J Physiol Volume 514, Number 3, 809-820, February 1, 1999
The Journal of Physiology (1999), 514.3, pp. 809-820
© Copyright 1999 The Physiological Society
Regulation of anion secretion by cyclo-oxygenase and prostanoids in cultured epididymal epithelia from the rat
P. Y. D. Wong, H. C. Chan, P. S. Leung, Y. W. Chung, Y. L. Wong, W. M. Lee *, V. Ng * and N. J. Dun ¹
Department of Physiology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, *Department of Zoology, University of Hong Kong, Pokfulam Road, Hong Kong and ¹Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA
MS 8590 Received 4 August 1998; accepted after revision 16 October 1998.
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ABSTRACT |
- The role of cyclo-oxygenase (COX) in the regulation of anion secretion (measured as short- circuit current, Isc) in cultured epididymal epithelia from immature rats was investigated.
- COX inhibitors attenuated the increase of anion secretion caused by bradykinin (LBK) but had no effect on that caused by PGE2, suggesting that prostaglandin synthesis mediates the secretory response of the tissues to LBK.
- The apparent IC50 values for indomethacin, piroxicam and L-745,337 in inhibiting the LBK-induced Isc were 0·14, 1·34 and 15·7 µM, respectively. This order of potency: indomethacin > piroxicam > L-745,337 >> DFU suggests the involvement of the COX-1 isozyme in the mediation of the secretory response to LBK.
- Among the COX products (prostaglandins, thromboxane and prostacyclins) tested, only PGE2 and, to a much lesser extent, PGF2
stimulated anion secretion by cultured rat epididymal epithelia.
- The effect of PGE2 was mimicked by 11-deoxyl PGE1, a specific prostaglandin E (EP)2/4 receptor agonist, but not by sulprostone, a specific EP1/3 receptor agonist, indicating that cyclic AMP-coupled EP2/4 receptors are involved in the LBK-stimulated anion secretion.
- A reverse transcriptase-polymerase chain reaction study detected the expression of COX-1 and COX-2 mRNA in intact rat epididymis and in cultured epididymal epithelia. The expression of COX-1 mRNA was reduced by LBK by 44 %.
- Immunohistochemical studies demonstrated the presence of COX-1 immunoreactivity in the basal cells of the intact rat epididymis. By comparision, COX-2 immunoreactivity was detected in the apical pole of the principal cells.
- The role of COX in the formation of the epididymal microenvironment and the implication of long term administration of non-steroidal anti-inflammatory drugs (NSAIDs) on male fertility are discussed.
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INTRODUCTION |
Prostanoids are paracrine hormones that have been shown to regulate many physiological processes. They are derived from arachidonic acid released from membrane lipids in response to a variety of humoral and neural signals. Prostaglandin synthetase catalyses the conversion of arachidonic acid to prostaglandin H2 (PGH2) which serves as the common precursor for the synthesis of prostaglandins, prostacyclins and thromboxanes. PGH2 synthesis by prostaglandin synthetase occurs as a two-step reaction, the conversion of arachidonic acid to PGG2 (cyclo-oxygenase activity) followed by the reduction of the hydroperoxide moiety of PGG2 to yield PGH2 (peroxidase activity), which is then transformed to the prostaglandins (PGE2, PGF2a, PGD2), prostacyclins (PGI2) and thromboxanes by the respective isomerases (see Herschman, 1996).
Previous work has provided evidence that the prostanoid biosynthetic pathway mentioned above plays an important role in the regulation of electrolyte secretion by the rat epididymis. Various vasoactive peptide hormones such as bradykinins (Cuthbert & Wong, 1986), angiotensins (Wong et al. 1990), arginine vasopressins (Lai et al. 1994) and endothelin (Wong et al. 1989) stimulate chloride and bicarbonate secretion (by inference water secretion) by the epididymal epithelium and, by doing so, contribute to the formation of the epididymal microenvironment. The secretory responses to these peptides were abolished if the tissues were pretreated with piroxicam or indomethacin, inhibitors of cyclo-oxygenase (COX) (see references above), suggesting that the effects of vasoactive peptides may be mediated by the formation of prostanoids.
Two forms of COX are now found to be expressed by mammalian cells (Vane et al. 1994). COX-1, which is ubiquitously distributed, mediates many physiological functions and is therefore thought to be involved in the normal processes of homeostasis (DeWitt et al. 1990). COX-2, which is expressed by the macrophages, synoviocytes and endothelial cells, is thought to mediate the process of inflammation (Seibert et al. 1994). Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit both isozymes to varying degrees and with varying specificities (Chan et al. 1995; Riendeau et al. 1997). It is now recognized that inhibition of COX-1 by the NSAIDs gives rise to the undesirable side effects such as ulceration and bleeding of the gastro-intestinal tract and impaired renal function, whereas inhibition of COX-2 contributes to the anti-inflammatory effects (Vane, 1994). Whilst the effects of chronic NSAIDs administration on male reproductive function remain largely unknown, we have investigated the role of cyclo-oxygenase and its products in the regulation of anion secretion by cultured rat epididymal epithelia.
