspended in Eagle 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 onto 22-mm glass coverslips in 35-mm Petri dishes. Cultures were incubated in 95% O2/5% CO2 for 3–6 days at 32°C. All experiments were carried out at room temperature, 21–24°C.
Spectral Analysis of Whole-Cell Currents
Spectral analysis of whole-cell current variance was carried out on continuous current recordings made at -50 mV before and after addition of lonidamine or AF2785. CFTR currents were activated by adding to the extracellular solution 8-Br-cAMP, a membrane-permeable cAMP analogue, at a concentration of 100 µM. Pipette resistances were 3–5 M
for whole-cell recordings. Patch pipettes were pulled from borosilicate glass pipettes (1.0-mm outside diameter, 0.5-mm inside diameter; Sutter Instrument Co., Novato, CA) using a horizontal puller (Sutter), and were polished before use. Current records were filtered at 5 kHz using an eight-pole Butterworth filter (Frequency Devices, Haverhill, MA), amplified using an Axopatch 200B amplifier (Axon Instruments, Union City, CA), digitized at 10 kHz using a DigiData 1200 interface (Axon Instruments), and then divided into nonoverlapping segments containing 8192 data points (819.2 milliseconds per segment). Power density spectra were then calculated for each segment using BioPatch Analysis software (BioLogic Science Instruments, Claix, France). Spectra from at least 100 segments were then averaged and fit by the sum of two or three Lorentzian functions of the form:
where Sf is the current variance per frequency unit at each frequency (f), S0 is the maximum value of S as f approaches zero, and fc is the corner frequency at which Sf = S0/2.
Inside-Out Patch Single Channel Recording
Subconfluent cell monolayers were incubated in Ca2+-free Hanks balanced salt solution (HBSS) containing 1 mM EGTA for 20 min to separate the cells. Single-channel CFTR current recordings were made using the excised, inside-out configuration of the patch clamp technique as described in detail previously [11]. Briefly, CFTR channels were activated following patch excision by exposure to 30–80 nM protein kinase A (PKA) catalytic subunit plus 1 mM MgATP. The pipette (extracellular) and bath (intracellular) solutions both contained 150 mM NaCl, 2 mM MgCl2, and 10 mM N-tris-(hydroxymethyl) methyl-2-aminoethanesulfonic acid. All solutions were adjusted to pH 7.4 with NaOH. Lonidamine or AF2785 were added to the patch clamp chamber from a stock solution made from the above solution and were initially solubilized in dimethyl sulfoxide at 100 mM. Pipette resistances were 6–8 M for single-channel recordings. Currents were filtered at 50 Hz using an eight-pole Butterworth filter (Frequency Devices), amplified using an Axopatch 1-D amplifier (Axon Instruments), and digitized at 1000 Hz using a DigiData 1200 interface (Axon Instruments). To investigate the voltage dependence, a voltage-step protocol (at a step of 20 mV) was used from the -80 mV to +80 mV range.
Single-Channel Data Analysis
Clampfit, Fetchan, and pStat (version 8.0, Axon Instruments) software programs were used for single-channel data analysis. At least 1 min of continuous recordings were used to construct the amplitude histograms and to make event lists for open-channel probability analysis. Single-channel open time was estimated using a 50% threshold detection method. Because the duration of blocked events appeared close to the limit of temporal resolution, closed time was not estimated. Basically, we performed two types of analyses. First, we measured the mean amplitude of control (Io) and blocked channels (I) from the amplitude histograms. The open-state probability (Po) was calculated as total channel open time divided by the total observed time. Bursts with a baseline shift or more than one channel opening were not used in Po calculations.
Statistical Analysis
Results are expressed as means ± SEM. Comparisons between groups of data were made with the Student t-test. A P value of < 0.05 was considered statistically significant.
