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Cellular Mechanisms of Carbachol-Stimulated Cl^– Secretion in Rat Epididymal Epithelium¹

School of Life Science, Sun Yat-sen University, Guangzhou 510275, People's Republic of China

ABSTRACT

Neurotransmitter-controlled Cl^– secretions play an important role in maintenance of the epididymal microenvironment for sperm maturation. This study was carried out to investigate the effect of carbachol (CCH) on the cultured rat epididymal epithelium and the signal transduction mechanisms of this response. In normal K-H solution, CCH added basolaterally elicited a biphasic Isc response consisting of a transient spike followed by a second sustained response. Ca²⁺ activated Cl^– channel blocker disulfonic acid stilbene (DIDS, 300 µM) only inhibited part of the CCH-induced Isc response, while nonselective Cl^– channel blocker diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced all, indicating the involvement of different conductance pathways. Both peaks of the CCH-induced Isc response could be significantly inhibited by pretreatment with an adenylate cyclase inhibitor, MDL12330A (50 µM). An increase in intracellular cAMP content upon stimulation of CCH was measured. All of the initial peak and part of the second peak could be inhibited by pretreatment with Ca²⁺-chelating agent BAPTA/AM (50 µM) and an endoplasmic reticulum Ca²⁺ pump inhibitor, Thapsigagin (Tg, 1 µM). In a whole-cell patch clamp experiment, CCH induced an inward current in the single cell. Two different profiles of currents were found; the first component current exhibited an outward rectifying I-V relationship in a time and voltage-dependent manner, and the current followed showed a linear I-V relationship. The carbachol-induced current was found to be partially blockable by DIDS and could be completely blocked by DPC. The above results indicate that the CCH-induced Cl^– secretion could be mediated by Ca²⁺ and cAMP-dependent regulatory pathways.

calcium, epididymis, male reproductive tract, neurotransmitters, signal transduction

INTRODUCTION

In secretory epithelium, epithelia activation of surface receptors triggers cascades of biochemical reactions leading to increased concentration of intracellular second messengers like adenosine 3',5'-cyclic monophosphate (cAMP) and/or calcium. These messengers act on different components of the secretory pathway to stimulate chloride secretion [1]. In epididymis, it has been shown that the secretory response to a number of secretagogues was mediated by a rise in intracellular cAMP ([cAMP]_i) [1, 2]. On the other hand, some of the putative Ca²⁺-mobilizing agents, including the calcium ionophore, have also been found to stimulate Cl^– secretion in endometrium [3]. In some cases, the stimulation of cAMP has been considered as consequent to increased phosphoinositide hydrolysis, intracellular Ca²⁺ ([Ca²⁺]_i) mobilization, and Ca²⁺/calmodulin activation of adenylyl cyclase activity [4]. On the other hand, recent studies have shown that in membranes of cells transfected with either the muscarinic acetylcholine receptor 1 (Chrm1) or 4 (Chrm4) gene, CCH can directly stimulate adenylyl cyclase activity, probably by promoting the coupling of the muscarinic acetylcholine receptors to the G protein Gs [5, 6]. Collectively, these data indicate multiple regulatory mechanisms of the muscarinic regulation of cAMP formation.

The activation of [cAMP]_i and [Ca²⁺]_i would stimulate the Cl^– secretion passing through cAMP-regulated Cl^– channel (CFTR) and Ca²⁺-activated Cl^– channel (CLCAs) in epididymal epithelium [1, 7]. CFTR is known as a key element in electrolytes and fluid secretion by epididymal epithelium [8]. As with other ion channels on the membrane, CFTR was controllable by pharmacological agents, such as noradrenalin [1], lysylbradykinin (LBK) [9], and PACAP [2]. However, the physiological functions of CFTR and CLCAs that are regulated by CCH have not been shown.

Previous studies reported that various neurohormonal agents, including adrenaline, bradykinin, gastrin-releasing peptide, and prostaglandin E₂ and F₂, are effective stimulants of electrogenic transport across epididymal epithelia of a number of species [9, 10]. The cellular mechanisms, e.g., transporters and ion channels, underlying these responses have been elucidated, but the mechanisms of CCH-induced Cl^– secretion by epididymis has not been shown. The present study was carried out to investigate the signal transduction pathway underlying these responses.

