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Epithelia lining the male reproductive duct modulate fertility by altering the luminal environment to which sperm are exposed. Although vas deferens epithelial cells reportedly express high levels of cyclooxygenases (Ptgs), and activation of bradykinin (BK) receptors can lead to upregulation of PTGS activity in epididymal epithelia, it remains unknown whether BKs and/or PTGSs have any role in modulating epithelial ion transport across vas deferens epithelia. Porcine and human vas deferens epithelial cell primary cultures and the PVD9902 cell line responded to lysylbradykinin with an increase in short circuit current (ISC; indicating net anion secretion), an effect that was 60%–93% reduced by indomethacin. The BK effect was inhibited by the B2 receptor subtype (BDKRB2) antagonist HOE140, whereas the B1 receptor subtype agonist des-Arg9-BK had no effect. BDKRB2 immunoreactivity was documented in most epithelial cells composing the native epithelium and on Western blots derived from cultured cells. Gene expression analysis revealed that the PTGS2 transcript is 20 times more abundant than its PTGS1 counterpart in cultured porcine vas deferens epithelia and that BDKRB2 mRNA is likewise highly expressed. Subsequent experiments revealed that prostaglandin E2, 1-OH prostaglandin E1 (prostaglandin E receptor 4 [PTGER4] agonist) and butaprost (PTGER2 agonist) increase ISC in a concentration-dependent manner, whereas sulprostone (mixed PTGER1 and PTGER3 agonist) produced no change in ISC. These results demonstrate that autacoids can affect epithelial cells to acutely modulate the luminal environment to which sperm are exposed in the vas deferens by enhancing PTGS activity, leading to the production of prostaglandins that act at PTGER4 and/or PTGER2 to induce or enhance anion secretion.
Autacoids modulate cellular processes, including ion transport across several epithelia, and data supporting such functional roles have been accumulating since these compounds were first described for their potent effect on vascular endothelia . Bradykinin (BK) can be synthesized in plasma by plasma kallikrein, and its analog lysylbradykinin (LBK) can be synthesized on the surface of target cells by tissue kallikrein . Receptors for BKs, specifically the B2 receptor subtype (BDKRB2), are present in tracheal and alveolar epithelia [3, 4] and in renal [5–8], corneal , and colonic epithelia [10, 11], where they reportedly stimulate electrogenic ion transport. In colonic epithelia, BDKRB2-stimulated ion transport is dependent on the cystic fibrosis transmembrane conductance regulator (CFTR)  channel that conducts both chloride and bicarbonate. BDKRB2 transcripts are present in Sertoli cells, epididymal epithelia, and prostatic epithelial cells of the rat [12, 13], and pharmacological data reveal BDKRB2 modulation of cytoplasmic calcium levels in canine prostatic epithelial cells . In cultured rat epididymal epithelial cells, LBK activates prostaglandin-endoperoxide synthases (PTGS [also known as cyclooxygenases 1 and 2]) and increases local prostaglandin (PG) synthesis [15, 16]. A primary culture system in which rat epididymal epithelial cells were sorted into two populations, basal and principal cells, revealed unique response profiles to LBK. A short-circuit current (ISC) increase was observed across basal cells but not across principal cells, unless these were recombined with basal cells. This report indicated that PTGS1 was expressed in basal cells but not in principal cells . Of greater interest to the current study are data revealing that Ptgs expression is highest in vas deferens epithelia . Semiquantitative protein and gene expression analysis revealed that vas deferens epithelia express both PTGS isoforms at the highest levels, when compared to other segments of the mouse reproductive tract. Likewise, intense PTGS2 immunoreactivity has been demonstrated in epithelial cells lining the human distal vas deferens and prostate . To date, it remains unknown whether BKs and/or PTGSs have a role in modulating epithelial ion transport across vas deferens epithelia.
The goal of this study was to determine whether BKs modulate ion transport across vas deferens epithelia. Outcomes demonstrate BK-induced ion transport and suggest that the effects are mediated by stimulation of PTGS activity with subsequent PG synthesis. Molecular identity, localization, and quantitative levels of molecular constituents of this pathway are described.
Porcine vas deferens were surgically excised immediately postmortem from sexually mature boars at a local swine production facility, placed in ice-cold Hank buffered salt solution (137 mM NaCl, 5.4 mM KCl, 0.4 mM KH2PO4, 0.6 mM Na2HPO4, and 5.5 mM glucose), and transported to the laboratory, where isolation of porcine vas deferens epithelial cells for primary culture (1°PVD) was performed as described previously . Distal human vas deferens (2 to 5 cm long) were obtained from a local hospital through procedures approved by both the university and hospital Institutional Review Boards. Isolation of human vas deferens epithelial cells for primary culture (1°HVD) has been described previously .