Chemicals
The catalytic subunit of PKA was purchased from Promega Ltd. (Madison, WI). HBSS, EMEM, fetal bovine serum, and nonessential amino acids were purchased from Gibco Laboratories (Grand Island, NY). MgATP, penicillin, streptomycin, sodium pyruvate, 5-dihydrotestosterone, trypsin, collagenase, 8-Br-cAMP, and EGTA were purchased from Sigma Chemical Company (St. Louis, MO).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The blocking effects of lonidamine and AF2785 on CFTR-Cl- channels [9, 10] have apparently similar characteristics to those of sulfonylureas, which have been studied and quantified using spectral analysis [12, 13]. To better quantify the kinetics of blockade we therefore performed a similar spectral analysis to assess lonidamine and AF2785 blockade of CFTR whole-cell current in epididymal cells (Figs. 1 and 2). Activation of CFTR currents at -50 mV was associated with an increase in current variance, especially at low frequencies, as shown in Figures 1B and 2B. The spectrum difference obtained by subtracting the background variance from that following full current activation with membrane-permeable cAMP was well fit by the sum of two Lorentzian components (Figs. 1C and 2C); a dominant, low-frequency component (fc [lonidamine] = 0.90 ± 0.25 Hz, fc [AF2785] = 0.72 ± 0.34 Hz; n = 3 different preparations), which may reflect CFTR-channel gating, and a second component with much lower power and very high frequency (fc [lonidamine] = 1000 ± 73 Hz, fc [AF2785] = 950 ± 97 Hz; n = 3 different preparations), similar to those described previously for human CFTR [13, 14]. Addition of lonidamine or AF2785 to the extracellular (bath) solution not only decreased current amplitudes (Figs. 1A and 2A) but also led to the introduction of a new component of current variance at intermediate frequencies (Figs. 1C and 2C). Difference spectra recorded following addition of lonidamine or AF2785 were fit by the sum of three Lorentzian components (see Materials and Methods); two with frequencies very similar to those seen before drug addition, and a third one, an intermediate-frequency component, which presumably reflects lonidamine and AF2785 block and unblock of the open channel. Furthermore, the fc of this intermediate Lorentzian component was sensitive to lonidamine and AF2785 concentrations (Figs. 1D and 2D).
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Previously [13], channel blocker-dependent changes in fc such as those shown in Figures 1D and 2D have been used to estimate blocker-on and blocker-off rates (kon and koff) according to the following simple kinetic scheme:
Although more complicated schemes may be envisioned [12, 15–17], this is the simplest one that can account for the effects of open-channel blockers such as lonidamine and AF2785. As predicted by such a scheme [13], 2
fc was a linear function of lonidamine or AF2785 concentration (Figs. 1D and 2D), with an intercept (
koff) of 69.01 sec-1 and a slope (kon) of 3.67 µmol-1/sec-1 for lonidamine; and with an intercept (koff) of 108 sec-1 and a slope (kon) of 3.27 µmol-1/sec-1 for AF2785. The ratio of these rates, koff;th:;thkon, suggests a Kd of 18.77 µM for lonidamine and 32.99 µM for AF2785 at -50 mV.
Single-Channel CFTR Current in Rat Epididymal> Epithelial Cells
Figure 3A shows a single-channel record (representative of three different patches from three different preparations) of CFTR activity in an excised inside-out membrane patch with a relatively large pipette opening (pipette resistances were 3–5 M). CFTR-Cl- current was typically activated almost instantaneously upon formation of the inside-out membrane patch when 1 mM MgATP and 30–80 nM PKA were present in the bath solution. Current activation generally reached a steady state level (4–5 channels opened) after 2–3 min. The time course of activation was consistent with the whole-cell current reported previously [10]. In the absence of blocker, CFTR channel activity was characterized by long, open bursts lasting from hundreds of milliseconds to seconds and interrupted by infrequent, brief intraburst closures, with longer interburst closures also lasting hundreds of milliseconds (Fig. 3A). The CFTR-Cl- current displayed a linear, symmetrical current-versus-voltage (I/V) relationship and was reversed at 0 mV under a symmetrical chloride concentration (154 mM) in the intracellular and extracellular solutions (Fig. 3, B and C). The unitary conductance was calculated to be 6 pS at -60 mV (Fig. 3C).