MATERIALS AND METHODS

Animals

Immature male Sprague-Dawley rats were purchased from the Animal Center of Sun Yat-sen University. Animals were housed and fed according to the guidelines of the Sun Yat-sen University Animal Use Committee; all procedures were approved prior to each experiment. Animals lived in a constant-temperature room (20°C) with a 12L:12D photoperiod and were allowed food and water ad libitum.

Materials

Eagle minimum essential medium (EMEM), fetal bovine serum (FBS), nonessential amino acids, penicillin/streptomycin, Hanks balanced salt solution, sodium pyruvate, and trypsin were purchased from Gibco Laboratories (New York). 5-dihydrotestosterone (5-DHT), collagenase IA, CCH, MDL12330A, BAPTA/AM, DIDS, and Thapsigagin (Tg) were from Sigma Chemical Co. (St Louis, MO). DPC was bought from Riedel-de-Haen (Germany). Fluo3/AM and pluronic F-127 were obtained from Molecular Probes, Inc. (Eugene, OR). The cAMP immunoassay kit was obtained from Assay Designs, Inc (Ann Arbor, MI). The bicinchoninic acid (BCA) protein assay kit was bought from Shenenergy, Inc. (Shanghai, China).

Cell culture of rat epididymal epithelium

The procedures of tissue culture have been described previously [11]. In brief, immature male Sprague-Dawley rats weighing 120–150 g were killed by CO₂ inhalation. Cauda epididymis 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 EMEM containing nonessential amino acid (0.1 mM), sodium pyruvate (1 mM), 5-DHT (1nM), 10% FBS, penicillin (100 IU/ml), and streptomycin (100 µg/ml) after 4–6 h primary culture, then seeded onto Millipore filters (0.45 cm²) floating on EMEM completed with other supplements. Cultures were incubated for 4 days at 32°C in 5% CO₂/95% O₂. Thereafter, the monolayers reached confluence, as monitored by measuring the transepithelial resistance [11], and were ready for the measurement of short-circuit current (Isc). The cells were also seeded onto a 12-well culture plate with a piece of cover glass after the primary culture. The cells were used in a patch-clamp experiment after 3 days of culture.

Short-circuit Current (Isc) Measurement

Confluent monolayers of rat epididymal cells were clamped vertically between the two halves of the Ussing chambers, and Isc measurement was made as described previously [12]. In brief, confluent epididymal monolayers were clamped between the two halves of Ussing chambers. The tissue was short-circuited by the use of a voltage-clamp amplifier (VCC MC6; Physiologic Instruments, San Diego, CA); the Isc results were obtained from the signal collection and analysis system (BL-420E, Chengdu Technology & Market Co. Ltd., China). Transepithelial resistance was obtained from Ohm's law by clamping the tissue intermittently at a voltage of 1 mV displaced from zero. In most situations, the monolayers were bathed on both sides with normal Krebs-Henseleit (K-H) solution (117 mM NaCl; 4.7 mM KCl; 2.5 mM CaCl₂; 1.2 mM MgCl₂; 24.8 mM NaHCO₃; 1.2 mM KH₂PO4; 11.1 mM glucose), gassed with 95%O₂/5%CO₂ at 32°C to attain a pH of 7.4.

Whole-cell Patch Clamp Recording

After 3 days of culture, epididymal cells were found to form colonies in dishes. The cells were incubated in Ca²⁺-free D-PBS solution containing 1 mM EGTA for 10–15 min to separate the cells. The cells then moved to a 1-ml chamber which fixed on an X-Y axis stage of an immersing lens microscopy system (BX51WI; Olympus, Japan). Recording was performed at room temperature using a Multiclamp700A amplifier and Digidata 1322 series interface (AXON Instrument, Foster City, CA). Signals were filtered at 10 KHz. Pclamp 9.0 software system (AXON Instrument) was used for data recording and analysis. Patch pipettes were pulled from 1.2 mm outside diameter, 0.5 mm inside diameter glass pipettes (local product) using a horizontal puller P-97 (Sutter Instrument Co., Novato, CA). The pipettes were filled with a solution containing 135 mM NMDG-Cl, 2 mM MgCl₂, 3 mM MgATP, and 10 mM HEPES, pH adjusted to 7.2 with Tris. The bath solution contained 135 mM NMDG-Cl, 4.7 mM CsCl, 2.5 mM CaCl₂, 1.2 mM MgCl₂, 1.2 mM NaH₂PO4, 10 mM glucose, and 10 mM HEPES (pH 7.4). The resistance of the patch pipettes is about 2–7 M.