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METHODS |
Culture of rat epididymal epithelium
The method used in preparing rat epididymal epithelial cultures has been described previously (Cuthbert & Wong, 1986; Wong, 1988a). Immature male Sprague-Dawley rats (150 g) were killed by asphyxiation with a rising concentration of CO2. The epididymides were dissected, cut into small fragments and placed in sterile Hanks' balanced salt solution (HBSS) containing 0·25 % trypsin. After gentle shaking for 30 min at 32°C, tissues were separated by low-speed centrifugation (800 g, 5 min). The supernatant was discarded and the pellet resuspended in HBSS containing collagenase (1 mg ml-1) for 75 min at 32°C, again with gentle shaking. Cells were separated by centrifugation at 800 g for 5 min. The pellet was resuspended in Eagle's minimum essential medium (EMEM) (ICN Biomedicals Inc., Costa Mesa, CA, USA) containing non-essential amino acids (0·1 mM), sodium pyruvate (1 mM), glutamine (4 mM), 5--dihydrotestosterone (1 mM), 10 % fetal calf serum, penicillin (100 i.u. ml-1), streptomycin (10 µg ml-1) and collagenase (0·25 mg ml-1). This suspension was allowed to stand for 4 h, during which time non-epithelial cells such as fibroblasts and smooth-muscle cells attached rapidly to the plastic surface while epididymal cells remained unattached. The resulting, decanted cell suspension freed from non-epithelial cells was then seeded into the wells of the Millipore filter assemblies (using a cell suspension of 105 cells ml-1) to form monolayers of epididymal cells (area 0·4 cm2) (Cuthbert & Wong, 1986; Wong, 1988a). Generally, monolayers reached confluency after 3 days in culture. Confluent monolayers were used for short-circuit current recording or for the determination of COX-1 or COX-2 mRNA as described below.
Measurement of electrogenic anion secretion
Confluent epididymal monolayers, area 0·4 cm2, were clamped between two halves of Ussing chambers with a 0·6 cm2 window as described previously (Cuthbert & Wong, 1986; Wong, 1988a). Epithelia were short-circuited with a dual voltage clamp (WPI, New Haven, CT, USA) and short-circuit current (Isc) was displayed continuously on pen recorders (Kipp and Zonen, Pelft, The Netherlands). Transepithelial conductance was measured by transiently commanding the clamp to set the voltage at 0·2 or 0·4 mV away from zero. The resulting changes in transepithelial current allowed calculation of conductance using Ohm's law. Monolayers were bathed on both sides with Krebs-Henseleit (K-H) solution (20 ml; composition given below), gassed with 95 % O2-5 % CO2, and warmed to 33°C. Drugs were added directly to the reservoirs connected to either the apical or basolateral surface of the tissue. Generally, when bathed in K-H solution, monolayers have a transepithelial potential of about 2 mV, apical side negative, and a transepithelial resistance of about 500 
cm2 (Wong, 1988a). The latter value was used to check the integrity of the monolayers. Monolayers with a transepithelial resistance less than this value were discarded.
To study the concentration-inhibition curves for the NSAIDs, epithelia from a batch of cells were used to test different concentrations of the inhibitor. A fresh epithelium was used each time to avoid deterioration resulting from repeated washing. The response following each concentration of the inhibitor was compared with the control response to LBK from the same batch of cells. The percentage inhibition was worked out for each dose and a concentration-inhibition curve was constructed. The IC50 value was estimated from the concentration-inhibition curve. This protocol was repeated 4-5 times using different batches of cells and the IC50 values obtained from individual curves were used to calculate the mean IC50 value for each inhibitor. To obtain the data shown in Fig. 2, the mean percentage inhibition at each concentration of the inhibitor was calculated from values obtained from four or five experiments.
