Estradiol (E2) accelerates egg transport by a nongenomic action, requiring activation of estrogen receptor (ER) and successive cAMP and IP3 production in the rat oviduct. Furthermore, E2 increases IP3 production in primary cultures of oviductal smooth muscle cells. As smooth muscle cells are the mechanical effectors for the accelerated oocyte transport induced by E2 in the oviduct, herein we determined the mechanism by which E2 increases IP3 in these cells. Inhibition of protein synthesis by Actinomycin D did not affect the E2-induced IP3 increase, although this was blocked by the ER antagonist ICI182780 and the inhibitor of phospholipase C (PLC) ET-18-OCH3. Immunoelectron microscopy for ESR1 or ESR2 showed that these receptors were associated with the plasma membrane, indicating compatible localization with E2 nongenomic actions in the smooth muscle cells. Furthermore, ESR1 but not ESR2 agonist mimicked the effect of E2 on the IP3 level. Finally, E2 stimulated the activity of a protein associated with the contractile tone, calcium/calmodulin-dependent protein kinase II (CaMKII), in the smooth muscle cells. We conclude that E2 increases IP3 by a nongenomic action operated by ESR1 and that involves the activation of PLC in the smooth muscle cells of the rat oviduct. This E2 effect is associated with CaMKII activation in the smooth muscle cells, suggesting that IP3 and CaMKII are involved in the contractile activity necessary to accelerate oviductal egg transport.
The canonical pathway by which estradiol (E2) affects its target cells comprises binding to estrogen receptors (ER) and modification of gene expression and protein synthesis (Nilsson et al. 2001). However, some E2 effects cannot be blocked by inhibitors of transcription or translation, or are too rapid to be due to changes in gene expression. These features do not appear compatible with the classical genomic actions and are termed nongenomic (Lössel et al. 2003, Lössel & Wheling 2003). E2 nongenomic actions often involve the activation of G protein-α inhibitory (Gαi), stimulation of intracellular signal transduction pathways, including the generation of second messengers such as cAMP and IP3, and activation of protein kinase A (PKA) or phospholipase C (PLC) in the E2-target cells (Nadal et al. 2001, Wyckoff et al. 2001, Acconcia et al. 2005, Hill et al. 2010).
In the rat, a single injection of E2 on day 1 of the cycle or pregnancy shortens oviductal transport of eggs from the normal 72–96 h to <24 h (Ortíz et al. 1979). We have previously demonstrated that RNA and protein synthesis inhibitors did not block E2-induced oviductal egg transport acceleration in unmated rats indicating that E2 accelerates oviductal egg transport by a nongenomic mechanism (Orihuela et al. 2001). This E2 nongenomic pathway involves a previous conversion of E2 to methoxyestradiols through the activation of catechol-O-methyltransferase (COMT) (Parada-Bustamante et al. 2007, 2010), ER and adenylyl cyclase (AC) (Orihuela et al. 2003), and sequential production of cAMP and IP3 (Orihuela et al. 2003, 2006, 2013).
The rat oviduct is mainly composed of an intrinsic layer of smooth muscle fibre, and an innermost highly folded mucosa formed by epithelial and stromal cells, the endosalpinx (reviewed in Croxatto (2002)). Transport of oocytes along the oviduct depends on the interaction between the secretory activity of the epithelial cells and the contractile activity of the smooth muscle cells (Moore & Croxatto 1988a,b, Ríos et al. 2007). The regulation of muscular motility is influenced by E2 and requires activity of adrenergic nerves (Helm et al. 1982), nitric oxide (Perez Martinez et al. 2000), endothelin (Parada-Bustamante et al. 2012), oxytocin (Jankovic et al. 2001) and prostaglandins (Wijayagunawardane et al. 2003). These factors activate intracellular signalling mainly associated with Ca2+, cAMP or IP3 (Jankovic et al. 2001, Barrera et al. 2004, Mohan et al. 2012). In this context, we have recently shown that, in the epithelial cells of the rat oviduct, E2 increased cAMP production between 3 and 6 h, although IP3 levels were not affected. Moreover, E2 increased cAMP in the oviductal epithelial cells by a nongenomic mechanism that requires coupling between ESR1 and Gαi and stimulation of AC (Oróstica et al. 2014). Previous research has also shown that E2 increased IP3 levels in primary cultures of smooth muscle cells from the rat oviduct (Oróstica et al. 2014).