1°PVD, 1°HVD, and PVD9902 (an immortalized epithelial cell line derived from porcine vas deferens ) were seeded on 25-cm2 tissue culture flasks or 24-well tissue culture plates and grown in Dulbecco modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA) and 1% penicillin and streptomycin (Invitrogen), with media changes every other day. Upon reaching confluency, cells were lifted with PBS containing trypsin and EDTA (Invitrogen), suspended, and seeded on 1.13- or 0.33-cm2 Transwell permeable supports (Corning-Costar, Cambridge, MA) to form polarized epithelial cell monolayers. Cells were cultured with media changes every other day until assayed in modified Ussing chambers and/or used for other experimental protocols, typically 11–14 days.
Epithelial cell monolayers were mounted in modified Ussing flux chambers (model DCV9; Navicyte, San Diego, CA), bathed symmetrically in Ringer solution (120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.83 mM K2HPO4, 1.2 mM CaCl2, and 1.2 mM MgCl2), maintained at 39°C for 1°PVD and PVD9902 or 37°C for 1°HVD, and bubbled with 5% CO2-95% O2 to provide mixing and pH stability. Monolayers were clamped to 0 mV, and ISC was measured continuously with a voltage-clamp apparatus (model 558C; University of Iowa, Dept. of Bioengineering, Iowa City, IA). Data were acquired digitally at 1 Hz with an Intel-based computer using an MP100A-CE interface and AcqKnowledge software (version 3.7.3; BIOPAC Systems, Santa Barbara, CA). Once recordings began, this system automatically generated a 5 mV bipolar pulse every 100 sec. The resulting change in ISC was used to calculate transepithelial electrical resistance (RTE) according to Ohm's law. To test for various experimental hypotheses, monolayers were exposed to different compounds: LBK, BK, des-Arg9-BK, HOE140 (all from Bachem, King of Prussia, PA), des-Arg10-HOE140 (Anaspec, San Jose, CA), PGE2, butaprost, indomethacin, amiloride, bumetanide (all from Sigma-Aldrich, Saint Louis, MO), 1-OH prostaglandin E1 (1-OH PGE1), and sulprostone (both from Cayman Chemical, Ann Arbor, MI).
1°PVD monolayers cultured on permeable supports were lysed and solubilized in buffer containing protease inhibitors (Calbiochem, San Diego, CA). After centrifugation at 14000 rpm and 4°C for 15 min, the supernatant protein isolates were transferred to a fresh tube and quantified by an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Protein sample aliquots were prepared for electrophoresis by addition of loading buffer (Boston Bioproducts, Worcester, MA) and β-mercaptoethanol (Sigma-Aldrich) and heated to 95°C for 5 min. SDS-PAGE was conducted with 4%–20% polyacrylamide gradient precast gels (Bio-Rad Laboratories, Hercules, CA) at 150 V for approximately 1.5 h or until optimal separation of prestained protein standards (Bio-Rad Laboratories) was obtained. Electro-transfer onto polyvinylidene fluoride membranes (Millipore, Billerica, MA) was carried out in methanol-containing transfer buffer (20% v/v) at 30 V for 3 h. Blots were blocked by blotting grade nonfat dry milk (5% w/v; Bio-Rad Laboratories) and exposed to 0.08 μg/ml of anti-BDKRB2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-BDKRB2 antibody preadsorbed by its immunizing peptide. A suitable horseradish peroxidase-conjugated secondary antibody was employed to develop chemiluminescence signals that were imaged by a FluorChem HD2 (Alpha Innotech Corporation, San Leandro, CA).
Segments (~1 cm) of both proximal and distal porcine vas deferens were snap-frozen in liquid N2 immediately after ducts were obtained and were transported to the laboratory, where they were kept frozen while being embedded in tissue-freezing medium (Richard-Allan Scientific, Kalamazoo, MI) for cryosectioning. Sections (18 μm) were collected onto poly-L-lysine-coated slides, fixed in 4% paraformaldehyde in PBS for 1 h, washed in PBS five times, and subjected to conventional immunocytochemistry labeling. In short, tissues were blocked with BSA (Sigma-Aldrich) in PBS and exposed to 2 μg/ml of anti-BDKRB2 antibody, anti-BDKRB2 antibody preadsorbed by its immunizing peptide, or vehicle only, overnight, in a humidified chamber. Tissues were washed in PBS five times and subsequently exposed to a suitable fluorophore-conjugated secondary antibody (Alexa Fluor 488; Invitrogen) and to a fluorescent chromatin dye (TO-PRO-3; Invitrogen) for 1 h. Following five washes in PBS, cover slips were mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL), and confocal microscopy was performed (LSM 510 META; Carl Zeiss Microimaging Inc., Thornwood, NY).
