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Cell Biology International (2006) 30, 741746 (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
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.
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 120–150
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; CaCl
2.4 RNA Isolation and RT-PCR
Total RNA was isolated using TRIzol reagent (Invitrogen, USA), and 2
Reactions were carried out with the following parameters: denaturation at 94
2.5 Data analysis
Results are expressed as means
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
Isc response to CCH. CCH (100
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
Isc response to repetitive stimulation of CCH. Monolayer was first stimulated with basolateral application of CCH (100
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
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
Effect of DPC on CCH-induced Isc. The monolayer of epididymal cells was pretreated before (A) and after (B) with an apical addition of 1
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 100
Effects of AchRs antagonists on Isc. The tissues were stimulated with CCH (100
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.
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.
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 HCO
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 (IP
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.
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 2006doi:10.1016/j.cellbi.2006.05.008