Detection of COX-1 and COX-2 mRNAs in epididymal cells by reverse transcription-polymerase chain reaction (RT-PCR)
RT-PCR was performed to detect expression of COX-1 and COX-2 in intact cauda epididymides from 150 g Sprague-Dawley rats and cultured epididymal cells. The two primers used for amplifying COX-1 were: 5'-TTTGCACAACACTTCACCCACCAG-3' (sense primer corresponding to nucleotides 659-681) and 5'-AAACACCTCCTGGGCCACAGCCAT-3' (antisense primer corresponding to nucleotides 911-934) (Feng et al. 1993), which generated a COX-1 PCR product of 277 bp. The two primers for COX-2 were: 5'-ACTTGCTCACTTTGTTGAGTCATTC-3' (sense primer corresponding to nucleotides 1336-1360) and 5'-TTTGATTAGTACTGTAGGGTTAATG-3' (antisense primer corresponding to nucleotides 1894-1918), which generated a 589 bp COX-2 PCR product. The ribosomal protein S-16 was used as an endogenous control and co-amplified in the PCR reactions. The two primers used for amplifying S-16 were: 5'-TCCGCTGCACTGGCTTCAAGTCTT-3' (sense primer corresponding to nucleotides 15-38) and 5'-GCCAAACTTCTTGGATTCGCAGCG-3' (antisense primer corresponding to nucleotides 376-399) (Chan et al. 1990), which yielded a S-16 product of 385 bp. Total RNA was extracted from rat primary epididymal cell culture using TRIzol Reagent (Gibco BRL). The quality and concentration of RNA was determined by gel electrophoresis and spectrophotometry at 260 nm. Two micrograms of total RNA and 1 µg of oligo(dT)-18 primer were denatured at 70°C for 5 min, annealed on ice for 5 min, and then reverse transcribed to cDNA by 40 U of MMLV (Moloney murine leukaemia virus) reverse transcriptase (Promega, Madison, WI, USA) in the presence of 28 U of RNasin (Promega), 2 µl dithiothreitol (0·1 M), 2 µl deoxynucleoside triphosphates (10 mM each of dATP, dCTP, dGTP and dTTP) in a final reaction volume of 25 µl for 2 h at 3°C. The reaction was terminated by heating to 70°C for 5 min and followed by RNase H treatment (0·1 U µl-1, Promega) at 3°C for 20 min. cDNA (0·5 µg) was used as a template for performing PCR using the following parameters: denaturation at 94°C for 1 min, annealing at 61°C for 2 min, and extension at 72°C for 3 min. A total of 20-23 cycles were performed. Under these conditions, the amplication of both the target gene and S-16 were in the linear range as demonstrated in a series of preliminary experiments by removing samples for analysis at cycles 16, 18, 20, 22, 24, 28 and 30. An aliquot of 5-10 µl sample was withdrawn from each PCR reaction tube, resolved in 0·8 % agarose gels in 0·5 × TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8·0 at 22°C) and visualized by ethidium bromide. The authenticity of the COX-1 and COX-2 PCR products was confirmed by nucleotide sequencing (AutoCycle Sequencing kit, Pharmacia LKB Biotechnology, Uppsala, Sweden) of the 277 bp and 589 bp fragments (ALF Express Autosequencer, Pharmacia LKB Biotechnology). For Southern blot analysis, PCR products resolved in agarose gel were transferred by capillary blotting to a nylon membrane (Amersham Life Science) and hybridized at 65°C in hybridization solution containing the cloned COX-1 or COX-2 fragment labelled with [-32P]dCTP by random priming as previously described (Lee et al. 1997). Radioactivity of the hybridized PCR fragments was measured by Phosphorimaging (STORM 860, Molecular Dynamics, Sunnyvale, CA, USA) and visualized by autoradiography using Kodak X-OMAT AR films.
Immunohistochemical detection of COX-1 and COX-2 proteins
Adult male Sprague-Dawley rats (Harlan, Indianapolis, IN, USA) weighing 150-200 g were used. Immunohistochemical procedures employed in this study were similar to those described previously (Dun et al. 1996). Rats were anaesthetized with ketamine (70 mg kg-1, I.P.) and perfused intracardially with chilled 0·1 M phosphate-buffered saline followed by freshly prepared 4 % paraformaldehyde. Epididymis was removed and postfixed for 3 h and cryoprotected in 30 % sucrose-phosphate-buffered saline. Tissues sectioned to 30 µm with the use of a cryostat were processed for COX-1- and COX-2-like immunoreactivity by means of the standard avidin- biotin complex techniques as described (Dun et al. 1996). Tissues were first treated with 3 % H2O2 to quench endogenous peroxidase, washed several times and blocked with 10 % normal goat serum. Tissues were then incubated in either prostaglandin H synthase 1 monoclonal antibody (1:100 with 0·4 % Triton X-100 in phosphate-buffered saline) or prostaglandin H synthase 2 rabbit polyclonal antisera (1:200 with 0·4 % Triton X-100 in phosphate-buffered saline) for 48 h at 4°C with gentle agitation. Both primary antibodies were from Cayman Chemical (Ann Arbor, MI, USA). After thorough rinsing, sections were incubated with biotinylated anti-mouse or anti-rabbit IgG for 2 h. Sections were rinsed with phosphate-buffered saline and incubated in avidin-biotin complex solution (1:100, Vector Laboratories) for 1 h. After several washes in Tris-buffered saline, sections were developed in diaminobenzidine-H2O2 solution and washed for at least 2 h with Tris-buffered saline. Sections were mounted on slides with 0·25 % gel alcohol, air-dried, dehydrated with absolute alcohol followed by xylene and coverslipped with Permount.