Total RNA was isolated from epithelial cell monolayers following Ussing chamber protocols, using an RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's recommendations. RNA quality was assessed with a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). RNA concentration within isolates was determined using an ND-1000 spectrophotometer, and all samples were diluted to 100 ng/μl. One-step RT-PCR was performed using a One-step RT-PCR kit (Qiagen) according to the manufacturer's recommendations, with 100 ng of RNA per reaction. Single-target reactions were conducted using primer sets designed upon previously published cDNA sequences when available. Porcine PTGER1, PTGER2, and PTGER4 coding sequences had not been published. Thus, sequence homology searches were performed by querying the porcine gene index from The Institute for Genomic Research with the respective human sequences. Such searches yielded at least one hit with relevant homology to the PTGER2 and PTGER4 cDNAs, and these candidate sequences were used in the design of primer sets. No porcine-candidate sequences were identified for the PTGER1, and the human PTGER1 sequence was used to design a primer set for the porcine target. Primer sequences, annealing temperatures, expected amplicon length, and sequence accession numbers are presented in Table 1. SYBR Green I (Molecular Probes, Eugene, OR) fluorescence signals emitted at each amplification cycle were detected and quantified by a SmartCycler (Cepheid, Sunnyvale, CA). Transcript abundance measurements in any given RNA sample were performed by reactions carried out in duplicates. Melting analysis was performed at the end of every amplification phase, and reaction products were subjected to agarose gel electrophoresis, sequencing, and computer-based identity analysis. Acquired threshold cycle (Ct) values for target genes were normalized to the reference porcine gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) according to the amplification efficiency method .
RT-PCR products resolved on agarose gels were excised and purified with a QIAquick gel extraction kit (Qiagen), and the resulting cDNA templates were sequenced with a CEQ8000 (Beckman Coulter, Fullerton, CA) according to the manufacturer's recommendations.
Paired and unpaired t-tests and ANOVA were performed as appropriate. These tests and the calculation of means and standard error of the mean (SEM) were performed with Microsoft Office Excel 2003 (version 11.8211.8202; Microsoft Corporation, Redmond, WA). A modified Michaelis-Menten (M-M) function, y = [ax]/[c + x], where y is the measured ΔISC, a is the maximum ΔISC (ΔISC-MAX), x is the concentration of agonist, and c is the concentration required for a half-maximal response (i.e., the apparent KD or kapp), was fitted to data collected from concentration-dependency experiments. All graphs and curve fittings were made with SigmaPlot (version 6.0; Systat Software Inc., Point Richmond, CA).
To investigate whether BKs modulate ion transport across vas deferens epithelia, cells derived from human and porcine vas deferens, as well as the vas deferens epithelial cell line PVD9902, were used. 1°PVD monolayers (n = 7) revealed basal ISC and RTE of 0.7 ± 0.2 μA cm−2 and 4400 ± 610 Ω cm2, respectively. LBK (500 nM), symmetrically added to the bathing media, caused a rapid bimodal increase in ISC that formed a first peak before it decreased briefly and generated a second peak (Fig. 1A). The ΔISC-MAX was 5.1 ± 1.0 μA cm−2. ISC then receded to a nadir and rose again to a plateau that lasted several minutes. Sustained ISC was partially sensitive to basolateral bumetanide (20 μM; data not shown), indicating that a portion of the ISC is attributable to Cl− secretion, as has been shown for other agonists affecting vas deferens epithelial cells [20, 24]. RTE rapidly but transiently declined upon exposure to LBK. The lowest value was consistently observed at the first voltage pulse following LBK exposure, and then RTE recovered towards pretreatment values. This is depicted in the deflections that are of maximal magnitude at the onset of responses and their subsequent and progressive narrowing. Maximal decrease in RTE (ΔRTE-MAX) was 3160 ± 500 Ω cm2. Experiments carried out with 1 nM LBK, the lowest concentration tested, revealed ΔISC-MAX of 4.5 ± 1.2 μA cm−2 (n = 5), which was not different from the results obtained with 500 nM. However, ISC tracings produced by 1 nM formed a single transient peak that relaxed to basal levels and then rose to a plateau (Fig. 1B). To assess whether LBK produced a sustained effect, ΔISC measured between 900 and 1000 sec after the onset of responses was determined: ΔISC was 1.1 ± 0.1 μA cm−2 and 