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a Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(100 µM) activated a whole-cell current that exhibited a linear I–V relationship as well as time- and voltage-independent characteristics. However, the current magnitude varied with different agonists. The agonist-activated current could be mimicked by an adenylate cyclase activator, forskolin (10 µM), and suppressed by an adenylate cyclase inhibitor, MDL12330A, suggesting the involvement of cAMP. Current characteristics remained the same after cation replacement, leaving Cl- as the major permeant ion species in the solutions. The reversal potential of the agonist-induced current was close to the equilibrium potential of Cl- in the presence of a Cl- gradient, indicating the activation of Cl- conductance. The agonist-induced current was inhibited by the Cl- channel blocker diphenylamine 2,2'-dicarboxylic acid (DPC), but not by the Cl- channel blocker 4,4'-diisothiocyanatostibene-2,2'-disulfonic acid (DIDS). The anion selectivity sequence of the current was NO3->Br->Cl->I-. The observed electrophysiological properties of the agonist-induced Cl- conductance were consistent with those reported for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel expressed in many epithelia. The expression of CFTR in the mouse endometrial cells was also demonstrated by Western blot analysis. It appears that neurohormonal regulation of the uterine fluid in the mouse endometrium converges on the cAMP-activated Cl- channel, presumably CFTR.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, a primary culture of mouse endometrial epithelial cells grown on permeable supports has been established [9]. It has been demonstrated that the cultured epithelium responds to adrenaline, noradrenaline, and prostaglandin (PG) E2 with increases in the short-circuit current that can be predominantly attributable to Cl- and HCO3- secretion [10, 11]. These secretory responses appear to be mediated by intracellular cAMP and inhibited by the Cl- channel blocker diphenylamine 2,2'-dicarboxylic acid (DPC), suggesting possible involvement of a cAMP-activated Cl- channel. Similar studies on intact rat uteri have also demonstrated the involvement of cAMP in ß-adrenoceptor-mediated stimulation of anion secretion [12–14].
In the present study, we further characterized the Cl- channel involved in mediating the secretory responses to adrenaline and to PGE2 and PGF2 in cultured mouse endometrial epithelial cells using the patch-clamp technique. The results indicate that neurohormonal regulation of the uterine fluid in the mouse endometrium appears to converge on a cAMP-activated Cl- channel that exhibits characteristics similar to those reported for CFTR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adrenaline was obtained from David Bull Laboratories (Victoria, Australia). Dulbecco's Modified Eagle medium (DMEM), F-2 nutrient mixture, penicillin-streptomycin, forskolin, 4,4'-diisothiocyanatostibene-2,2'-disulfonic acid (DIDS), PGF2, and PGE2 were purchased from Sigma Chemical Company (St. Louis, MO). DPC was obtained from Riedel-de Haen Chemicals (Hannover, Germany).
Cell Isolation and Culture
Endometrial epithelial cells were enzymatically isolated from the mouse uterus according to the method described by McCormack and Glasser [15], with slight modifications [9]. For each culture preparation, samples of uteri were obtained from 40 immature ICR mice (3.5–4 wk) to avoid complications of the estrous cycle. The sliced uteri were incubated in PBS supplemented with 5 mg/ml trypsin and 25 mg/ml pancreatin at 0°C for 60 min and then at room temperature for another 60 min. After the enzyme digestion, the test tube containing PBS and the tissues was shaken gently for 30 sec. Uterine tissues were carefully removed, and the crude cell solution was passed through a 70-µm fluorocarbon mesh filter (Spectra/Mesh, Spectrum, Houston, TX). The filtrate was centrifuged at 1000 x g for 5 min. The cell pellet was resuspended in 12 ml PBS and centrifuged again at 1000 x g for 5 min. The washing procedures were then repeated once more. After centrifugation, the cell pellet was resuspended in DMEM/Ham's F-12 culture medium containing 10% fetal bovine serum, 1% non-essential amino acid (NEAA), 100 U/ml penicillin, and 100 mg/ml streptomycin. The isolated endometrial cells were plated in Petri dishes and incubated at 37°C and 5% CO2:95% O2 for 3–4 days.
Patch-Clamped Whole-Cell Current Measurement
After 3–4 days in culture, the cells formed colonies. To isolate single cells for the patch-clamp study, the cells were immersed in a bath NaCl solution containing a low Ca2+ concentration (120 nM) for 10–20 min. The dissociated cells were then allowed to recover from the low Ca2+-treatment in a solution with the same composition except that 1 mM Ca2+ was used, for another 10–15 min before patching.