In control experiments, randomly selected epididymis sections from each rat were processed without the primary antibodies.
Solutions and drugs
Krebs-Henseleit solution used in the Isc study had the following composition (mM): NaCl, 118; KCl, 4·7; CaCl2, 2·5; MgSO4, 1·8; KH2PO4, 1·8; NaHCO3, 25·0; and glucose, 14·0. This solution had a pH of 7·3-7·4 when bubbled with 95 % O2-5 % CO2. Most of the solutions and chemicals used in culture were obtained from the usual commercial sources. The origins of the more unusual materials were as follows: lysylbradykinin (LBK) and indomethacin were from RBI Research Biochemicals; piroxicam, PGE2, PGF2 and PGD2 were from Sigma; U-46619, L-745,337, cicaprost, 11-deoxyl PGE1 and sulprostone were gifts from Professor R. Jones (Department of Pharmacology, The Chinese University of Hong Kong); 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone (DFU) was gift from Merck Frosst Canada Inc. (Quebec, Canada)
Where possible, concentrated solutions of these agents were made in distilled water, but piroxicam and DFU were dissolved in DMSO; indomethacin and L-745,337 in methanol; and PGE2, PGF2, PGD2, 11-deoxyl PGE1 and sulprostone in ethanol. In all cases, solvent alone did not affect the Isc.
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RESULTS |
Effect of NSAIDs on bradykinin-stimulated anion secretion
Stimulation of anion (chloride and bicarbonate) secretion in the epididymis by the vasoactive peptides has been shown to be blocked by pretreating the tissues with piroxicam or indomethacin (see Introduction for references), which are inhibitors of cyclo-oxygenase. In order to investigate which COX isozyme is involved in the anion secretory response to the peptides, experiments were conducted with four NSAIDs with known IC50 values for COX-1 and COX-2 obtained from standard assays (Riendeau et al. 1997).
Figure 1A-D shows the short-circuit current (Isc) records of four 4-day-old cultured rat epididymal epithelia. In each epithelium, LBK (0·5 µM) added to the basolateral bathing solution caused an inward-flowing current which has been shown to be attributed to an increase in electrogenic anion secretion (Cuthbert & Wong, 1986). Indomethacin (A), piroxicam (B), L-745,337 (C) and DFU (D) caused a dose-dependent inhibition of the Isc response to LBK. The concentration-inhibition relationships of the four NSAIDs are shown in Fig. 2. The control response to LBK was 4·02 ± 0·17 µA cm-2 (n = 4) for indomethacin, 3·69 ± 0·18 µA cm-2 (n = 5) for piroxicam, 3·45 ± 0·17 µA cm-2 for L-745,337 (n = 5) and 3·58 ± 0·18 µA cm-2(n = 5) for DFU, respectively (values not significantly different). The concentration-inhibition curves for indomethacin, piroxicam and L-745,337 are parallel, with apparent IC50 values of 0·14 ± 0·01 µM (n = 4), 1·34 ± 0·08 µM (n = 5), and 15·7 ± 1·13 µM (n = 5), respectively. (Each value is significantly different from its immediate neighbour at P < 0·01.) DFU was least efficacious in inhibiting the LBK-induced Isc response; less than 30 % inhibition was achieved at 1000 µM. Thus, the relative efficacy of the four NSAIDs in inhibiting the LBK-induced response is indomethacin > piroxicam > L-745,337 >> DFU.
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Figure 1. Effects of indomethacin (A), piroxicam (B), L-745,337 (C), and DFU (D) on bradykinin (LBK)-induced Isc in cultured rat epididymal epithelia
All agents were added to the basolateral side of the epithelium. Each record is representative of 4-5 experiments.