As smooth muscle cells are the mechanical effectors for the accelerated oocyte transport induced by E2 in the oviduct (Croxatto 2002), this work determined the mechanism by which E2 increases IP3 in primary cultures of rat oviductal smooth muscle cells. Thus, we examined the effect of E2 on the IP3 levels in the smooth muscle cells under conditions in which protein synthesis, ER, GαI or PLC activity were blocked by selective inhibitors. The subcellular localization of ESR1 and ESR2 as well as the effect of selective agonists for ESR1 or ESR2 on the IP3 level was evaluated in the smooth muscle cells. Furthermore, expression of GαI in the oviductal smooth muscle cells was also determined. Finally, the effect of E2 on the activity of the enzyme associated to muscle contraction calcium–calmodulin protein kinase II (CaMKII) was determined in the primary cultures of smooth muscle cells.
Materials and methods
Locally bred Sprague-Dawley rats weighing 200–260 g were used. Animals were kept under controlled temperature (21–24 °C), and lights were on from 0700 to 2100 h. Water and pelleted rat chows were supplied ad libitum. Female mature rats were used in the estrous stage. The phases of the estrous cycle were determined by daily vaginal smears (Turner 1961) and all females were used after showing 2 consecutive 4-day cycles. The Ethical Committees of the Universidad de Santiago de Chile and the National Fund of Science (CONICYT-FONDECYT 1110662) approved the protocols for the care and manipulation of the animals.
Culture of primary smooth muscle cells from rat oviducts
For each replicate, 12 oviducts from six rats were excised and placed in pre-warmed Hanks's solution (Sigma Chemical) at pH 7.4. The whole oviduct was cut into small (4–8 mm2) pieces in Hanks's solution and then the smooth muscle cells were mechanically removed from the rest of the tissue and treated with Collagenase, Type I (Invitrogen) for 1 h to further disaggregation of the cells. The cell suspension was centrifuged at 1200 g during 5 min, washed, and seeded into six-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ, USA) in DMEM/High Modified medium with 4.0 mM l-glutamine and 4.5 g/l glucose free of Phenol Red (Cat. No. SH30284.02, HyClone, Thermo Scientific, Waltham, MA, USA) supplemented with 10% (v/v) foetal bovine serum (Cat. No. SH30396.03, HyClone), 1 mM sodium pyruvate and 100 UI/ml penicillin and 100 μg/ml streptomycin. Smooth muscle cells were incubated at 37 °C in an atmosphere of 5% (v/v) CO2 for at least 7 days to reach 75–80% confluence and their purity verified by immunofluorescence staining for cytokeratin (marker of epithelium cells), vimentin (marker of fibroblasts) or α-actin (marker of smooth muscle cells) antibodies.
Primary cultures of smooth muscle cells were changed to medium without serum for 15 h before each treatment. Then, they were treated with 10−9 M E2 (Sigma) or 0.01% ethanol as vehicle. Other primary cultures of smooth muscle cells were also incubated with the protein synthesis inhibitor 1 μg/μl Actinomycin D (ActD, Goldberg et al. 1962, Sigma), the ER antagonist 25 μg/μl ICI 182780 (Gagliardi & Collins 1993, Tocris Bioscience, Bristol, UK), the PLC inhibitor 1 μM ET-18-OCH3 (Powis et al. 1992, Calbiochem, La Jolla, CA, USA) or the Gαi protein inhibitor pertussis toxin 1 μg/ml PTX (Lee et al. 2002, Sigma) as appropriate to each experiment. Furthermore, the ESR1 agonist 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (10−7–10−5 M PPT, Sigma) or ESR2 agonist Diarylpropionitrile (10−7–10−5 M DPN, Sigma) were added to the primary smooth muscle cells. The concentrations of PPT and DPN used in this work were based considering that both drugs have lower affinity for ESR1 and ESR2 than E2 (Harris et al. 2002, Frasor et al. 2003, Harrington et al. 2003). DMSO at 0.01% was used as a vehicle for the inhibitors and agonists because it is more efficient than ethanol at dissolveing nonpolar or semi-polar drugs.