0.9 ± 0.3 μA cm−2 for responses acquired with 500 nM and 1 nM LBK, respectively. At 1 nM, ΔRTE-MAX was 2170 ± 670 Ω cm2 and, similar to that observed with 500 nM, RTE was reduced to the lowest value with the onset of each response and then recovered gradually towards pretreatment values. Still in the context of the first experiments, PVD9902 monolayers (n = 6) were assessed with 500 nM LBK. From basal ISC and RTE at 0.4 ± 0.1 μA cm−2 and 6900 ± 1400 Ω cm2, respectively, these monolayers responded with ΔISC-MAX of 5.6 ± 1.0 μA cm−2 and ΔRTE-MAX of 2720 ± 650 Ω cm2 (Fig. 1C). ISC tracings from PVD9902 revealed a sharp and rapid rise in ISC to form a single peak that declines toward baseline in about 300 sec. The magnitude of responses acquired from 1°PVD and PVD9902 at the tested concentrations were indistinguishable (P < 0.05). Subsequent experiments tested for BK modulation of ion transport across 1°HVD monolayers. A first 1°HVD monolayer was tested with 1 nM LBK, which produced a small ΔISC-MAX (~0.8 μA cm−2). After 400 sec, a second and cumulative LBK addition brought the concentration to 10 nM, and the recorded ΔISC-MAX was 6.4 μA cm−2 for this monolayer. Based on this single observation, all subsequent experiments with 1°HVD were conducted with 10 nM LBK. 1°HVD epithelial monolayers (n = 6) were, at first, exposed to 10 μM amiloride, to which they consistently responded with a decrease in ISC (data not shown, ). These monolayers were then allowed sufficient time to establish a new baseline, at which point they exhibited basal ISC of 3.9 ± 1.2 μA cm−2 and basal RTE of 550 ± 180 Ω cm2. Upon LBK exposure (10 nM), 1°HVD monolayers responded with a rapid increase in ISC that formed one single peak (Fig. 1D), in a pattern similar to 1°PVD responses acquired following exposure to 1 nM LBK. 1°HVD responses produced a ΔISC-MAX of 9.4 ± 2.2 μA cm−2 and a ΔRTE-MAX of 205 ± 75 Ω cm2. Taken together, the results presented in Figure 1 demonstrate similar ion transport responses to LBK for vas deferens epithelial cells from three sources, an immortalized cell line and primary cultures of cells isolated from both porcine and human tissues.
A subset of four assays was performed to determine which BK receptor subtype is present in and linked to ion transport across vas deferens epithelia by exposing paired 1°PVD monolayers to selective agonists and/or antagonists of BK 1 receptor subtype (BDKRB1) and BDKRB2. Each assay employed cells derived from one boar that gave rise to six monolayers. LBK and BK are BDKRB2-selective agonists, whereas des-Arg9-BK is a BDKRB1-selective agonist. Results presented in Figure 2 demonstrate that BDKRB2, but not BDKRB1, agonists stimulated ISC. Furthermore, concurrent exposure to a BDKRB2 antagonist, HOE140, precluded an effect of BK on paired monolayers. The BDKRB1 antagonist, des-Arg10-HOE140, despite being used at a supermaximal concentration (5 μM), caused only a modest inhibition of the BK-induced change in ISC, which did not achieve statistical significance (n = 4). These results provide compelling evidence that BDKRB2s mediate the effect of BK and/or LBK on ISC across vas deferens epithelial cells.
Western blot analysis targeted expression of BDKRB2 in protein samples isolated from 1°PVD monolayers. Outcomes reveal a most prominent signal, with mobility consistent with the theoretical molecular weight (41 kDa), calculated for the human BDKRB2 (Fig. 3A). Four other bands of reduced intensity were present. All bands had reduced intensity in the lane that had less protein loaded. The identity of these bands is unknown, although all signals were absent when the primary antibody was preadsorbed by the immunizing peptide regardless of the amount of protein loaded (Fig. 3B).
Immunocytochemistry and confocal microscopy analysis were carried out to investigate expression and localization of BDKRB2s in transverse sections of freshly excised porcine vas deferens. BDKRB2 immunoreactitivy was localized to cells lining the duct. Receptor immunoreactivity was present throughout the epithelium, whereas nonepithelial cells were devoid of labeling (Fig. 3, C and E). BDKRB2 immunoreactitivy was present in the same labeling pattern on sections of both proximal and distal vas deferens epithelia (not shown). Tissue sections made in a serial manner and exposed to preadsorbed primary antibody exhibited only very modest labeling (Fig. 3D), and sections probed with the secondary antibody only exhibited no labeling (not shown). BDKRB2 immunoreactitivy was also present at the endothelium of capillaries present in the vicinity of the epithelium and at the endothelium of veins located outside the vas deferens muscular layer (not shown).