Positive pressure was added in the patch pipette before it was immersed in the bath solution, and when the tips of the pipette attached to the cell surface, the pressure was withdrawn, normally a giga-seal (>2G) between pipette and cell membrane was formed. After being sucked from the pipette, the whole cell configuration was confirmed. The epididymal cell was held at its resting potential –30mV in the episodic recording mode, and the voltage was clamped from –120mV to +120mV, step +20mV. In the gap-free recording mode, the cells were held at –70mV throughout the period of recording.

Measurement of Intracellular cAMP

The cultured epididymal epithelial cells were seeded onto 24-well tissue plates with a density of 1.5x10⁶ cells per ml. After 4 days of culture, the cells were washed and incubated in K-H solution at 32°C for 15 min. Drugs were added to the wells and incubated for various time periods. Afterward, each well of cells was washed with K-H solution and extracted with 0.5 ml of lysing buffer (0.1M HCl and 1% triton X-100) for 10 min. Lysed cells were mixed with the buffer solution and centrifuged at 2000 rpm for 4 min at room temperature. The cAMP content of cleared supernatants was assayed using a commercially available enzyme immunoassay kit. In each experiment, the protein content of three wells was measured by using the bicinchoninic acid (BCA) protein assay kit. Protein determination allowed detection of cAMP at pmol/mg [13].

Measurement of Intracellular Calcium

Cells were prepared and loaded with the fluorescence dye Fluo3/AM, as described [14]. Briefly, the cells were grown in culture medium on 12-well tissue plates at 32°C. After 24 h cultured, the cells were washed in normal physiological saline solution (N-PSS) that contained 140 mM NaCl, 1 mM KCl, 1 mM MgCl₂, 1 mM Ca²⁺, 5 mM HEPES, and 10 mM glucose (pH 7.4). For loading of Fluo3/AM, cells were incubated for 1h in the dark at room temperature with 10 µM membrane-permeant Fluo3/AM and 0.02% pluronic F-127 in N-PSS. Then the cells were washed three times in new N-PSS before taking Ca²⁺ measurements. The fluorescence signal was monitored and recorded by a Laser Scanning Confocal Imaging System (TCS SP2; Leica Microsystems, Germany). The data analyses were processed with Origin 6.1. The change of fluorescence intensity after drug treatments was normalized with the initial intensity.

Statistical Analysis

Results are expressed as means ± SEM. In Isc experiments, comparisons between groups of data were made by the unpaired Student t-test. The mean Isc responses to the drug-treated cells were compared with response in untreated controls obtained from different pieces of cultured cells; In [cAMP]_i measurement experiments, the comparisons between drug-treated and untreated cells were also made by unpaired Student t-test. These cells were obtained from one batch of culture. A P value of less than 0.05 was considered significant.

RESULTS

Characteristics of CCH-Induced Whole-cell Cl^– Current

Whole-cell currents could be activated upon addition of 100 µM CCH. The current recordings were obtained using pipette and bath solutions containing a symmetrical Cl^– concentration (containing intracellular 135 mM NMDG-Cl and extracellular 135 mM NMDG-Cl, respectively). The resting membrane potential of cells successfully forming tight seals were found to be about –30mV [7]. Addition of 100 µM CCH to the bath caused an increase in inward current at –70 mV (Fig. 1A). The current increased within 1 min and reached peak level after about 3 min. The maximal response to CCH was found to be highly variable but averaged 800 pA (n = 7). The CCH-stimulated inward current at –70 mV was always sustained.