Extraction and measurement of IP3
Primary smooth muscle cell cultures were sonicated in 100 μl of ice-cold 1 M trichloroacetic acid (TCA) and an aliquot was taken to measure protein concentration by the Bradford assay using BSA dissolved in 1 M TCA as standard (Bio-Rad). The remaining homogenate was then centrifuged for 10 min at 1000 g at 4 °C. The pellet was discarded and the supernatant was incubated for 15 min at room temperature. TCA was removed from the supernatant with 0.5 ml of a solution 1,1,2-Trichloro-trifluoroethane (TCTFE, Sigma)-Trioctylamine (Sigma), 3:1 (v/v). Levels of IP3 were determined using IP3 [3H] radioreceptor assay Kit, Cat. No NEK064 (NEN Life Science Products, Boston, MA, USA). This kit is based on competition between non-radioactive IP3 and a fixed quantity of [3H]-IP3, for a limited number of calf cerebellum IP3 receptor binding sites. This allows the construction of a standard curve and the measurement of IP3 levels in unknown samples.
Polyclonal antibodies that recognize the phosphorylated state of CaMKII on Thr286 (anti-phospho-CaMKII, Cell Signaling Technology, Beverly, MA, USA) or total CaMKII (anti-CaMKII, Abcam, Cambridge, UK) were used to assess activation of CaMKII. Smooth muscle cells were processed by duplicate to determine the activity of the CaMKII protein. Cells were lysed in lysis buffer (20 mM Tris–HCl, pH 8.0, 137 mM NaCl, 1% Nonidet P-40 and 10% glycerol) supplemented with a protease inhibitor cocktail (Roche Diagnostics). The lysate was centrifuged at 4 °C for 10 min at 10 000 g and the pellet was discarded. Protein concentrations in the supernatant were measured by the Bradford assay (Bio-Rad). After boiling for 5 min, proteins (20 μg) were separated on 10% SDS–PAGE slab gels in a Mini PROTEAN electrophoretic chamber (Bio-Rad). Proteins resolved in the gels were electroblotted onto nitrocellulose membranes (Bio-Rad). The membranes were blocked 3 h in TTBS (100 mM Tris–HCl pH 7.5, 150 mM NaCl and 0.05% Tween-20) that contained 5% nonfat dry milk and were incubated overnight with 0.4 μg/ml rabbit anti-phospho-CaMKII (Cell Signaling Technology). The immunoreactive band was visualized by incubation for 1 h with 0.04 μg/ml goat anti-rabbit IgG antibody (Chemicon International, Temecula, CA, USA) conjugated to HRP, followed by the Enhanced Western Lighting Chemiluminescence reaction (PerkinElmer Life Sciences, Boston, MA, USA). Blots were stripped in 100 mM β-mercaptoethanol, 2% SDS and 62.5 mM Tris–HCl, pH 6.7 at 60 °C for 30 min, and reprobed with 0.2 μg/ml rabbit anti-CaMKII antibody and developed in a similar manner to ensure even loading. All blots were then digitalized and the relative level of phospho-CaMKII was normalized against total CaMKII. Oviductal samples without anti-phospho-CaMKII or anti-CaMKII antibody were included as negative controls.