Semiquantitative RT-PCR analysis of RNA isolated from 1°PVD monolayers derived from three animals revealed that the BDKRB2 mRNA is expressed at a level equivalent to 16.45% ± 0.45% of the total number of transcripts expressed from the housekeeping gene GAPDH (n = 6). The BDKRB1 transcript was detected only once in a series of eight reactions (two reactions per RNA sample; each sample was isolated from monolayers derived from one of four animals). Ct values for the porcine BDKRB2 and porcine GAPDH mRNAs were 20.59 ± 0.24 and 17.90 ± 0.09, respectively. The BDKRB1-positive reaction had a Ct of 38.70, which shows that even when detected, BDKRB1 mRNA is >105 less abundant than the BDKRB2 mRNA. Sequencing of the amplicons derived from these reactions was performed, and all products proved to be of expected identity.
To determine whether the BDKRB2-induced ΔISC includes an increase in PTGS activity, monolayers were exposed to indomethacin or vehicle control for 10 to 15 min prior to LBK exposure. Indomethacin was without effect on ISC or RTE. Subsequent exposure to LBK (500 nM) caused a ΔISC-MAX and ΔRTE-MAX of 0.9 ± 0.5 μA cm−2 and 1630 ± 390 Ω cm2, respectively. These ΔISC responses in the presence of indomethacin are less than 20% when compared to vehicle-treated controls (Fig. 4). The possibility remained that although the ΔISC-MAX was reduced by indomethacin, net flux, measured as area under the ISC curve, might not have changed. Thus, the magnitude of responses was also defined over the course of 300 sec and revealed an indomethacin-suppressive effect (Fig. 4C). Both ΔISC-MAX and the net change in flux over 300 sec were affected to a similar extent by indomethacin. In addition, the effect of LBK on RTE was approximately 50% less in the presence of indomethacin (Fig. 4D).
Monolayers derived from the PVD9902 cell line and subjected to the same protocol (n = 6) displayed no effect of exposure to indomethacin (500 nM). Once exposed to LBK (500 nM), these monolayers responded with ΔISC-MAX and ΔRTE-MAX of 0.39 ± 0.06 μA cm−2 and 440 ± 200 Ω cm2, respectively, which is less than 7% of the ΔISC-MAX and less than 17% of the ΔRTE-MAX exhibited by paired monolayers not treated with indomethacin (Fig. 5).
1°HVD cultures were assessed for an effect of LBK (10 nM) in the absence or presence of indomethacin (2 μM). Monolayers of 1°HVD were assessed twice for effects of LBK in the absence and presence of indomethacin: either first with LBK only then replaced in culture for 3 to 5 days and evaluated a second time in the presence of indomethacin, or in the reverse order. Baseline values for indomethacin-treated 1°HVD monolayers (six monolayers derived from six donors) were 2.4 ± 1.5 μA cm−2 and 721 ± 382 Ω cm2. Exposure to indomethacin produced no measurable changes in ISC or RTE. Responses to LBK within the indomethacin-treated group constituted ΔISC-MAX and ΔRTE-MAX of 3.7 ± 0.5 μA cm−2 and 196 ± 108 Ω cm2, respectively. This ΔISC-MAX is significantly less than the untreated group (Fig. 5C), but 1°HVD exhibited no difference in terms of ΔRTE-MAX (Fig. 5D). Taken together, these results support the inference that in vas deferens epithelial cells, BK modulates ion transport via activation of cyclooxygenases that likely contribute to PG synthesis.
Indomethacin inhibits both PTGS1 and PTGS2. Thus, experiments were conducted to determine the relative expression of mRNA coding for the PTGS isoforms in 1°PVD cells. Semiquantitative RT-PCR was carried out with RNA isolated from monolayers derived from five animals. Ct values were 18.72 ± 0.60 for GAPDH, 21.25 ± 0.35 for PTGS2, and 25.68 ± 0.22 for PTGS1. Normalization of targeted PTGS mRNA to GAPDH reveals that the PTGS2 transcript abundance was more than 20 times greater than the PTGS1 transcript and as much as 20% of GAPDH (Fig. 6). Resulting amplicons had expected mobility on agarose gels, and subsequent cDNA sequencing proved expected identity (data not shown).