The whole-cell patch-clamp technique as described by Hamill and colleagues [16] was employed. Current recordings were obtained using a patch-clamp amplifier (Axopatch-200 or Axopatch-1D; Axon Instruments, Inc., Forster City, CA). Patch pipettes, made from borosilicate glass (Vitrex, Modulohm I/S, Herlev, Denmark), were prepared as previously described [17]. After formation of a whole-cell configuration, the series resistance was measured and cell capacitance was compensated. The control of command voltages was carried out using an IBM-AT-compatible computer equipped with an interface (TL-1–125; Axon Instruments, Inc.) and utilizing the software pClamp Version 6. The output current signals, after being filtered through an 8-pole Bessel filter (AI-2040; Axon Instruments, Inc.) at a cutoff frequency of 10 kHz, were displayed on a chart recorder (Graphic, Yokohama, Japan).
The following pipette solutions (mM) were used: 140 KCl (or N-methyl-D-glucamine [NMDGCl]), 1 MgSO4, 1.2 NaH2PO4, 10 HEPES, 16 glucose, and 0.1 EGTA (pH 7.2). The bath solutions (mM) contained 140 NaCl (or NMDGCl), 1 MgSO4, 1.2 NaH2PO4, 10 HEPES, 16 glucose, and 1 CaCl2 (pH 7.4). At times, 40 KCl was used in the bath solution to generate a Cl- gradient across the membrane. Osmolarity of the solutions was raised to an isotonic level (300 mOsm) by addition of mannitol, using a vapor pressure osmometer (Wescor 5500, Logan, UT).
Western Blot of CFTR Protein
Proteins of endometrial cells were extracted with PBS, pH 7.4, containing 10 mM EDTA and 1 mM PMSF. The homogenate was spun down at 1000 x g, and the supernatant was then centrifuged at 30 000 x g for 30 min at 4°C. Proteins from the resultant pellet were separated by SDS-PAGE. Separated proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane. The electroblotted PVDF membrane was saturated with 5% skimmed milk in PBS containing 0.1% Tween 20. The membrane was incubated with monoclonal anti-human CFTR serum (Genzyme, Cambridge, MA; 1:1000) overnight at 4°C followed by a peroxidase-labeled antibody (1:500). After thorough washing, the positive band was detected using ECL Western blotting reagents and exposed to autoradiography film for 1.5 min (Amersham, England).
Statistical Analysis
Results are expressed as mean ± SE. Comparisons between groups of data were made by Student's unpaired t-test. A p value of less than 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The adrenaline-activated whole-cell current profile elicited by a series of commending voltages exhibited time- and voltage-independent characteristics with a linear I–V relationship (Fig. 1, n = 5). In the presence of a Cl- gradient, 140 mM in pipette solutions and 40 mM in bath solutions, adrenaline (1 µM) activated a whole-cell current that had a reversal potential of 24 ± 0.5 mV (Fig. 1C), close to the calculated Cl- equilibrium potential, 30 mV. When permeant cations (K+, Na+) in the pipette and bath solutions were replaced by impermeant NMDG+, leaving Cl- as the major permeant species, current with similar characteristics was also stimulated by adrenaline (n = 3, data not shown), indicating the activation of Cl- conductance.
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The adrenaline-activated whole-cell Cl- current was mimicked by forskolin (10 µM), an adenylate cyclase activator. In symmetrical Cl-, as well as in the presence of a Cl- gradient (pipette 140 mM: bath 40 mM), the observed forskolin-activated current was time-independent with a linear I–V relationship (Fig. 2). The forskolin-activated current reversed at 26 ± 2 mV (Fig. 2C, n = 7), which was also close to the calculated equilibrium potential for Cl-, 30 mV, further indicating the activation of Cl- conductance.
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Anion Selectivity Sequence of the cAMP-Dependent Cl- Conductance
Ion channels can be characterized by their ion selectivity sequences. In the present study, the anion selectivity sequence was studied by examining the relative permeability of a number of monovalent anions to that of Cl- (PX/PCl). The shift in reversal potential was measured upon replacement of the test anion in a bath solution (n = 6, Fig. 3), and PX/PCl was calculated from the bi-ionic equation. The anion selectivity sequence of the cAMP-dependent Cl- conductance thus obtained was NO3- (3.07) > Br- (1.95) > Cl- (1.0) > I- (0.20), consistent with that reported for the cAMP-activated Cl- channel or CFTR in a number of epithelia [17–21].