A post-embedding immunogold-labelling method that preserves cellular integrity and maintains ER immunogenicity was used (Kessels et al. 1998, Qualmann et al. 2000, Orihuela et al. 2009). Primary smooth muscle cell cultures were fixed in 4% freshly depolymerised paraformaldehyde, 0.5% glutaraldehyde in 1 M phosphate buffer pH 7.4 containing 0.1 M saccharose, 1% DMSO and 1% CaCl2 for 2–4 h at room temperature. The fixed samples were washed three times with phosphate buffer, dehydrated in a graded ethanol series and infiltrated with LR Gold (Plano, München, Germany). Subsequently, the samples were transferred to gelatin capsules filled with 0.8% (w/v) benzoyl peroxide in LR Gold and kept for polymerization at a pressure of 500 mmHg. The blocks were cured for 1–2 days at room temperature before sectioning with a Sorvall-2000 ultramicrotome using a diamond knife. The sections (50–80 nm) were mounted on formvar-coated nickel grids and incubated on droplets of 0.1 M glycine in PBS pH 7.6, and subsequently blocked with 1% bovine fetal serum for 2 h at room temperature. The grids were then incubated for 2 h with 4.0 μg/ml rabbit anti-ESR1 (MC-20, Santa Cruz Biotechnology) or 4.0 μg/ml anti-ESR2 (clone 68-4, Chemicon International). After washing with PBS, the preparations were incubated for 1 h with 0.3 μg/ml goat anti-rabbit immunoglobulin conjugated to 10 nm gold particles (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). Sections were washed and contrasted with Reynolds stain (Reynolds 1963). All sections were examined using a Phillips-TECNAI 12 BIOTWIN EM Microscope (FEI Company, Hillsboro, OR, USA) at 80 kV. As negative control for ESR1 and ESR2, the primary antibody was replaced by preimmune serum. At least ten areas of 63 μm2 from different smooth muscle cells from the primary cultures were photographed and the photomicrographs were digitalized in an iBook computer (Apple Computer, Cupertino, CA, USA), and gold particles present only in the cells were counted using the image analysis Software Adobe Photoshop 7.0 (Adobe Systems) by an observer blinded to the different groups. The results of the immunolabelling are presented as the quotient of the number of gold particles present divided by the area and cell number analysed in three different cultures (Orihuela et al. 2009).
Smooth muscle cells were fixed in cold 4% paraformaldehyde in PBS pH 7.4–7.6 for 2 h, transferred to 10% (w/v) saccharose in PBS for 60 min at 4 °C and 30% (w/v) saccharose in PBS at 4 °C overnight. Then, they were blocked with 1% PBS–BSA for 120 min, and incubated with 0.8 μg/ml mouse anti-cytokeratin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 2.5 μg/ml mouse anti-vimentin (Santa Cruz Biotechnology), 0.4 μg/ml mouse anti-α-actin (Santa Cruz Biotechnology) or 5.0 μg/ml mouse anti-Gαi (Santa Cruz Biotechnology) antibodies. After washing with PBS, the preparations were incubated for 2 h with 0.5 μg/ml Alexa fluor 588-conjugated goat anti-mouse IgG (Invitrogen). Sections were washed and counterstained with 1 μg/ml of Hoechst 33342 (Thermo Scientific, Rockford, IL, USA) washing again and then mounted in Fluoromount G. As negative controls, the primary antibody was replaced by preimmune serum. As positive control for Gαi we used samples of whole oviducts from rats on day 1 of the estrous cycle (Oróstica et al. 2013, 2014). All sections were visualized with an Optiphot Epifluoresence Microscope (Olympus).
Total RNA from primary secretory cell cultures was isolated using Trizol Reagent (Invitrogen). One microgram of total RNA of each sample was treated with Dnase I Amplification grade (Invitrogen). The single-strand cDNA was synthesized by reverse transcription using the Superscript III Reverse Transcriptase First Strand System for RT-PCR (Invitrogen), according to the manufacturer's protocol. The Light Cycler instrument (Roche Diagnostics) was used to quantify the relative gene expression of the E2-target genes c-fos (Nilsson et al. 2001) in the oviductal smooth muscle cells; Gapdh was chosen as the housekeeping gene for load control. The SYBR Green I double-strand DNA binding dye (Roche Diagnostics) was the reagent of choice for these assays. Primers for c-fos were 5′-CCG AGA TTG CCA ATC TAC TG 3′ (sense) and 5′-AGA AGG AAC CAG ACA GGT CC 3′ (antisense) and for Gapdh were 5′-ACC ACA GTC CAT GCC ATC AC 3′ (sense) and 5′- TCC ACC ACC CTG TTG CTG TA 3′-(anti sense). All real-time PCR assays were performed in duplicate. The thermal cycling conditions included an initial activation step at 95 °C for 5 min, followed by 40 cycles of denaturizing and annealing-amplification (95 °C for 15 s, 59 °C for 30 s and 72 °C for 30 s) and finally one cycle of melting (60° up to 95 °C). The expression of c-fos was determined using the equation: Y=2−ΔCp (16) where Y is the relative expression, Cp (crossing point) is the cycle in the amplification reaction in which fluorescence begins to be exponential above the background base line, −ΔCp is the result of subtracting Cp value of c-fos from Cp value of gapdh for each sample. To simplify the presentation of the data, the relative expression values were multiplied by 103 (Livak and Schmittgen 2001).