Changes in ISC exhibited by 1°PVD were recorded by a modified Ussing chamber as monolayers were exposed to cumulative concentrations of subtype selective prostaglandin E receptor agonists: sulprostone (PTGER1 and PTGER3), butaprost (PTGER2), and 1-OH PGE1 (PTGER4). PGE2, the primary product of PTGS activity, although a nonselective PTGER agonist, was also used in this concentration response study. These assays were carried out in a paired manner so that cells from each animal had at least one monolayer exposed to each compound. Results are summarized in Figure 7. Concentration-dependent stimulation of ISC was observed with PGE2, 1-OH PGE1, and butaprost, whereas sulprostone was without a remarkable effect, even at the greatest concentration tested. Effects at the highest concentration tested (1 μM) were not different for PGE2, 1-OH PGE1, or butaprost. However, quantitative analysis of the concentration dependence revealed some differences. Responses to 1-OH PGE1 and butaprost were well-fitted by a modified M-M function with derived macroscopic dissociation constants (kapp) of 20.2 ± 6.5 nM and 37.7 ± 18.4 nM, respectively. Visual inspection revealed that concentration-dependent responses to PGE2 were not fitted well by the M-M function. Nonetheless, at every concentration tested, the response to PGE2 was greater than the response to any of the receptor-selective agonists. Thus, the rank order of potency was PGE2 > 1-OH PGE1 > butaprost sulprostone, suggesting that both PTGER4 and PTGER2 can contribute to the response.
Semiquantitative RT-PCR revealed a direct correlation between the pharmacological data described above and transcript abundance for PTGER subtypes measured in RNA isolated from 1°PVD monolayers. A comprehensive readout of PTGER expression levels is summarized in Figure 8. PTGER4 mRNA was approximately four times more abundant than PTGER2 mRNA and more than 60 times more abundant than PTGER3 mRNA. Reactions for PTGER1 failed to detect this transcript in six attempts. Single amplicons of expected mobility derived from reactions targeting PTGER2, PTGER3, and PTGER4 mRNAs were sequenced and revealed expected identity (data not shown). Thus, both pharmacological and molecular data support the conclusion that PGs acting at PTGER4 and PTGER2 contribute to ion transport that is stimulated by LBK.
Results presented here demonstrate for the first time that BKs with preferential affinity to the BDKRB2 elicit an increase in ISC that is indicative of anion secretion by cells lining the vas deferens. Molecular and functional data derived from both porcine and human epithelial cell models are reported that describe a role for BK and the BDKRB2 in vas deferens epithelial physiology. 1°PVD cells respond to BK in an HOE140-sensitive manner and, likewise, PVD9902 respond to BKs. Native porcine vas deferens tissues exhibit epithelial BDKRB2-immunoreactivity. LBK also induces a ΔISC that is indicative of anion secretion in 1°HVD. Data derived from these three epithelial models, along with BDKRB2 immunoreactivity in native tissue, support the inference that activation of BDKRB2 upregulates PTGS activity, mostly PTGS2, to synthesize PGs that ultimately bind to PTGER4 and/or PTGER2 to initiate pathways leading to anion secretion across the vas deferens epithelium. It is expected that such cellular events change the vas deferens luminal fluid: its volume, ion composition, and pH.
The rapid BK-induced increase in ISC was accompanied by a similarly rapid and drastic reduction in RTE, which is suggestive of the opening of ion channels, likely CFTR. Functional hallmarks of this ion channel have been reported previously for these cell-based systems [20–22, 25]. Such observations are in keeping with the BK effects observed in epithelia elsewhere in the male reproductive tract, most notably in epididymal epithelia [15–17, 26]. Epithelial systems in which the qualitative aspect of BK-induced anion secretion have been at least partially elucidated, as in the kidney [5–8] and colon [10, 11], provide further support for the proposed link between BDKRB2 activation and an anion secretory effector such as CFTR in vas deferens epithelia. Both porcine and human vas deferens epithelial cells respond to submicromolar concentrations of BK with substantial anion secretion, although the concentrations employed are approximately 10 to 100 times greater than the circulating plasma levels of BK . Within this context, it should be noted that epithelia lining the male reproductive tract, specifically epididymal epithelia, have been shown to express tissue kallikrein immunoreactivity , which suggests that BK synthesis can occur at the apical membrane of epithelium lining other portions of the male excurrent duct.