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PG-Stimulated Whole-Cell Cl- Current
To determine whether the cAMP-dependent Cl- conductance was also activated in response to other hormonal agents, the effect of PGE2 and PGF2 on the whole-cell current in mouse endometrial cells was also studied. PGE2 (5–10 µM) stimulated a whole-cell current similar to that stimulated by adrenaline, exhibiting time- and voltage-independent characteristics with a linear I–V relationship (Fig. 4). In the presence of a Cl- gradient (140:40, pipette:bath), the measured reversal potential for the PGE2-activated current was 26 ± 3 mV (n = 8), close to the value of 30 mV for the Cl- equilibrium potential. However, the current magnitude at 120 mV was significantly smaller than that observed with adrenaline stimulation, 25.7 ± 3.1 pA/pF (n = 8) as compared to 39.0 ± 5.2 pA/pF (n = 5, p < 0.04). To elicit significant current activation, a much higher concentration of PGF2 (100 µM) was used. As shown in Figure 5, a small increase in whole-cell current was obtained upon stimulation with PGF2, and yet the current characteristics remained similar to other agonist-stimulated whole-cell currents. A further current activation by forskolin after stimulation with PGF2, with similar current characteristics, was observed, suggesting the involvement of the same Cl- conductance.
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Figure 6 shows a comparison of current magnitudes at 120 mV elicited by different agonists, indicating an order of relative effect: forskolin > adrenaline > PGE2 >> PGF2.
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Sensitivity to Cl- Channel Blockers and Adenylate Cyclase Inhibitor
It has been reported that the cAMP-activated Cl- channel in many epithelial cell types exhibits a sensitivity to the Cl- channel blocker, DPC, but not DIDS [21]. The present study also examined the sensitivity of the cAMP-activated Cl- conductance to these two Cl- channel blockers. While DIDS at 100 µM, a concentration known to block other Cl- channels, did not have a significant effect on the agonist-induced current (n = 4, not shown), DPC (1 mM) blocked 99.8 ± 0.8% (n = 3) of the adrenaline-induced Cl- current (Fig. 7A). A similar effect of DPC on the forskolin-induced and PGE2-induced currents was also observed (Fig. 7B).
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The involvement of cAMP in activating Cl- conductance was further investigated using an adenylate cyclase inhibitor, MDL12330A. As shown in Figure 8, MDL12330A (20 µM) suppressed the PGE2-induced current. Similar suppression of the current by the protein kinase A inhibitor H-8 was also observed (not shown), further indicating the involvement of cAMP.
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Demonstration of CFTR Expression by Western Analysis
The expression of CFTR protein was demonstrated by Western blot analysis. The anti-CFTR monoclonal antibody detected a protein band, the immunoreactivity of which increased as the amount of protein used increased. The molecular mass of the protein band was between 113 kDa and 198 kDa (Fig. 9), which is close to the predicted value of 165 kDa reported for CFTR polypeptide.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study has demonstrated the activation of Cl- conductance upon stimulation with the neurohormonal agents adrenaline, PGE2 and PGF2 in mouse endometrial epithelial cells. The evidence supporting the involvement of Cl- in mediating the agonist-activated whole-cell current includes 1) a reversal potential of the current close to the equilibrium potential of Cl-, 2) the lack of effect on the current by replacement with an impermeant cation in the solutions, and 3) the inhibition of the current by the Cl- channel blocker DPC. The agonist-activated whole-cell current also exhibited characteristics similar to those reported for the cAMP-activated Cl- conductance found in many epithelial tissues [17–21]. These characteristics include a relatively linear I–V relationship, time- and voltage-independence, and sensitivity of the current to DPC but not DIDS. Given these characteristics and an anion selectivity sequence of NO3->Br->Cl->I-, the agonist-activated current in mouse endometrial epithelial cells appears to be a cAMP-activated Cl- conductance similar to that found in other epithelia. It should be noted that the characteristics associated with the cAMP-activated Cl- conductance observed in the present study are distinct from those associated with the Ca2+-activated Cl- conductance previously observed in the same endometrial epithelial cells [26] and in other epithelial cell types [17–21].