View larger version (19K): [in this window] [in a new window]   FIG. 1. Characteristics of CCH-induced whole-cell currents. A) Outline of the whole-cell current recording performed by holding the cell potential at –70 mV (n = 7), dashed line represents current level prior to stimulation; at the time indicated, CCH (100 µM) was added to the bath. Whole-cell currents profile changed with time after addition of CCH (100 µM): t = 0 min (zero current) (B); t = 0.5 min (C); t = 6 min (D). E) I-V relationships obtained from C and D on the same cell. Current were elicited by voltage pulses from a holding potential of –30 mV to potential between –120 mV to +120 mV, 20 mV increment (representative of four experiments)

Fig. 1B–D shows the whole-cell membrane current in response to square voltage pulses from a holding potential at –30 mV to potential between –120 mV to +120 mV, 20 mV one step. A delayed activation and rectifying outward current was first observed during the depolarization in less than 1 min. And a time-independent and linear current was found in about 6 min after the addition of CCH. The two types of the currents I-V relationships were shown in Fig. 1E. The observed changes in CCH-activated whole-cell current profiles with time could be due to different Cl^– conductance activated at different times.

The reversal potentials of the CCH-induced currents in symmetrical Cl^– solutions were close to the Cl^– equilibrium, 0 mV. In order to further identify whether the CCH-activated whole-cell currents were mediated by Cl^–, and not through any nonselective conductance, we performed experiments in which Cl^– concentration in the bath was changed from 140 mM to 70 mM while a pipette containing 135 mM NMDG-Cl was used. As shown in Fig. 2, the reversal potential was shifted to a value close to the new equilibrium for Cl^–, 22 ± 0.5 mV (n = 4) as compared to the theoretic value of 18.7 mV. The results suggested that currents activated by extracellular CCH were mediated by Cl^–.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Demonstration of the involvement of Cl– in CCH-induced currents. Whole-cell recordings obtained 6 min after CCH stimulation from a cell patched with pipette containing 140 mM NMDGCl and bathed in 140 mM NMDGCl or 70 mM NMDGCl with corresponding I-V relationships. Note that reversal potential was shifted from 0 to 22 mV (22 ± 0.5 mV, n = 4), close to the new ECl = 18.7 mV. The records are representative of four experiments

Effect of DPC and DIDS on the CCH-Stimulated Cl^– Secretion and Cl^– Conductance

The CCH-induced current was blocked by Cl^– channel blockers, DIDS and DPC. Fig. 3A shows the control Isc response induced by CCH. Before the CCH was added in the bath, the monolayer was respectively pretreated with DIDS (300 µM, apical; Fig. 3B) and DPC (1 mM, apical; Fig. 3C) for about 20 min. DIDS, a Ca²⁺-activated Cl^– channel blocker, could partially block the CCH-induced current from a control level of 2.5 ± 0.21 (first peak; 1.75 ± 0.25, second peak) to 1.33 ± 0.11 (first peak; 0.97 ± 0.14, second peak; n = 3, P < 0.05). And DPC, a nonselective Cl^– channel blocker could almost block the CCH-induced Isc response from control to 0.21 ± 0.04 (first peak; 0, second peak; n = 4, P < 0.05).

View larger version (19K): [in this window] [in a new window]   FIG. 3. Effect of DPC and DIDS on CCH-stimulated Isc currents. A) Addition of CCH (100 µM) to the basolateral side of the monolayer culture caused a rise in Isc. This response was found to be partially inhibited by adding DIDS (300 µM, ap) (B) and almost inhibited by DPC (1 mM, ap) (C). Summary of the effect of these two Cl– channel blockers were also shown (D). Columns and bars are mean ± SEM (*P < 0.05; **P < 0.01, compared with control). The records are representative of four experiments. ap, apical side; bl, basolateral side

In patch-clamp experiments, similarly to the Isc results, DIDS and DPC could block the CCH-induced whole-cell currents. The control (100 µM CCH-induced current, bath) is shown in Fig. 4A. Fig. 4B and C show the effects of DIDS (100 µM, bath) and DPC (500 µM, bath), DIDS was added to the bath 3 min after the addition of CCH, and DPC was added 6 min after CCH. The I-V relationship before (control) and after addition of blockers is shown in Fig. 4D. The above results show that the CCH-stimulated currents may pass through two types of Cl^– channels.