Data for IP3 and CaMKII assays from cultured oviductal cells were replicated five times for each treatment (for each culture experiment, oviductal cells were recovered from a pool of six different rats). Statistical analysis was performed using a GraphPad Prism 5.0 Software program. All data are presented as mean±s.e.m. These data followed a non-normal distribution (Kolmogorov–Smirnov test) and significant differences between groups were determined through the use of variance analysis by Friedman's test with subsequent post-hoc Wilcoxon signed-rank test. Significance was accepted at P<0.05. On the other, the quantitative analysis of the ESR1 or ESR2 distribution was subjected to Kruskal-Wallis test, followed by Mann-Whitney's U tests for pairwise comparisons when overall significance was detected. Significance was accepted at P<0.05.
E2 increased IP3 production by a nongenomic action in the smooth muscle cells from the rat oviduct
Primary cultures of smooth cells from rat oviducts, with a purity of 90–95% (Fig. 1), were divided into the following treatment groups: i) ethanol+DMSO, ii) E2+DMSO, iii) ethanol+ActD and iv) E2+ActD. At 0, 1, 3, 6 or 9 h after treatment, cultured cells were processed to measure the concentration of IP3 as described in the ‘Materials and methods’ section.
Figure 2 shows that in the vehicle group, IP3 production ranged from 114.1±30.3 to 123.2±26.7 fmol/μg of protein while in the E2-treated group it was increased at 6 h (523.2±66.7 fmol/μg of protein) but not at 1, 3 or 9 h. Administration of ActD alone or concomitant with E2 did not affect the time-course of IP3 production in the control or in the E2-treated group.
E2 increased IP3 production through ER and PLC activation in the oviductal smooth muscle cells
In each experiment, primary cultures of smooth muscle cells from rat oviducts were divided into the following treatment groups: i) ethanol+DMSO, ii) E2+DMSO. iii) ethanol+inhibitor and iv) E2+inhibitor. At 0, 1, 3, 6 or 9 h after treatment, cultured cells were processed to measure the concentration of IP3 as described in the ‘Materials and methods’ section.
Figure 3 shows that in the control groups, the IP3 level ranged from 151.6±26.3 to 193.5±37.3 fmol/μg of protein while in the E2-treated groups it ranged from 488.3±59.7 to 599.4±71.3 fmol/μg of protein. Administration of ICI 182780 or ET-18-OCH3 alone did not affect the basal IP3 production although blocked the E2-simulated IP3 increase at 6 h.
ESR1 and ESR2 are localized in association with the plasma membrane of the oviductal smooth muscle cells
Primary cultures of smooth muscle cells from rat oviducts with no treatment were processed by immunoelectron microscopy using specific antibodies for ESR1 and ESR2.
Figure 4A shows that immunoreactivity for ESR1 and ESR2 was found associated to the plasma membrane, cytoplasm and nucleus in the oviductal smooth muscle cells. Furthermore, Fig. 4B shows that the quantitative analysis of the ESR1 or ESR2 distribution was a higher number of ESR1 and ESR2-reacting gold particles in the nucleus than in the plasma membrane or cytoplasm of the smooth muscle cells.
Activation of ESR1 but not ESR2 mimic the effect of E2 on the IP3 production in the oviductal smooth muscle cells
Primary cultures of smooth muscle cells from rat oviducts were treated with DMSO, PPT (10−7–10−5 M) or DPN (10−7–10−5 M) during 0, 1, 3, 6 or 9 h and processed to measure the concentration of IP3 as described in the ‘Materials and methods’ section. Other smooth muscle cells cultures were also treated with DMSO or DPN 10−7 M and 6 h later processed by Real-Time PCR to determine the mRNA level of c-fos.
