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Cell Biology International (2006) 30, 741–746 (Printed in Great Britain)
Involvement of muscarinic acetylcholine receptors in chloride secretion by cultured rat epididymal epithelium
Jian‑Yang Du, Wu‑Lin Zuo, Min‑Hui Chen, Hui Xiang and Wen‑Liang Zhou*
School of Life Science, Sun Yat-Sen University, 135 Xingang West Road, Guangzhou 510275, PR China


Abstract

The aim of our present study was to investigate the short-circuit current response to carbachol in cultured rat cauda epididymal epithelia and the signal transduction mechanisms involved. Carbachol added basolaterally induced a concentration-dependent increase in short-circuit current (Isc) across the epididymal epithelium consisting of a rapidly rising phase and a long term sustained response. The response was almost abolished by removing Cl from the extracellular medium and blockable by pretreating the tissues with DPC, indicating a substantial contribution of Cl secretion to the carbachol-induced response. The muscarinic acetylcholine receptor antagonist atropine inhibited the response, but the nicotinic acetylcholine receptors antagonist curarine had no effect, suggesting that only the muscarinic acetylcholine receptors mediated the secretory response of the basolateral side of rat cauda epididymal epithelium to carbachol. Addition of carbachol to the apical side of the tissue was found not to elicit an Isc response. These results suggested that muscarinic receptors are present in the basolateral side of rat cauda epididymal epithelium. Activation of these receptors by acetylcholine released from the nerve endings regulates epididymal transepithelial Cl secretion. Cholinergic stimulation therefore contributes to the formation of luminal fluid microenvironment.


Keywords: Muscarinic acetylcholine receptors, Carbachol, Cl secretion, Epididymal epithelium.

*Corresponding author. Tel./fax: +86 20 84110060.


1 Introduction

Transepithelial chloride secretion in some epithelia has been shown to be stimulated by cholinergic agonists. Activation of AchRs on epithelial cells activates cascades of intracellular reactions leading to increased concentrations of intracellular second messengers like adenosine 3′,5′-cyclic monophosphate (cAMP) and/or Ca2+. These messengers act on different cellular signal transduction pathways to stimulate chloride secretion (Donowitz and Welsh, 1986). It is believed that the signal transduction mechanisms are specific to the types of AchRs involved.

It has been demonstrated that the secretory function of the mammalian epididymal epithelium is richly controlled by the nervous system. Nerve fibers are connected to the epithelial regions in epididymis (Richins and Kuntz, 1953; El-Badawi and Schenk, 1967a,b). Previous studies have also shown the different distribution of the three regions (caput, corpus and cauda) in epididymis (El-Badawi and Schenk, 1967a,b; Suarez-Garnacho et al., 1989). These nerves fibers might serve a secretomotor function (Wong et al., 1992).

Previous work has shown that the epididymal epithelium is subject to multiple regulations by neurohumoral agents (Leung and Wong, 1992; Leung et al., 1992; Leung and Wong, 1994; Zhou et al., 1997; Chan et al., 1995a,b) coupled to different cellular signaling pathways (Chan et al., 1995a,b). The present study investigated the effect of carbachol (CCH) on transepithelial Cl secretion (Bajnath et al., 1992; Kachintorn et al., 1993) using the short-circuit current (Isc) technique (Cuthbert and Wong, 1986) with a view to identifying the type of cholinergic receptors and the underlying signal transduction mechanisms involved.

2 Materials and methods

2.1 Medium and drugs

Eagle's minimum essential medium (EMEM), fetal bovine serum, non-essential amino acids, penicillin/streptomycin, Hank's balance salt solution, sodium pyruvate and trypsin were purchased from Gibco Laboratories (New York, USA). 5α-Dihydrotestosterone (5α-DHT), collagenase IA, carbachol, atropine and curarine were from Sigma Chemical Co. (St Louis, USA). Diphenylamine-2-carboxylate (DPC) was bought from the Riedel-de-Haen (Germany). RT-PCR kit was purchased from Promega (USA).

2.2 Cell culture

The procedures of tissue culture have been described previously (Cuthbert and Wong, 1986; Wong, 1988). In brief, immature male Sprague–Dawley rats weighting 120–150g 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 Eagle's minimum essential medium (EMEM) containing non-essential amino acid (0.1mM), sodium pyruvate (1mM), 5α-dihydrotestosterone (1nM), 10% fetal bovine serum, penicillin (100IU/ml) and streptomycin (100μg/ml), after 4–6h primary culture, then seeded onto Millipore filters (0.45cm2) floating on EMEM complete with other supplements. Cultures were incubated for 4 days at 32°C in 5% CO2/95% O2. Thereafter, the monolayers reached confluence and were ready for the measurement of Isc.