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Inhibition of Single CFTR-Chloride Channel Current> by Lonidamine and AF2785
The effects of lonidamine or AF2785 on unitary CFTR currents were examined using inside-out membrane patches excised from epididymal cells with smaller pipette openings (6–8 M). Figure 4 shows the effect of lonidamine on a PKA-activated CFTR-Cl- current (representative of three patches from three different preparations). Visual inspection of single-channel records showed that addition of lonidamine (50 and 100 µM) to the intracellular solution led not only to frequent, brief interruptions in the open-channel current giving rise to a concentration-dependent decrease in mean open time, but also to a decrease in single-channel open probability at a hyperpolarized voltage (-50 mV). In contrast, at these concentrations, lonidamine had no apparent effect on unitary current amplitude.
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To quantify the effects of lonidamine or AF2785 at the single-channel level, we measured single-channel open time, current amplitude (I), and open probability (Po). At least 1 min of continuous recording was used for analysis. Because the kinetics of blockade appear close to the limit of resolution at the filter frequencies used, channel-closed time was not quantified from these single-channel records. Figure 5 shows the effects of lonidamine and AF2785 on the open time of single CFTR channels (representative of three patches from three different preparations) obtained from recordings at -50 mV, such as those shown in Figure 4. Because 100 µM lonidamine and AF2785 dramatically decrease the channel open-burst duration, only 10 and 50 µM data were used for open-time analysis. Application of lonidamine or AF2785 to the intracellular side of the membrane of epididymal cells significantly decreased the mean open time of CFTR channels as shown in Figure 5D. The time constants are 590 ± 105 milliseconds (control), 55 ± 12 milliseconds (10 µM lonidamine), 8 ± 5 milliseconds (50 µM lonidamine), 75 ± 18 milliseconds (10 µM AF2785), and 13 ± 6 milliseconds (50 µM AF2785) using pStat analysis by fitting a single exponential function (n = 3 different preparations).
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The effect of lonidamine on CFTR single-channel current amplitude and open probability (Fig. 6, A–C) indicates that lonidamine did not decrease CFTR single-channel current amplitude, which is at -0.25 pA at a membrane potential of -50 mV. However, the number of events for the channel-open state gradually decreased, and the number of events for the channel-closed state increased with an increase in lonidamine concentration (Fig. 6, A–C). Mean open probabilities calculated from traces such as those shown in Figure 6, A–C, as well as the traces with 10 µM lonidamine, are shown in Figure 6D. At a lower concentration, lonidamine does not significantly affect the open-channel probability with a Po of 0.75 ± 0.15 for control and a Po of 0.73 ± 0.18 for 10 µM lonidamine (n = 3 different preparations); however, at higher concentrations (50 and 100 µM), lonidamine showed a flickery block, and it also caused a decrease in channel-open probability with Pos of 0.43 ± 0.11 and of 0.09 ± 0.08, for 50 µM and 100 µM lonidamine, respectively (n = 3 different preparations; Fig. 6D).