Figure 5 shows that in the control group, the IP3 production ranged from 120.3±44.1 to 148.5±50.6 fmol/μg of protein while treatment with PPT increased IP3 level in a dose-dependent manner at 6 h without any effect at 0, 1, 3 or 9 h. On the other hand, administration of DPN had no effect on the IP3 level at any time or concentration studied (Fig. 5), although it increased the mRNA level of c-fos (control: 51.4±11.3 vs DPN: 210.7±36.2 relative expression, n=5).
GαI protein is not required for the IP3 production increase induced by E2 in the oviductal smooth muscle cells
Primary cultures of smooth cells from rat oviducts were divided into the following treatment groups: i) ethanol+DMSO, ii) E2+DMSO. iii) ethanol+PTX and iv) E2+PTX. At 0, 1, 3, 6 or 9 h after treatment, cultured cells were processed to measure the concentration of IP3 as described in the ‘Materials and methods’ section.
Figure 6A shows that in the control group, IP3 production ranged from 124.9±38.1 to 133.5±36.9 fmol/μg of protein while in the E2-treated group it was increased at 6 h (601.4±98.5 fmol/μg of protein) but not at 1, 3 or 9 h. Administration of PTX alone or concomitant with E2 did not affect the time-course of IP3 production in the control or in the E2-treated group.
GαI protein is not expressed in the smooth muscle cells from the rat oviduct
Primary cultures from smooth muscle cells from rat oviducts were processed by immunofluorescence in order to assess the immunoreactivity of GαI in these cells. In addition, we used sections of whole oviducts from rats on day 1 of the oestrous cycle to corroborate presence of Gαi in the oviductal tissues. This experiment was replicated five times.
Figure 6B shows that immunoreactivity of GαI was not found either in the primary cultures of the oviductal smooth muscle cells or in the myosalpinx layer of the whole oviduct. However, GαI was expressed in the endosalpinx of the rat oviduct as previously reported by Oróstica et al. (2014).
E2 induced activation of CaMKII in the oviductal smooth muscle cells
Primary cultures from rat oviductal secretory cells were treated with ethanol or E2 10−9 M and 6.5 h later the level of phosphorylated CaMKII (p-CaMKII) was assessed by immunoblot. As E2 increases the IP3 level at 6 h after treatment, we consider that 6.5 h is a reasonable time to evaluate activation of CaMKII downstream of the IP3 increase.
Figure 7 shows that E2 increased the level of p-CaMKII in comparison with the vehicle group.
The contribution of the different cell phenotypes of the rat oviduct on the E2 nongenomic pathway associated with the cAMP-IP3 signalling and involved in the accelerated egg transport is recently being disclosed (reviewed in Orihuela et al. (2013)). Here we show that E2 increased IP3 levels in the oviductal smooth muscle cells by a nongenomic mechanism because suppression of mRNA and protein synthesis by ActD did not prevent the effect of E2 on the IP3 level. Moreover, the E2 nongenomic pathway that increases IP3 requires activation of ER and PLC since blockade of ER by ICI 182780 and PLC by ET-18-OCH3 reverted the E2-induced IP3 increase in the oviductal smooth muscle cells. Previous works have shown that some E2 nongenomic pathways are associated with changes in the turnover of inositol lipids that generates IP3 from the hydrolysis of phosphatidylinositol 4,5-biphosphate in several cell systems (Kisielewska et al. 1996, 1997, Razandi et al. 1999, Ariazi et al. 2010). Our findings show for the first time that a nongenomic action of E2 associated with PLC-IP3 signalling is also present in the smooth muscle cells of the mammalian oviduct. The effect of E2 on the IP3 level occurred from 6 h and declined at 9 h, indicating a transient action on the PLC-IP3 signalling in the smooth muscle cells. A rapid turnover of IP3, inactivation of PLC or down-regulation of ER could explain the lack of effect E2 on the IP3 level at 9 h; however, this remains to be determined.