Results suggest that the BDKRB2 is solely responsible for the ion transport observed upon BK exposure. Stimulation was fully blocked by the BDKRB2 antagonist HOE140. Furthermore, BK, which is a BDKRB2 selective agonist, stimulated ISC, whereas the BDKRB1 selective agonist des-Arg9-BK caused no appreciable change in ISC. The BDKRB1 antagonist des-Arg10-HOE140 was associated with a reduction in BK-stimulated ISC, although this difference did not achieve statistical significance. This observation is consistent with reports that BDKRB1 antagonists developed from the des-Arg-fragment of HOE140 exert potent antagonistic effect on the BDKRB1 while exhibiting weak antagonistic effects on the BDKRB2 .
The 1°PVD system expresses the BDKRB2 at the protein level, as indicated by Western blot analysis. The porcine coding sequence is not fully cloned to date, but the theoretical mobility of the human BDKRB2 is 41 kDa. In these assays, the most prominent band had mobility between the protein standards 37 and 50 kDa. In addition, other signals of lesser intensity were present. Two different signals were present at and below the 37-kDa marker, and two other signals were present below and above the 50-kDa marker. Though it becomes difficult to account for the origin of these signals, it should be noted that a signal with mobility greater than the theoretical value has been reported for the human BDKRB2  and that all signals were substantially reduced or eliminated by preadsorption of anti-BDKRB2 with the immunizing peptide. BDKRB2 immunoreactivity was observed in the majority of cells composing the intact porcine vas deferens epithelium. Principal, basal, clear, narrow, and dark (pencil) cells are reported as cell types within this epithelium, although principal cells account for the vast majority of the cells that line the duct [29, 30]. Micrographs obtained following BDKRB2 immunolabeling indicated that this receptor is present throughout most cells composing the vas deferens epithelium. The labeling pattern allows, with a reasonable degree of certainty, for the inference that principal cells express the BDKRB2 in vivo. At the transcriptional level, BDKRB2 is highly expressed in 1°PVD monolayers, which seems to place this cell model apart from others in this regard. G-protein coupled receptors—BDKRB2 included—are generally expressed at low levels in mammalian cells despite their wide participation in cellular signal transduction in several tissues. This has made necessary the development of cellular expression systems, largely based on insect cells, in order to obtain reasonable yields of BDKRB2 for various applications [31–33]. The BDKRB1 transcript is, however, virtually absent from the transcriptome of 1°PVD monolayers. As described in Results, this transcript was amplified only once out of eight attempts using 1°PVD RNA, and that amplification generated a 38.7 Ct value, which indicated at least 100000-fold fewer copies than BDKRB2 mRNA. These data provide further support for an anion secretion pathway that is stimulated by the BDKRB2 and not the BDKRB1 in vas deferens epithelia. The BDKRB2 is present in several tissues and is constitutively expressed under physiological conditions, while the BDKRB1 is an inducible form of the BK receptor that is expressed in the context of inflammation and/or trauma [2, 34]. The possibility for BDKRB1 modulation, however, does remain. BDKRB1 was transcribed, translated, and expressed at the basolateral membrane of colonic epithelia within 3 h after being mounted in Ussing chambers . The 1°PVD monolayer from which that RT-PCR amplification of BDKRB1 mRNA was derived remained in the Ussing chamber for approximately 45 min. That single observation where the BDKRB1 transcript was amplified could reflect rapid induction, although this possibility remains to be tested.
Exposure to the nonselective PTGS inhibitor indomethacin significantly reduced LBK responses in all three vas deferens epithelial cell models tested. This observation leads to the conclusion that, similar to what has been demonstrated in epididymal epithelia [15–17], BKs activate PTGS and upregulate PG synthesis in vas deferens epithelia. PGs, in turn, bind PTGERs to initiate a pathway (or pathways) leading to anion secretion. In 1°PVD, PTGS2 expression is nearly 20 times greater than PTGS1 expression. This observation is consistent with a previous report that defined relative levels of these transcripts in murine vas deferens . PTGSs, and most prominently PTGS2, have been the subject of substantial pharmaceutical development. Recently, selective inhibitors of PTGS2 (rofecoxib, celecoxib, and valdecoxib) have been actively marketed as nonsteroidal anti-inflammatory drugs, although rofecoxib was withdrawn. Since then, attention has turned to side-effects occurring in the kidney , where PTGS2 is constitutively expressed. The high levels of PTGS2 expression in the vas deferens as well as its role in modulating ion transport make it reasonable to believe that therapeutic use of these compounds may impact fertility at the level of epithelia lining the male excurrent duct.