View larger version (20K): [in this window] [in a new window]   FIG. 4. Inhibition of the CCH-activated whole-cell currents by Cl– channel blockers. Current recording before (A) and after exposure of single voltage-clamped cells to 300 µM DIDS (B) (3 min after addition of CCH) and 500 µM DPC (C) (6 min after addition of CCH). Experimental conditions are similar to those described in Fig.1. D) I-V relationships obtained from B and C on the same cell. DIDS and DPC significantly reduced the CCH-activated currents. The records are representative of four experiments

Effect of [cAMP]_i on the Chloride Secretion

The involvement of different types of Cl^– channels in the CCH-induced Cl^– secretion indicated different regulatory pathways and suggested Ca²⁺ and cAMP-dependent regulatory pathways were involved in mediating the response. So the involvement of cAMP was examined by investigating the inhibitory effect of adenylate cyclase inhibitor MDL12330A on the Isc reponse to CCH (100 µM), as shown in Fig. 5. Preincubation of the epididymal cells for 25 min with 50 µM MDL12330A resulted in a 90% (first peak) and 98% (second peak) (n = 4, P < 0.01) inhibition of the Isc response elicited by 100 µM CCH. The above results showed that the intracellular cAMP was a very important factor involved in the CCH-induced Cl^– secretion in epididymis.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Effect of MDL12330A on the CCH-induced Isc response. The monolayer of epididymal cells was before (A) and after (B) pretreated with an apical addition of 50 µM MDL 12330A 25 min prior to the addition of CCH (100 µM, bl). C) Summary of the effects of MDL12330A on CCH-induced Isc. Columns and bars are mean ± SEM (**P < 0.01, compared with control). The records are representative of four experiments. ap, apical side; bl, basolateral side

To further characterize the cAMP-dependent pathway involved in the CCH-induced response, we measured the intracellular cAMP content. As shown in Fig. 6A, the intracellular cAMP content under basal conditions was 5.31 ± 1.39 pmol/mg protein (n = 5). After incubation with CCH (100 µM), cAMP levels increased significantly to 39.92 ± 13.14 pmol/mg protein (n = 3, P < 0.05); the response was close to the forskolin (10 µM, 54.59 ± 3.42 pmol/mg protein, n = 5) and was higher than that of IBMX (100 µM, 13.84 ± 6.08 pmol/mg protein, P > 0.05, compared with control, n = 5). To test whether decreased cAMP degradation contributed to increased cAMP levels, the broad, specific phosphodiesterase inhibitor IBMX (100 µM) was added to the cells, and the resulting effects on CCH-responsive cAMP levels were evaluated. As shown in Fig. 6B, IBMX (100 µM) alone caused an increase in cAMP levels to 13.84 ± 6.07 pmol/mg protein, which is not significantly different from control (n = 5, P > 0.05). After incubation with 100 µM CCH in the presence of IBMX (100 µM), there was a significant increase in cAMP levels, 32.46 ± 10.5 pmol/mg protein (n = 3, P < 0.05). In the presence of forskolin (10 µM) and IBMX (100 µM), CCH 100 µM could not increase cAMP content (n = 3, data not shown). The data suggest that CCH could increase the intracellular cAMP levels significantly, and the degradation of intracellular cAMP by phosphodiesterase may not alter the process of CCH-induced Cl^– secretion.

View larger version (20K): [in this window] [in a new window]   FIG. 6. Effect of CCH on intracellular cAMP level in epididymal cells. A) Cells before and after being pretreated with IBMX (100µM) or CCH (100µM) or forskolin (10µM). Compared with control (untreated cells), CCH and forskolin induced a significant increase of cAMP content respectively. B) cAMP elevations was compared following addition of IBMX (100 µM), CCH (100 µM) with IBMX (100 µM), and forkolin (10 µM) with IBMX (100 µM). Columns and bars are mean ± SEM (*P < 0.05)

Effect of [Ca2+]_i on the Chloride Secretion

To study the involvement of [Ca²⁺]_i in the Isc response to CCH, the monolayer was loaded with BAPTA/AM (a cell permeant Ca²⁺ chelator) and Tg (an endoplasmic reticulum Ca²⁺ pump inhibitor) before stimulation. The Isc response to CCH before (Fig. 7A) and after (Fig. 7B) the cells were loaded with 100 µM BAPTA/AM for 25 min is shown in Fig. 7. The initial spike triggered by CCH was all abolished by pretreatment with BAPTA/AM, and the second component was partly inhibited. The same results were found when the cells were pretreated with 1µM Tg (Fig. 7C), suggesting that the transient increase part of the Isc response was controlled by the rise of [Ca²⁺]_i.