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Effect of piroxicam on the PGE2-stimulated Isc
The inhibitory effect of the NSAIDs on the LBK-stimulated anion secreton shown in Fig. 1 could be attributed to the inhibition of the enzyme cyclo-oxygenase, whose major product is prostaglandin E2 which has previously been shown to stimulate anion secretion in the rat epididymis (Wong, 1988a). For this reason, the effects of NSAIDs on PGE2-induced Isc were investigated. Figure 3 shows an epithelium stimulated successively by LBK (0·1 µM) and PGE2 (0·5 µM). The response to either agent consisted of a rapid rise to a peak followed by a decline to a lower but sustained level. The responses to LBK and PGE2 were not additive (Fig. 3). The 
Isc produced by LBK (0·1 µM) alone was 4·75 ± 0·19 µA cm-2 (n = 6) and that by PGE2 (0·5 µM) was 5·03 ± 0·21 µA cm-2 (n = 6). The Isc achieved when both agents were present was 4·92 ± 0·22 µA cm-2 (n = 6) (not significantly different from either LBK or PGE2 alone). Pretreatment of the tissue with piroxicam (10 µM) abolished the response to LBK but not that to PGE2. The effect of piroxicam on LBK was reversible upon washing out the drug (Fig. 3).
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Figure 3. Effects of piroxicam on Isc response in a single epididymal monolayer
Epithelium was stimulated first with LBK (0·1 µM) then with PGE2 (0·5 µM), both added to the basolateral side. After washing, both agents were added again in the presence of piroxicam (10 µM) added to the basolateral side. Finally, after washing, control responses were repeated. The record is representative of 4 experiments.
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Identification of the COX products eliciting the LBK response
It is known that COX converts arachidonic acid into PGG2 then to PGH2 which is the common precursor for the prostanoids, namely the prostaglandins, thromboxane and prostacyclins. To identify which COX product(s) may be responsible for the anion secretory response to the vasoactive peptides, we have studied the effects on chloride secretion by cultured rat epididymal epithelium of externally applied PGE2, PGD2, PGF2, U-46619 and cicaprost, which are specific agonists for prostaglandin E (EP), prostaglandin D (DP), prostaglandin F (FP), thromboxane (TP) and prostacyclin (IP) receptors (Coleman et al. 1994). The results are shown in Fig. 4. U-46619, cicaprost and PGD2 at two consecutive doses did not affect the Isc of the epididymal epithelia, although the tissues responded to basolaterally applied PGE2 (0·1 µM) with an increase in inward current (Fig. 4A-C). This effect of PGE2 is attributable to an increase in anion (Cl- and HCO3-) transport from the basolateral to the apical compartment, since addition of diphenylamine-2-carboxylate (DPC; 1 mM), an anion channel blocker (Wong, 1988b), to the apical side caused an inhibition of the current. PGF2 added basolaterally produced a dose-dependent increase in the Isc, although the magnitude of the response was much smaller than that of PGE2 (Fig. 4D).
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Figure 4. Effects of U-46619 (A), cicaprost (B), PGD2 (C), PGE2 (A-C) and PGF2 (D) on anion secretion by cultured rat epididymal epithelia
All agents were added to the basolateral bathing solution except DPC which was added apically (ap). Each record is representative of 4 experiments. TP, thromboxane receptor; IP, prostacyclin receptor; DP, prostaglandin D receptor; FP, prostaglandin F receptor; EP, prostaglandin E receptor.
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The results of these experiments suggest that the active COX product responsible for the stimulation of anion secretion by LBK is PGE2 acting on the prostaglandin E (EP) receptors. A systematic study of PGE2 on anion secretion was undertaken. Figure 5 shows the concentration- response relationship of PGE2 added basolaterally and apically. Basolateral application caused greater responses, with an apparent EC50 at 0·04 µM and the threshold concentration at 0·01 µM. The maximal effect was achieved at 0·1 µM. Responses to apical application were smaller, with an apparent EC50 at 2 µM, and the maximal response was only 40 % of that produced by basolateral PGE2. The effect of basolaterally applied PGF2 is also shown in Fig. 5. The maximal response was only 20 % of that produced by PGE2.
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Figure 5. Concentration-response curves for PGE2 and PGF2
PGE2 was added basolaterally and apically and PGF2 was added basolaterally. Each point shows the mean ± S.E.M. of 5 experiments.
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Identification of prostaglandin E (EP) receptor subtypes
Having established that the active COX product which mediates the secretory response to LBK is likely to be PGE2, further experiments were performed to investigate the type of EP receptor subtypes that may be involved. EP receptors are known to be classified into at least four subtypes, viz. EP1, EP2, EP3 and EP4. Effects of the specific EP2/4 receptor agonist 11-deoxyl PGE1 and the specific EP1/3 agonist sulprostone were evaluated here. 11-Deoxyl PGE1 (0·2 µM) caused an increase in Isc (Isc = 3·92 ± 0·23 µA cm-2, n = 4, Fig. 6A) with a time course similar to that caused by PGE2 and LBK. The response was biphasic consisting of an initial peak followed by a secondary plateau phase. During the latter, addition of DPC (1 mM) to the apical bathing solution caused an inhibition of the current to the basal level. Sulprostone (0·1 or 0·2 µM) did not affect the Isc in rat epididymal epithelia (Fig. 6B).