2.3 Short-circuit current measurement

Confluent monolayers of rat epididymal cells were clamped vertically between the two halves of the Ussing Chambers and Isc measurement was made using a voltage-clamp amplifier (VCC MC6, Physiologic Instruments, San Diego, USA) as described previously (Ussing and Zerahn, 1951), and the data were from the signal collection and analysis system (BL-420E, Chengdu Technology and Market Co. Ltd, China). The normal Krebs–Henseleit solution used had the following composition (mM): NaCl, 117; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 24.8; KH2PO4, 1.2; glucose, 11.1. The solution was gassed with 95% O2/5% CO2 at 32°C to attain a pH of 7.4. In Cl free experiment, ambient Cl was replaced by gluconate. When HCO3 was removed, the solution was bubbled with 100% O2.

2.4 RNA Isolation and RT-PCR

Total RNA was isolated using TRIzol reagent (Invitrogen, USA), and 2μg was used for first strand cDNA synthesis using random hexamer primers and M-MLV Reverse Transcriptase (Promega, USA). The resulting first strand cDNA was directly used for polymerase chain reaction (PCR) amplification. Primers against m1–m4 muscarinic receptor subtypes (Drescher et al., 1992; Sharma et al., 1996) and against GAPDH (Scofield et al., 1995) were synthesized in the Sangong (Shanghai, China). Primers sequences, corresponding base sites and the sizes of the PCR products were as follows: m1sense, 5′-CTGGTTTCCTTCGTTCTCTG-3′(593–612) and m1antisense, 5′-GCTGCCTTCTTCTCCTTGAC-3′ (1233–1214) (D641); m2sense, 5′-GGCAAGCAAGAGTAGAATAAA-3′ (633–653) and m2antisense, 5′-GCCAACAGGATAGCCAAGATT-3′ (1184–1164) (D552); m3sense, 5′-GTGGTGTGATGATTGGTCTG-3′ (591–610) and m3antisense, 5′-TCTGCCGAGGAGTTGGTGTC-3′ (1380–1361) (D790); m4sense, 5′-AGTGCTTCATCCAGTTCTTGTCCA-3′ (543–566) and m4antisense, 5′-CACATTCATTGCCTGTCTGCTTTG-3′ (1052–1029) (D510); GAPDHsense, 5′-CGGGAAGCTTGTGATCAATGG-3′ (258–277) and GAPDHantisense, 5′-GGCAGTGATGCCATGGACTG-3′ (614–595) (D357).

Reactions were carried out with the following parameters: denaturation at 94°C for 10s, annealing at 60°C for 10s, and extension at 72°C for 45s, for a total of 35 cycles. PCR products were analyzed by agarose gel electrophoresis and visualized by staining with ethidium bromide. The PCR products of the expected sizes were confirmed by sequencing.

2.5 Data analysis

Results are expressed as means±S.E.M. Comparisons between groups of data were made by the Student t-test. Multiple comparisons were made using one-way ANOVA. A P value of less than 0.05 was considered significant.

3 Results

3.1 Stimulation of Isc by CCH

When bathed in normal K–H solution, the epididymal monolayer exhibited a transepithelial potential difference of 3.0±0.15mV (n=20 cultures), a basal Isc of 6.2±0.26μA/cm2 (n=12 cultures) and a transepithelial resistance of 475.2±20Ωcm2 (n=30 cultures). Fig. 1 shows the effects of CCH on Isc when it was added to the apical or basolateral side of the epithelium. Addition of 100μM CCH to the basolateral side led to an increase in Isc (Fig. 1A), while the effect of apically applied drugs was negligible (Fig. 1B), indicating that the effect of CCH on Isc was restricted to the basolateral aspect of the cells. In all subsequent experiments, CCH was added to the basolateral aspect of the cells. In normal K–H solution, the CCH elicited a biphasic response consisting of an initial spike followed by a delayed, but more sustained response. Fig. 2A shows the responses to increasing concentrations of CCH. The concentration–response curve (Fig. 2B) revealed an apparent EC50 value of 83.4μM and a maximum Isc response of 10.0±0.26μA/cm2 (first peak) and 7.84±0.74μA/cm2 (second peak). Experiments were also carried out to study the effects of repetitive stimulation with CCH on the Isc. Fig. 3 demonstrates that basolateral CCH (100μM) elicited an increase in Isc when first applied, but no effect was observed when the same concentration of the drug was added for the second time, possibly due to the desensitization of the receptors involved in mediating the CCH response.