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Properties of Lonidamine Block at Positive Voltage
Previously, we demonstrated that lonidamine block of CFTR was voltage-dependent [10]. To investigate the voltage dependence of lonidamine and AF2785 inhibition of single-channel CFTR currents, we examined their effects at +50 mV with lower and higher (only lonidamine was examined) concentrations. Figure 7 compares the inhibitory effect of lonidamine and AF2785 on single-channel CFTR activity at positive and negative membrane potentials. The records are representative of three different preparations. As shown in Figure 7, A and B, the flickery block of CFTR by lonidamine or AF2785 (both at 10 µM) was observed only at the negative voltage. When a higher concentration (100 µM) of lonidamine was applied, the flickery block of CFTR was also observed at +50 mV within a long, single-channel burst (Fig. 7C). Furthermore, a sublevel conductance was observed within the burst (Fig. 7C). Because the seal of these excised patches at positive membrane potentials was not very stable, further quantitation of the effects was not undertaken.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several drugs can interact directly with CFTR at multiple sites to modulate channel activity. These include phloxine B [32], genistein [33], apigenin [34], and Au(CN)
[11]. Phloxine B, genistein, and apigenin stimulate CFTR-Cl- current at low micromolar concentrations, and inhibit it at higher concentrations by occluding the pore and causing a reduction in channel-open probability [32–34]. In contrast, Au(CN) significantly reduced CFTR channel-open probability at low concentrations (<10 µM) and caused an apparent reduction in unitary current amplitude at higher concentrations [11]. In each of these cases separate drug binding sites within the pore, or cytoplasmically accessible ports of the channel protein, or both, were proposed to exist. However, unlike phloxine B, genistein, and apigenin, lonidamine and AF2785 did not have any apparent stimulatory effects on CFTR-Cl- currents. Lonidamine and AF2785 also differ from Au(CN) in that they did not reduce CFTR channel-open probability at low concentrations. Lonidamine and AF2785 show higher affinity for pores than for cytoplasmically accessible ports of the channel protein (NBDs and RDs), whereas phloxine B, genistein, apigenin, and Au(CN) show higher affinity for those parts of the CFTR molecule that affect channel gating, either stimulating (phloxine B, genistein, and apigenin) or inhibiting (Au(CN)) CFTR open probability. Further experiments are needed to investigate the possibility that lonidamine and AF2785 bind to sites within NBDs and RDs.
Some differences were observed between the present results and those previously reported for lonidamine and AF2785 block of CFTR in rat epididymal cells [9, 10]. First, in a previous study employing whole-cell recording with perfusion, the apparent IC50s of lonidamine and AF2785 for CFTR have higher values than the Kd values calculated here by the analysis of current variance [10]. Second, intracellular lonidamine and AF2785 were found to be less effective in blocking CFTR than following external application [10]. Third, AF2785, which contains one more ethene group than lonidamine, was found to be more effective in blocking CFTR than lonidamine [9, 10]. These discrepancies are likely to be attributed to the different methods used in the two studies (e.g., whole-cell versus inside-out patch recording, intracellular dialysis of lonidamine and AF2785 versus direct application to the intracellular face of the membrane, fast perfusion system with short time [seconds] of drug application versus longer application [minutes] of the drugs, and CFTR activation by cAMP versus PKA plus MgATP).
Inhibition of CFTR may prove to be of value in the treatment of diseases such as secretory diarrhea and polycystic kidney disease, which may involve abnormal increases in CFTR-Cl- channel activity [35, 36]. By the same token, blockers of CFTR could be used to purposely disrupt the epididymal fluid environment, hence offering a new method of contraception for men. In fact, lonidamine and its analogues have been proposed as potential male contraceptives by virtue of their interference with spermatogenesis [37, 38]. The CFTR blockers available today are neither selective nor potent [39]. Furthermore, to inhibit fluid secretion by the epididymis, these blockers would have to gain access to the apical membrane where CFTR is located. It is uncertain whether they can cross the blood-testis or blood-epididymis barrier to reach the lumen of the epididymis. Pharmacokinetic studies, however, revealed that lonidamine and its analogues are capable of permeating the blood-testis/blood-epididymis barrier to attain high concentrations within the male reproductive tract [40–42]. Furthermore, it is already known that unlike the steroidal contraceptives, indazole compounds generally have low systemic toxicity and lack undesirable hormonal effects [43, 44]. Therefore, systematic screening of indazole compounds for their inhibition of CFTR in the male reproductive tract can provide a new avenue for research in our quest for novel male contraceptives.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 852 26035022; patrickwong{at}cuhk.edu.hk
Received: 16 May 2002.
First decision: 10 June 2002.
Accepted: 2 July 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  H.-F. Huang, R.-H. He, C.-C. Sun, Y. Zhang, Q.-X. Meng, and Y.-Y. Ma Function of aquaporins in female and male reproductive systems Hum. Reprod. Update, November 1, 2006; 12(6): 785 - 795. [Abstract] [Full Text] [PDF]
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