The fact that the agonist-activated current could be mimicked by the adenylate cyclase activator forskolin but suppressed by the adenylate cyclase inhibitor MDL12330A further suggests the involvement of cAMP. This is consistent with our previous findings on receptor-coupled signal transduction pathways in the mouse endometrial epithelium [10, 11]. We have previously demonstrated that the effect of adrenaline and noradrenaline on anion secretion is predominantly mediated by ß-adrenoceptors, which are well-known G protein-coupled receptors leading to activation of adenylate cyclase and production of cAMP. Studies on intact rat uteri also indicate the involvement of cAMP in the adrenaline-induced secretory response [12–14]. Our previous studies have also demonstrated that PGE2-stimulated anion secretion could be inhibited by an adenylate cyclase inhibitor, suggesting possible involvement of the cAMP-linked EP2/EP4 receptors [11]. It has been reported that PGF2 may also interact with EP2/EP4 receptors involved in a cAMP-dependent pathway [27]. Thus, the secretory responses elicited by the cAMP-activating neurohormonal agents tested appear to converge on the cAMP-activated Cl- conductance in mouse endometrial epithelial cells. It should be noted that the current magnitude elicited by different agonists varies, suggesting that different degrees of secretory response could be achieved by various neurohormonal factors. However, the high concentrations of agonist used in order to obtain detectable responses in the current patch-clamp study may raise concern about the physiological relevance of these agonists. It should be noted that concentration dependence of these stimuli in activating the cAMP-dependent anion secretion, as measured by the short-circuit current, across polarized endometrial monolayers has been previously examined [10, 11]. Effective concentrations are in nanomolar ranges. The discrepancy in the sensitivity to stimuli may be due to the difference in cell culture conditions. The cells used in the present study for patch-clamping were grown in plastic dishes instead of on permeable supports that allow the cells to form polarized monolayers as used in the short-circuit current experiments. Therefore, the number of receptors expressed may be different in the two cases, and it may have required higher concentrations to activate the same conductance in the present study. In addition to the possible influence of different cell culture conditions, the perturbation of cells by direct patching with dialysis cytosolic components into pipette solution may be another explanation for the observed difference in sensitivity.
An interesting finding of the present study is the similarity in the electrophysiological properties found between the cAMP-activated Cl- conductance and CFTR. This raises the possibility that many of the neurohormonal responses in the mouse endometrium may be mediated by CFTR. The demonstration of CFTR expression by Western blot analysis in the mouse endometrial cells is consistent with this notion. CFTR has also been shown to be expressed in the uterus of a number of species including humans and rodents [1–5]. Furthermore, CFTR expression is found to be regulated during development [3, 4] and during the estrous cycle [2], and has been shown to be influenced by estrogen [6] and progesterone [7]. The highly regulated CFTR expression in uterine tissues suggests that CFTR may play an important role in the normal function of the uterus. This possibility may gain support from the present finding that hormone-regulated secretory responses converge on the cAMP-activated Cl- channel, presumably CFTR. The importance of CFTR in uterine functions may be emphasized by the observation that women with cystic fibrosis (CF), the most lethal genetic disorder caused by mutations in the CFTR gene [28, 29], are less fertile than those not afflicted with CF [30–32]. However, the reasons for this observation are not well understood. While the reduced fertility in CF women is generally attributed to a dense, thick cervical mucus that forms a barrier for sperm penetration, the endometrium is not considered to be affected by CF. In contrast to this notion, the present study does indicate an important role of cAMP-activated Cl- conductance in mediating a variety of neurohormonal responses in mouse endometrial epithelial cells. The precise role of the Cl- conductance, presumably CFTR, in uterine functions remains to be elucidated.
In summary, the present study is the first to provide direct evidence of a cAMP-activated Cl- conductance, presumably CFTR, in mouse endometrial epithelial cells. An important role of the Cl- conductance in uterine functions is implied by the observed convergence of a number of neurohormonal responses on the conductance and its similar characteristics to those of CFTR. Future investigations into the regulation of the channel during various reproductive events may help further to elucidate its role in the physiology and pathophysiology of the uterus.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 825 2603 5022; hsiaocchan{at}cuhk.edu.hk
Accepted: September 18, 1998.
Received: March 19, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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