Fig. 1

Isc response to CCH. CCH (100μM) was added to the apical side (A) and to the basolateral side (B) of the cultured epididymal epithelial monolayers. Summary of the effect is shown in (C), columns and bars are means±SE. (**P<0.01, compared with control, n=7).


Fig. 2

Effect of various concentrations of CCH on Isc response. (A) Representative Isc recordings from separate monolayers challenged with basolateral CCH at the concentration shown. (B) Concentration–response curves of CCH. Each data point is the means±SE. Each record is representative of four different experiments.


Fig. 3

Isc response to repetitive stimulation of CCH. Monolayer was first stimulated with basolateral application of CCH (100μM). When the current had returned to basal level, the monolayer was stimulated with a second basolateral addition of CCH (100μM, n=3).




3.2 Effect of Cl removal and Cl channel blocker on the Isc response to CCH

The Isc response to CCH was studied in the absence of extracellular Cl (replaced by gluconate). As shown in Fig. 4A and B, when epididymal monolayers were incubated in Cl-free solution, the Isc response to CCH was almost completely abolished (0.5±0.03μA/cm2, n=4, P<0.01). To investigate further the involvement of Cl secretion in mediating the Isc response to CCH, experiments were carried out to study the effects of the Cl channel blocker, DPC. Fig. 5 shows the Isc response to CCH was almost completely abolished after the tissue had been pretreated with 1mM DPC for 15min. These results suggested that the Isc response to CCH was largely mediated by Cl secretion.


Fig. 4

Cl-dependence of the Isc response to CCH. Isc recordings obtained from monolayer bath in normal K–H solution (A), in Cl-free solution (B), (C) summary of the effect under the different conditions (**P<0.01, compared with respective controls, n=4).


Fig. 5

Effect of DPC on CCH-induced Isc. The monolayer of epididymal cells was pretreated before (A) and after (B) with an apical addition of 1mM DPC 25min prior to the basolateral addition of CCH (100μM, n=4). (C) Summary of the effect of the DPC. Columns and bars are means±SE (**P<0.01, compared with control).



3.3 AchRs involved in the culture of rat epididymis

In order to find which CCH receptors are involved in the CCH-stimulated Isc response, we used the mAchRs antagonist atropine and nAchRs antagonist curarine, pretreated the cultured epididymis cells and then added 100μM CCH to the basolateral bathing solution. Fig. 6A shows the control Isc response without receptor antagonists pretreatment (5.0±0.21μA/cm2, n=7). Fig. 6B shows the effect of the atropine on Isc; addition of 1μM atropine to the basolateral bathing solution can block almost all the CCH-induced Isc response (0.1±0.002μA/cm2, n=4, P<0.001). However, curarine does not cause the same Isc response (3.3±0.20μA/cm2, n=4) (Fig. 6C).


Fig. 6

Effects of AchRs antagonists on Isc. The tissues were stimulated with CCH (100μM, basolateral) in the absence (A) and presence (B) of atropine (1μM, basolateral) or the presence (C) of curarine (5μM, basolateral). The tissues were pretreated with the two AchRs antagonists for about 15min, the addition of adrenaline (2.5μM, basolateral) lastly was in order to check the cells were still alive. (D) Summary of the effect of atropine and curarine. Columns and bars are means±SE. Each record is representative of at least four separate experiments (**P<0.01, compared with control).


The RT-PCR result (Fig. 7) shows the mAchRs subtypes in the cultured rat epididymal cells. The total RNA was obtained from the cauda epididymis. The DNA band corresponding to the m1 and m4 AchRs mRNA subtypes was amplified, showing that the m4 AchRs subtype gene has the predominant expression in rat cauda epididymis.


Fig. 7

RT-PCR analysis of mAchR mRNA in the rat epididymis. PCR products are shown in reactions using oligonucleoside primer pairs for mAchRs and GAPDH mRNA targets. DNA size markers are indicated on the right.