View larger version (13K): [in this window] [in a new window]   FIG. 7. Effect of Tg and BAPTA/AM on the CCH-induced Isc response. The monolayer of epididymal cells was before (A) and after being pretreated with an apical addition of 100 µM BAPTA/AM (B) or 1µM Tg (C) 25 min prior to the addition of CCH (100 µM, bl). ap, apical side; bl, basolateral side

To further investigate the intracellular Ca²⁺ involved in the CCH-induced response, we measured the intracellular Ca²⁺ level by Fluo3 in the presence of extracellular Ca²⁺. Fig. 8 shows the effect of CCH (100 µM) on the [Ca²⁺]_i in single epididymal cells. The cells were bathed in N-PSS containing 1 mM free Ca²⁺. It can be seen that 100 µM CCH stimulation gave rise to a Ca²⁺ spike. Subsequent Tg (5 µM) stimulation led to an additional transient rise in [Ca²⁺]_i. The results suggested that CCH stimulation led to [Ca²⁺]_i increase and the Ca²⁺ response was functionally smaller than that by Tg.

View larger version (24K): [in this window] [in a new window]   FIG. 8. Effects of CCH on intracellular Ca2+ in epididymal cells. Intracellular Ca2+ measurements in single epididymal cells using Fluo3 as a probe. Addition of CCH (100 µM) significantly increased the intracellular Ca2+. The response was less than the addition of Tg (5 µM) that followed. The records are representative of 15 experiments

DISCUSSION

The results showed the CCH-stimulated Cl^– secretion in the cultured rat epididymal epithelial cells. The response consisted of an initial spike and a long-term component. Earlier studies have demonstrated that adrenergic stimulation of Isc in the rat epididymal epithelium is largely due to electrogenic Cl^– secretion [1, 11, 15, 16]. In the Cl^–-free extracellular solution, the initial spike disappeared, and the second component was reduced to about 30% of its prior strength. This residual current seen in Cl^–-free solution has been attributed to HCO₃^– secretion, since this current can be completely inhibited by agents affecting HCO₃^– transport [1]. In our studies, it demonstrated that CCH-induced anion secretion is a main contribution of Cl^– (Du and Zhou, unpublished).

Activation of muscarinic acetylcholine receptor, although not directly open Cl^– channels, may initiate a Ca²⁺-dependent opening of such channels leading to efflux of Cl^– and a resulting depolarization contributing to further influx of Ca²⁺ via voltage-dependent calcium channel [17]. In many tissues, CCH has the capacity to activate multiple signaling pathways in various cell types. In airway cells, activation of phospholipase C (PLC) via the intermediary heterotrimeric G protein Gq is the predominant pathway through which the muscarinic acetylcholine receptor 3 (CHRM3) regulates important airway cell functions [18]. PLC activation induces protein kinase C (PKC) activation and inositol 1,4,5-trisphosphate (IP₃) generation, which serve to increase intracellular Ca²⁺ and sensitize and activate the cell's contractile machinery [19, 20]. The activation of muscarinic acetylcholine receptor by Ach in tracheal smooth muscle cells induced a Ca²⁺ independent Cl^– current [21]. In an exocrine avian salt gland cell preparation, CCH stimulated intracellular Ca²⁺, then evoked a voltage-activated K⁺ current and a linear Ca²⁺-activated Cl^– current [22]. And in Xenopus oocytes, expression of the muscarinic acetylcholine receptor 2 (CHRM2) opened Cl^– channels by activation of PI3-kinase, generation of PIP₃, and stimulation of PKC [23]. Activation of muscarinic acetylcholine receptors may also modulate PKA-regulated Cl^– current in guinea pig ventricles [24]. In rat SCG, the synergistic action of Ca²⁺ and DAG induced a delayed Ca²⁺ independent Cl^– current, and the PKC inhibitor can block the response [25].

In the human colon carcinoma cell line, HT-29, two different types of Cl^– channels were found to be involved in the CCH-stimulated response by using the short-circuit current technique. One channel was Ca²⁺-dependent and activated transiently; the other exhibited a more sustained response along with the activation by PKC [26]. And it has been shown that cAMP and Ca²⁺ activate two different types of whole-cell Cl^– conductance in the colonic epithelial cell line T84 [27].