In contrast to other reports that showed a rapid increase of IP3 by E2 in rat vaginal epithelial cells and HEPG2 cells (Singh & Gupta 1997, Marino et al. 1998), we found that E2 has a time of latency of 6 h to exert its effects on the IP3 production in the smooth muscle cells from the rat oviduct. Differences in the expression of the ER isoforms or in the signalling pathways between the different cell phenotypes may explain the delayed response to E2 in the smooth muscle cells. We postulate that the E2 nongenomic action that increases IP3 appears as a secondary response to intracellular changes localized upstream of PLC activation in the oviductal smooth muscle cells. According to this assumption, we have recently shown that E2-induced IP3 increase is preceded by a cAMP decrease in smooth muscle cells of the rat oviduct (Oróstica et al. 2014). Alternatively, E2 may be first metabolized into 2-methoxyestradiol to increase IP3 production in the oviductal smooth muscle cells. In this context, various biological effects of E2 including regulation of egg transport in the rat oviduct or modulation of the antihypertensive and neuroprotective effects of E2, requires previous conversion from E2 to 2ME in its target organs (reviewed in Dubey & Jackson (2001) and Parada-Bustamante et al. (2015)).
Inhibition of the ER activity did not affect basal IP3 production in the oviductal smooth muscle cells, indicating that other ER-independent signalling pathways are acting to state basal IP3 level. In according with this idea, various signalling pathways such as Angiotensin-II, arachidonic acid, endothelin-1 and norepinephrine regulate production of IP3 (reviewed in Bolton (2006)). On the other hand, ET-18-OCH3 alone had no effect on the IP3 production in the oviductal smooth muscle cells. Since 13 mammal PLC subtypes have actually been reported (Rhee 2001), it is probable that basal IP3 production depends on an ET-18-OCH3-insensitive PLC. In this context, it has been found that ET-18-OCH3 is more effective at inhibiting membrane-associated PLC-β1 than PLC-γ1 localized in the cytosol of human fibroblasts (Powis et al. 1992).
Physiological effects of E2 are mainly influenced by the differential distribution of ESR1 and ESR2 in its target organs. Our results showing localization of ESR1 and ESR2 in the cell membrane, cytoplasm and nucleus indicate compatible localization of both ER subtypes with genomic and nongenomic actions of E2 in the smooth muscle cells of the rat oviduct. These findings corroborate previous studies on the presence of ER in nuclear and extranuclear regions in the rat oviductal epithelium cells (Orihuela et al. 2009) and reinforces the concept that signalling pathways associated with E2 nongenomic and genomic actions operate in the mammalian oviduct (Parada-Bustamante et al. 2010, 2012, Orihuela et al. 2013). The fact that a lower proportion of ESR1 and ESR2 was found in the plasma membrane compared with those present in the cytoplasm and nucleus of the oviductal smooth muscle cells is consistent with the notion that only a small pool of ER is responsible for the E2 nongenomic actions (reviewed inWatson & Gametchu (2003) and Wehling et al. (2006)).
The functional relevance of ER subtypes on the E2-induced IP3 increase in the oviductal smooth muscle cells was investigated utilizing selective agonists for ESR1 (Harris et al. 2002) or ESR2 (Frasor et al. 2003). We found that activation of ESR1 by PPT mimicked the effect of E2 on the IP3 levels while activation of ESR2 by DPN had no effect. This suggests that the nongenomic pathway by which E2 increases IP3 in the oviductal smooth muscle cells operates through ESR1 activation. This is in keeping with previous works showing that the nongenomic actions of estrogenic compounds require activation of ESR1 (reviewed in Marino et al. (2006), Moenter & Chu (2012) and Watson et al. (2014)). On the other hand, the E2 nongenomic pathways associated with activation of ESR2 in the smooth muscle cells of the rat oviduct needs to be disclosed.