4 Discussion

The present study demonstrated that CCH stimulated an inward current in short-circuited cultured epididymal epithelia clamped in an Ussing chamber. This Isc response was likely caused by an increase in electrogenic chloride secretion from the basolateral side to the apical side of the epithelium. The supposition is that the Isc response to CCH was abolished by Cl channel blocker DPC. Extracellular HCO3 appeared to be little involved in the genesis of the Isc, as removing extracellular chloride without changes to HCO3 almost completely abrogated the CCH-stimulated current. In order to observe whether Na+ absorption from the apical to the basolateral side of the epithelium was involved in the CCH-induced Isc response, we added the epithelial Na+ channel blocker, amiloride, to the apical side and found this did not change the CCH-stimulated Isc response (data not shown). The above results show that the response of the cultured rat epididymal epithelia to basolateral application of CCH was largely attributable to Cl secretion.

We also investigated the sidedness of the CCH response. Only basolateral application but not apical application of CCH stimulated Cl secretion. This demonstrated that the AchRs mediating the response resided on the basolateral side of the epithelium where they may interact with Ach released from nerve endings or from the circulating blood. The present study also showed that the mAchRs but not the nAchRs were involved in the Isc response as atropine, blocker of mAchR but not curarine, blocker of the nAchR, abolished the response. To investigate further the mAchRs subtypes involved, RT-PCR was used to detect mRNA for the receptor subtypes. In that we found m1 and m4 AchRs expressed in the rat cauda epididymal cells in culture. Our results differ from that of Marostica et al.'s (2001) work. In their work, m2 and m3 AchRs were detected in the cauda epididymis. There may be two reasons for this discrepancy: firstly, Wistar rats were used in Marostica's work, but SD rats were used in our work; secondly, the RNA was extracted from acute-dissected entire epididymis in Marostica's work, but it was extracted from cultured epididymal epithelium in our work.

Each of these two receptor subtypes may mediate one of the two phases (fast vs sustained) of the Isc response to CCH. The phenomenon that each receptor subtype mediates one of the components of the biphasic Isc response has been observed for the adrenaline-stimulated Cl secretion in the epididymis (Leung et al., 1992).

In many other epithelia, such as the rat olfactory bulb (Onali and Olianas,1995, CCH directly stimulates basal adenylyl cyclase activity, hence elevates intracellular camp, which activates cAMP-dependent Cl channel. In T84 cells (Dickinson et al., 1992; Kachintorn et al., 1992; Barrett et al., 1998) and airway epithelium (Nahorski et al., 1997; Zaagsma et al., 1997; Billington and Penn, 2002), mAChRs regulate the activation of phospholipase C (PLC), which induces protein kinase C (PKC) activation and inositol 1,4,5-trisphosphate (IP3) generation. The latter serves to increase intracellular Ca2+, which opens the Ca2+-activated Cl channels. Some other neurotransmitters, such as adrenaline, have been shown to increase intracellular cAMP and Ca2+ concentration concurrently through beta- and alpha-adrenergic receptors, respectively, giving rise to a biphasic stimulation of chloride secretion (Leung et al., 1992; Leung and Wong, 1994; Wong, 1988; Huang et al., 1992; Huang et al., 1993; Huang et al., 1994).

Previous studies have indicated that the receptors for neurotransmitters of the autonomic nervous system play important roles in the modulation of different physiological functions. The present studies demonstrate that there are two receptor subtypes for Ach on the basolateral side of the epididymis. Each one can stimulate secretion by activating different chloride channels (e.g. CFTR or Ca2+-activated chloride channels). It would be of interest to investigate whether the m1 receptor, which is linked to Ca2+ signaling, is responsible for the fast initial response, whereas the m4 receptor, which is linked to cAMP signaling, is responsible for the sustained response. Ach released from nerve terminals may act on m1 and m4 AChRs on the epithelium to stimulate Cl secretion (El-Badawi and Schenk, 1967a,b). The neurohumoral control of Cl secretion may play an important role in the maintenance of a unique and specific microenvironment on which the maturation and storage of spermatozoa depend (Jenkins et al., 1980). Thus, mAChRs may play an important role in the maintenance and/or regulation of epididymal and sperm functions.

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 their suggestions. This work was supported by the National Natural Science Foundation of China (No. 3037 0540).

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Received 5 March 2006/30 March 2006; accepted 11 May 2006

doi:10.1016/j.cellbi.2006.05.008


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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