Expression of COX-1 and COX-2 in rat epididymal epithelia
RT-PCR was used to assess whether COX-1 and COX-2 are expressed in the epididymis. A 277 bp PCR product corresponding to the expected size of COX-1 and a 589 bp PCR product corresponding to COX-2 were found in the intact rat cauda epididymides (Fig. 7) and in cultured rat epididymal cells (Fig. 8). The lung tissue was used as the positive control. The authenticity of these PCR products was confirmed by Southern blot analysis (Figs 7 and 8) and verified by direct nucleotide sequencing in a separate experiment (data not shown). To assess the effect of bradykinin on cultured epididymal cells, expression levels of COX-1 and COX-2 were quantified by phosphor imaging analysis and normalized by S-16 expression detected in the same PCR reaction. Treatment with bradykinin (0·5 µM for 10 min) was found to inhibit the COX-1 mRNA to 55·75 ± 6·07 % of the basal level (P < 0·01, Student's t test). The COX-2 mRNA was not significantly affected by bradykinin (Fig. 8).
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Figure 7. Expression of COX-1 and COX-2 in rat cauda epididymides
RT-PCR was performed using specific primers to give a 277 bp COX-1 fragment and a 589 COX-2 fragment (upper panel). COX-1 and COX-2 were co-amplified with S-16. Lung tissue was used as a positive control since it was known to express both COX-1 and COX-2. The authenticity of the fragments was confirmed by Southern blotting (middle and lower panels) and base sequencing (data not shown). M, 100 bp DNA ladder; Epid, epididymis tissue; Con, PCR control; RTCon, reverse transcription control with no reverse transcriptase added.
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Figure 8. Effect of bradykinin (LBK) on COX-1 and COX-2 steady-state mRNA levels in cultured rat epididymal cells
RT-PCR of COX-1 and COX-2 were performed on cultured epididymal cells. COX-1 and COX-2 were co-amplified with S-16 (A, upper panel). The authenticity of the fragments was confirmed by Southern blotting (A, middle and lower panels). The amount of COX-1 and COX-2 expression was measured by phosphor imaging and normalized against S-16 detected in the same reaction. B shows mean ± S.E.M. percentage COX expression from 3 independent experiments. M, 100 bp DNA ladder; +LBK or -LBK, with or without addition of LBK, repectively.
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Localization of COX-1 and COX-2 in the intact rat epididymis
To gain insight into the distribution of COX-1 and COX-2, epididymides from five rats (corresponding in weight to the animals used in the Isc studies) were processed for either COX-1 or COX-2 immunohistochemistry. COX-1- and COX-2-like immunoreactivities were localized to the epithelium but they appeared to differ in their cellular localization (Fig. 9). COX-1-like immunoreactivity was concentrated in the cells at the periphery of the epithelium, identifed as basal cells (Fig. 9A-C), while COX-2-like immunoreactivity appeared to be localized in the apical border of the principal cells (Fig. 9D-F). This pattern of distribution of COX-1 or COX-2 immunoreactivity was noted throughout the caput, corpus and cauda.
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Figure 9. Photomicrographs of sections through the rat epididymis labelled with COX-1 or COX-2 antisera
A, low magnification of a section of the rat cauda epididymis labelled with COX-1 antibody; immunoreactivity is limited to the epithelial cells. B and C, higher magnification showing the presence of COX-1-like immunoreactivity in basal cells. D, low magnification of a rat caput section labelled with COX-2 antisera; immunoreactivity is concentrated in epithelial cells. E and F, higher magnification showing the presence of COX-2-like immunoreactivity at the luminal pole of the principal cells. Calibration bars: 500 µm for A and D; 100 µm for B and E; 25 µm for C and F.
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In control experiments where the antibodies were omitted in the staining procedures, positive labelling was not detected in the epithelial cells (not shown).
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DISCUSSION |
Regulated secretion of electrolytes and fluid by the epithelial cells lining the epididymal tubule is an important process that leads to the formation of a correct milieu for sperm maturation and storage (Wong et al. 1992). In common with the other secretory epithelia, fluid secretion in the epididymis is driven by anion secretion which involves the interplay of ion channels, ion pumps and transporters, among which an apical cAMP-activated anion channel or cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role (Huang et al. 1992; Chan et al. 1994; Leung & Wong, 1994). In cystic fibrosis, because of the mutation of the CFTR gene, the CFTR protein is aberrantly expressed, resulting in abnormal channel operation and regulation (Leung et al. 1996). Since CFTR is expressed by the fetal epididymis (Harris et al. 1991; Pollard et al. 1991), decreased fluid secretion by the Wolfian duct due to CFTR defect has been implicated in the agenesis or absence of the epididymis/vas deferens seen in CF men (Wong, 1998).