The previous studies in rat caudal epididymal epithelial cells using the short-circuit current technique, fluorescence measurement, and whole-cell patch clamp technique have indicated the possible involvement of different regulatory pathways in mediating adrenergic-stimulated Cl^– secretion by epididymal cells. cAMP and Ca²⁺, which activated two different types of whole-cell Cl^– conductance, were found in cultured epididymal epithelial cells [15, 28]. The cAMP-mediated whole-cell current exhibits time independence profile and the characteristics of the cAMP-mediated conductance was similar with CFTR [28]. Previous studies have shown that CFTR is expressed by the rat and human epididymal cells [29, 30]. Furthermore, the expression of CFTR showed region-specific characteristics. CFTR was weak expressed in the initial segment of epididymis, leading to an increase in the concentration of sperm [31–34]; however, CFTR was richly expressed in cauda epididymis. There, when cells were stimulated by agents, an increase of [cAMP]_i followed, which activated CFTR, driving Cl^– efflux into lumen. The Cl^– and fluid secretion in the cauda region would maintain the fluid microenvironment of epididymal lumen, where most of the testicular fluid would have been reabsorbed [8]. The Ca²⁺ ionophore increasing Cl^– conductance in a time-dependent manner of rat epididymal epithelium was also reported [15].

However, the CCH-stimulated Cl^– conductance has not been shown in epididymal epithelial cells. The present studies observed the CCH-induced Cl^– secretion in the epididymis, similar to the mechanism of Cl^– secretion in the other epithelial cells which were illustrated above. The increase of intracellular Ca²⁺ and cAMP could induce a biphasic whole-cell Cl^– conductance, the first transient component being time-dependent and outwardly rectified in accordance with the characters of Ca²⁺-activated Cl^– conductance (CLCAs); the second slow component is a time-independent and linear current (3 min after the addition of CCH), which could be taken as cAMP-activated Cl^– conductance (CFTR). In order to confirm the Cl^– channel involved in the CCH-activated response, DIDS (Ca²⁺-activated Cl^– channel blocker) was added to the bath, current was partially inhibited, and then the DPC (nonselective Cl^– channel blocker) was added; almost all the CCH-induced response was blocked, indicating the involvement of cAMP-activated Cl^– channel (CFTR) in the action of CCH. The same results were acheived in Isc experiments. BAPTA/AM, Tg and MDL12330A were used to further study the intracellular signal transduction pathways. All of them can inhibit the CCH-induced Isc response. Respectively, BAPTA/AM and Tg could inhibit all the Isc transient peak and part of the second peak. These observations suggested that CCH activates its receptors on the epididymal epithelium, causing Ca²⁺ release from an internal store or from extracellular solution, hence the initial spike in Isc. And MDL12330A could inhibit most of the Isc response. It may be suggested that the effects of intracellular Ca²⁺ and cAMP overlap with each other, and the intracellular cAMP may act as a main factor in the CCH-induced Cl^– secretion on the epididymal epithelium. In order to confirm the effects of intracellular Ca²⁺ and cAMP, we also directly measured the intracellular Ca²⁺ and cAMP levels after the cells were pretreated with CCH. The results showed that CCH could induce a significant increase of intracellular Ca²⁺ and cAMP.

The above findings demonstrate that CCH stimulated its receptors, which are located on the epididymal epithelial cells membrane [35]. The activation of the muscarinic acetylcholine receptors triggers cascades of biochemical reactions leading to increased concentrations of intracellular second messengers like cAMP and/or Ca²⁺. These messengers act on different components of the secretory pathway to stimulate chloride secretion. The role of G-protein and protein kinase awaits further observation.

ACKNOWLEDGMENTS

The authors are grateful to Prof. P.Y.D. Wong and Dr. K.H. Cheung at The Chinese University of Hong Kong for technical support and suggestions.

FOOTNOTES

¹ Supported by National Natural Science Foundation of China (contract grant numbers: 30370540 and 30570229).

² Correspondence: FAX: 86 20 8411 0060; wenliangzhou{at}yahoo.com

Received: 20 March 2006.

First decision: 16 April 2006.

Accepted: 31 May 2006.

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