It has been documented that the Gαi subclass is the G protein most often linked with the E2 nongenomic actions coupled with a presumptive ESR1 localized in extranuclear sites (Wyckoff et al. 2001, Kumar et al. 2007, Lin et al. 2011, Watson et al. 2012). However, the ADP-ribosylating agent pertussis toxin did not block the effect of E2 on the IP3 production in the smooth muscle cells, suggesting that heterotrimeric Gi/o-type proteins are not required for this E2 nongenomic action. This is corroborated by the fact that the Gαi protein was not expressed either in the primary cultures of smooth muscle cells or in the myosalpinx layer (mainly composed of smooth muscle fibers) of whole oviducts from estrous rats. Gαs-, Gq- or Gβγ-coupled receptors and tyrosine kinase receptors also activate PLC subtypes in a variety of cell systems (Zhu & Birnbaumer 1996, Yang et al. 2013). Furthermore, small Rho and Ras GTPases participate in the activation of the PLC-ϵ (Yang et al. 2013). Since differential expression of PLC subtypes and G proteins is unknown in the smooth muscle cells from the rat oviduct, further studies are necessary to establish the mechanism by which the E2-ER complex activate PLC in these cells.
Previous reports have shown that the cytokine Tumour Necrosis Factor-α (TNF-α) stimulated production and release of contractile mediators as prostaglandins, endothelins and nitric oxide in the bovine oviduct (Wijayagunawardane & Miyamoto 2004, Szóstek et al. 2011). Moreover, TNF-α modulates the effect of E2 on the oviductal egg transport and TNF-α receptors are present in the rat oviduct (Oróstica et al. 2013). It is probable that the E2 nongenomic pathway that increases IP3 could be associated with TNF-α signalling in the oviductal smooth muscle cells, but this needs to be further explored.
It is well known that binding of IP3 to its specific receptors (types 1, 2 and 3) localized on the intracellular compartments (nucleus, plasma membrane or endoplasmic reticulum) participates in Ca2+ signalling by mediating intracellular Ca2+ release, which results in activation of proteins associated with smooth muscle contraction (Kamm & Stull 1985, Kim et al. 2000). In this context, CaMKII is a serine/threonine kinase that is activated by Ca2+ and Calmodulin and it is implicated in regulation of vascular tone through phosphorylation of contractile proteins, including myosin light chain kinase (MLCK), the 20-kD myosin light chain (LC20), phospholipase-α2 and the α-subunit of Ca2+ channel (reviewed in Kim et al. (2008)). Furthermore, the synthetic estrogen, estradiol benzoate, is able to stimulate CaMKII activity in mouse hippocampus by a nongenomic mechanism (Sawai et al. 2002). Similarly, we found that E2 increased CaMKII phosphorylation in the smooth muscle cells of the rat oviduct. To our knowledge, this is the first time that an effect of E2 on the activity of CaMKII in the mammalian oviduct is reported. As E2 activated CaMKII presumably downstream of the IP3 increase, it is probable that this enzyme could be part of the PLC-IP3 signalling cascades induced by estrogens to activate nongenomic actions in smooth muscle cells.
Several works have reported that E2 exerts its vasoprotective actions regulating the contractile tone of the vascular, airway and myometrium smooth muscle cells (Kisielewska et al. 1996, Townsend et al. 2010, 2012, Cairrão et al. 2012, Holm et al. 2013). These effects are mainly associated with changes in the intracellular Ca2++ mobilization associated with cAMP and IP3 signalling pathways (Kisielewska et al. 1996, 1997, Towsend et al. 2010, 2012). Since E2 increases the frequency of myosalpinx contractions that accelerate oviductal egg transport in the rat (Moore & Croxatto 1988a,b), we can speculate that this E2 effect involves IP3 production and activation of CaMKII in the smooth muscle cells. This E2 effect on the oviductal egg movement could be involved in the very early maternal–embryo interactions that occur in the uterine tissues necessary to the embryo implantation (Gomez & Muñoz 2015).
In summary, we have found that E2 increases IP3 by a nongenomic action operated by ESR1, and that involves activation of PLC in the smooth muscle cells of the rat oviduct. Furthermore, E2 activates CaMKII presumably downstream of the IP3 increase in the oviductal smooth muscle cells, suggesting that IP3 and CaMKII are involved in the contractile activity of the oviduct. These findings provide new evidence to understand the molecular mechanisms underlying the role of smooth muscle cells on the E2 nongenomic action that accelerates egg transport in the rat oviduct.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
This work received financial support from grants FONDECYT # 1110662, Proyecto BASAL FBO-07 and Proyectos Basales y Vicerrectoría de Investigación, Desarrollo e Innnovación.
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