Secretion of anions and fluid by the epididymal epithelial cells is controlled by vasoactive peptides such as angiotensins, endothelin, arginine vasopressin and bradykinins (for references, see Introduction). These peptides function in a paracrine fashion to regulate electrolyte and fluid secretion by the epididymal epithelium. A common feature shared by these peptides is that their effects can be blocked by the non-steroidal anti-inflammatory drugs (NSAIDs), which are inhibitors of prostaglandin synthesis, suggesting that prostaglandins are the common mediators of the peptide responses. The male reproductive tract is known to be a rich source of prostaglandins (Badr, 1976) and a role for prostaglandins in male reproduction has been well documented. Prostaglandin E and prostaglandin F have been shown to affect sperm metabolism and functions (Breitbart et al. 1995; Herrero et al. 1997; Kelly & Critchley, 1997), and enhance the contractility of the smooth muscle layer surrounding the epididymal tubule (Hib & Oscar, 1978; Cosentino et al. 1984). Intratesticular injection of prostaglandins (Rej & Chatterjee, 1980) or Silastic implants bearing indomethacin (Ratnasooriya & Wadsworth, 1987) have been shown to suppress fertility in rats by mechanisms not entirely known. Our present experiments demonstrated that among the prostanoids tested, only PGE2, and to a much lesser extent PGF2, stimulate electrogenic anion secretion (driving force for water secretion) by the epididymis. An increased blood-to-lumen flow of fluid produced by these agents affects the physico-chemical properties of the milieu in which sperm are bathed (Wong, 1986). The prostaglandins, through regulation of the luminal microenvironment, may therefore have an important influence on sperm maturation and fertility.
To delineate which COX isozyme is involved in the mediation of the secretory responses to the peptides, we conducted experiments with indomethacin, piroxicam, L-745,337 and DFU with known apparent selectivities towards COX-1 and COX-2 from a variety of assay systems (Riendeau et al. 1997). Generally, indomethacin is an effective COX-1 inhibitor (see Vane, 1994), whereas L-745,337 (Chan et al. 1995) and DFU (Riendeau et al. 1997) are relatively COX-2 specific. All four compounds inhibited the LBK-induced Isc response in the cultured rat epididymal epithelia. Indomethacin, piroxicam and L-745,337 produced parallel concentration-inhibition curves (Fig. 2) with apparent IC50 values of 0·14, 1·34 and 15·7 µM, respectively. DFU, a new, selective COX-2 inhibitor (Riendeau et al. 1997) was the least effective in inhibiting the LBK-induced Isc response, with only 30 % inhibition observed at 1000 µM (Fig. 1). These IC50 values lie within an order of magnitude of the IC50 reported for the respective agents for inhibiting COX-1 but are a few orders of magnitude away from the IC50 for inhibiting COX-2, when enzyme activities were measured by the inhibition of arachidonic acid-dependent PGE2 production in Chinese hamster ovarian (CHO) cells stably transfected with human COX-1 and COX-2 (Riendeau et al. 1997). The concentration-inhibition curve for DFU obtained in our study also mirrored that for DFU in the CHO cell assay for COX-1 (Riendeau et al. 1997). These findings suggest that COX-1 is likely to be the isoform mediating the secretory response to LBK and probably other peptides, and are therefore in line with the premise that COX-1 is involved in many physiological processes of homeostasis. Regulation of the epididymal microenvironment by the prostaglandins is undoubtedly one of the important homeostatic processes of reproduction.
Having established that it is the COX-1 isoform which mediates the anion secretory response to the peptides, we then elucidated which COX product(s) is (are) responsible, and, furthermore, through which receptors. We have studied the effects on chloride secretion by cultured rat epididymal epithelia of externally applied PGE2, PGD2, PGF2, U46619 and cicaprost, which are specific agonists for prostaglandin E, prostaglandin F, thromboxane and prostacyclin receptors, respectively. Among them only PGE2 and, to a much lesser extent, PGF2 could mimick the effects of the hormones. The results therefore show that the active prostanoid is likely to be PGE2 acting on the prostaglandin E (EP) receptors. The small response to PGF2 may be due to PGF2 acting on the FP or the EP receptors. At present it is not possible to discriminate between these possibilities, but suffice it to say that the magnitude of the PGF2 response is probably too small to be of significance.