Concentration-dependent responses to PGs are exhibited by 1°PVD epithelial cell monolayers. The outcomes suggest that the PTGER4 and/or PTGER2 account for the response to PGE2 in the vas deferens. Butaprost is the most selective PTGER2 agonist available among synthetic prostaglandins and possesses a reported kapp of 110 nM, while 1-OH PGE1 has PTGER4 selectivity with a reported kapp of 190 nM . 1°PVD responded to both of these selective agonists at concentrations at or slightly below their reported kapp. PGE2 is a powerful nonselective agonist of all PTGER subtypes, and its concentration-dependent changes in ISC were not fitted well by the M-M function. This is consistent with the notion that two PTGER subtypes can modulate ISC across vas deferens epithelia. Nonetheless, butaprost, 1-OH PGE1, and PGE2 produced virtually the same ΔISC at the greatest concentration tested, which suggests the existence of a response-limiting factor, most likely the ion-transporting proteins carrying out their function at a maximal rate when the greatest agonist concentration was effectively in the system. Both PTGER4 and PTGER2 are GS-protein coupled receptors, suggesting substantial adenylyl cyclase activation. Previously reported data from this laboratory demonstrated cAMP synthesis in 1°PVD upon exposure to agonists other than PG . PTGER2 and PTGER4 reportedly modulate ion transport induced by PGE2 elsewhere in the male reproductive tract, specifically in epididymal epithelia . The relative abundances of mRNA for prostaglandin E receptor subtypes described here for vas deferens epithelia seem to set the male excurrent duct apart from other urogenital epithelia. Gene expression analysis performed on two mice strains reveals that all EP receptor transcripts are expressed in the kidney of both strains but that PTGER4 mRNA is least abundant while PTGER1 mRNA is the most abundant . In the second mouse strain, kidney levels of PTGER4 expression are comparable to that of PTGER1 and PTGER3 and well below the levels of medullary PTGER2 mRNA. Further evidence is available that supports high expression of the PTGER1 and PTGER3 transcripts in the kidney but modest expression of the PTGER4 and PTGER2 transcripts . These PTGER expression profiles place the kidney as an organ system where the expected effect of PGE2 is via inositol thriphosphate and reduction in cellular cAMP levels, as opposed to signaling through phosphatidylinositol 3 (PI3)-kinase and increase in cAMP levels that, based upon our observations, is expected in the vas deferens.
PGs modulate acid and base transport. In the duodenum, PGs are required for the modulation of net bicarbonate secretion and cause alkalinization of the duodenal lumen. In this segment of the gastrointestinal tract, bicarbonate secretion is upregulated through activation of PTGER3 and PTGER4 [41–43]; cAMP-, calcium-, and PI3-kinase-dependent pathways ; and by modulating activity of apically expressed bicarbonate exchangers such as SLC26A6 . Although further investigation is needed to elucidate the qualitative aspects of anion secretion induced by BKs and PGs in vas deferens epithelium, this laboratory demonstrated that anion secretion in this tissue is bicarbonate dependent . Using a variety of ion substitution conditions and selective permeabilization of either the apical or basolateral membranes of 1°PVDs, bicarbonate was necessary for the maintenance of sustained changes in ISC upon forskolin stimulation and was required for changes in ISC to occur at the isolated basolateral membrane. Additionally, mRNA coding for a collection of bicarbonate cotransporters and exchangers is expressed in porcine vas deferens epithelia, including SLC4A4, SLC26A3, SLC26A4, and SLC26A6 [22, 24]. Sperm bicarbonate uptake, while still in the male reproductive tract, is required for adequate sperm capacitation and the development of fertilizing capacity . Taken together, these data make it reasonable to conclude that the vas deferens epithelium might be a substantial, if not the main, cellular contributor of bicarbonate secretion onto the environment to which sperm is exposed.
In conclusion, BKs act on the BDKRB2 to upregulate PTGS activity and the synthesis of PGs that then act on PTGER4 and/or PTGER2 to ultimately induce anion secretion across vas deferens epithelia. Anion secretion most likely modifies the male duct luminal environment to which sperm are exposed and thus may modulate sperm function and, ultimately, male fertility.
The authors extend sincere thanks to Mr. Joel Sanneman and the KSU-COBRE Center for Epithelial Function in Health and Disease for resources provided through its Confocal Microscopy and Molecular Biology Core Facilities. Appreciation is also expressed to Henrys Ltd., Dr. Pradeep Malreddy, and Dr. John Devine for their assistance with tissue procurement.
1Supported by the Cystic Fibrosis Foundation (SCHULT06PO) and National Institutes of Health (RR-17686). This manuscript represents contribution number 08–154-J from the Kansas Agricultural Experiment Station.