The mechanisms by which LBK and the other peptides increase PGE2 production is unknown. LBK can stimulate PGE2 production either by stimulating the receptor-coupled phopholipase A2 (PLA2) to increase the intracellular pool of arachidonic acid, a substrate of COX, or by increasing COX activity through upregulating the COX gene. Our experiments, however, showed that the expression of COX-1 mRNA was not increased but actually decreased by LBK (Fig. 8), suggesting that the rate-limiting step in the biosynthesis of PGE2 in the epididymis is the formation of arachidonic acid by the PLA2-mediated breakdown of membrane phospholipids. The downregulation of COX-1 mRNA by LBK (by about 40 %) is unknown but may reflect a feedback inhibition on the enzyme by endogenous PGE2 or cAMP. The present study showed that the intact rat epididymis and cultured rat epididymal cells express COX-1 mRNA and also COX-2 mRNA (Fig. 7) and that in the intact rat epididymis COX-1 immunoreactivity appears to be located in the basal cells (Fig. 9A-C), whereas the COX-2 immunoreactivity is present in the apical pole of the principal cells (Fig. 9D-F). The constitutive expression of COX-2 mRNA and protein is not peculiar to the epididymis as expression of COX-2 is also found in the lung, testis, brain and prostate under basal conditions (Tippetts et al. 1988; O'Neill & Ford-Hutchinson, 1993). At present, the role of the constitutive COX-2 in these organs, including the epididymis, remains to be established.
Using specific EP receptor agonists, we further showed that the physiological responses to the peptides and PGE2 were reproduced by 11-deoxy PGE1, a specific EP2/4 receptor agonist, but not by sulprostone, a specific EP1/3 receptor agonist (Fig. 6). Given that the former receptors are linked to the cAMP-protein kinase A signal transduction pathway and the latter to the Ca2+-IP3 pathway, respectively (see citations in Coleman et al. 1994), our results indicate that the peptide hormones regulate electrolyte and fluid secretion by the epididymis via the cAMP signal transduction pathway. This contention was supported by previous reports from our laboratory that LBK and PGE2 increase intracellular cAMP in cultured rat epididymal cells (Wong & Huang, 1990). Furthermore, the Isc responses to PGE2 and LBK mimick that of cAMP in that they exhibit a biphasic time course consisting of an initial rise to a peak level followed by a decline to a plateau level. The initial peak has been attributed to chloride secretion whilst the sustained phase is attributed to bicarbonate secretion (Chan et al. 1996).
Based on the results, we put forward a model to explain the biochemical pathway of the regulation of anion (fluid) secretion by the vasoactive peptides (Fig. 10). According to the model, the peptides first interact with specific receptors on the basal cells (based on the localization of COX-1 immunoreactivity in basal cells, Fig. 9) and possibly also principal cells to activate PLA2 (probably through G proteins) with release of arachidonic acid (AA) from membrane phospholipids (PL). AA is converted to PGG2 and PGH2 by the cyclo-oxygenase 1 (COX-1) and then to PGE2 by the specific isomerase. PGE2 diffuses out of the cells and acts on the prostaglandin receptors, notably the EP2/4 subtypes, on the basolateral membrane of the principal cells to increase intracellular cAMP which activates an apical anion channel (CFTR) resulting in secretion of anions and, secondarily, water. In cystic fibrosis (CF) and in congenital bilateral absence of the vas deferens (CBAVD), the gene encoding the CFTR protein is disrupted by mutation, resulting in the loss of cAMP-driven secretion. Since the pathways of stimulation of secretion by the peptide hormones (paracrine factors) and adrenergic agents (adrenergic nerves) all converge on the CFTR as the final effector of secretion, it is expected that fluid secretion by the epididymis would be severely affected in CF or in CBAVD (Wong, 1998). Another potentially important question relates to the effects of long term treatment with NSAIDs on male fertility. As NSAIDs are being aggressively prescribed to combat inflammation, intervention of the COX pathway is expected to lead to perturbation of the epididymal microenvironment. Future research in the role of COX isoforms in reproduction and the effects of long term treatment with NSAIDs on male fertility will therefore be of considerable importance.
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Figure 10. Model to explain the regulation of anion secretion by bradykinin and other vasoactive peptides in the epididymis
See text for explanation. PC, principal cells; BC, basal cells; PKA, protein kinase A; AA, arachidonic acid; PL, phospholipids; PLA2, phospholipase A2; AVP, arginine vasopressin.
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Acknowledgements
This work was supported by the Research Grants Council of Hong Kong and the International Consortium on Male Contraception, Population Council, New York. The authors thank Professor R. Jones and Dr H. Wise for their invaluable advice on the study of eicosanoids.
Corresponding author
P. Y. D. Wong: Department of Physiology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong.
Email: patrickwong{at}cuhk.edu.hk
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Introduction
