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Neuroscience Research Institute, State University of New York, College at Old Westbury, Old Westbury, New York 11568
Address requests for reprints to: Dr. G. B. Stefano, Neuroscience Research Institute, State University of New York, College at Old Westbury, Old Westbury, New York 11568-0210. E-mail: gstefano{at}sunynri.org.
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Abstract |
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Introduction |
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Justification for this hypothesis is based on earlier reports from our laboratory demonstrating that neural tissues in the marine mollusk M. edulis contain endogenous morphine, opiate receptors, opioid peptides, and their precursors as well as endocannabinoid signaling processes that are similar, or identical, to those found in human tissues (8, 9, 10, 11, 12). Furthermore, these reports also demonstrate that endogenous opiate alkaloid and endocannabinoid signaling are coupled to cNOS-derived NO release, demonstrating the conservation of these signaling processes during evolution. If estrogen processes are found in this organism, which evolved 500 million years before man, it would mean this is an ancient signaling mechanism that was important enough to conserve, underscoring its significance. Moreover, M. edulis also proves to be an excellent invertebrate experimental organism in which to study these estrogen processes.
The central nervous system of Mytilus is comprised of three ganglia, cerebral, visceral, and pedal, which are located in different parts of the animal. As such, this constitutes a diffuse central nervous system, which on an anatomical level does not have a vertebrate counterpart. The counterparts are simply the presence of these signaling families, which now appear to have evolved earlier than previously thought, i.e. before centralizing neural tissues into a brain. We examined M. edulis pedal ganglia to: 1) investigate whether acute estrogen exposure could stimulate rapid NO release via cNOS activation; 2) determine whether such 17ß-estradiol-stimulated NO release was dependent on the expression of an ER; and 3) determine whether estrogen itself was present.
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Materials and Methods |
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Tissue preparation
Pedal ganglia (50/group) and hemolymph (10 ml of each group) were extracted and ganglia were washed extensively with PBS. In addition, samples of seawater as well as incubation water were evaluated as blank controls and for potential contamination during the various procedures. The tissues were prepared in small fragments and then homogenized in a 1 N HCl water solution, 0.5 g wet weight of tissue per milliliter HCl. The resulting homogenates were then extracted using 5 ml chloroform/isopropanol in a 9:1 preparation. After 5 min at room temperature, the homogenates were centrifuged at 4000 rpm for 15 min. The observed three phases were separated as follows: the lowest layer corresponding to the organic phase, intermediated phase corresponding to precipitated proteins, and top aqueous supernatant phase. The aqueous and organic phases were collected and dried using a Centrivap console (Labconco, Kansas City, MO). The dried extract was stored at -20 C before experimentation.
HPLC purification
The HPLC system was a 626 pump (Waters, Milford, MA) equipped with a 2847 UV detector (Waters). The separation was performed using a Symmetry C8 column (Waters). The samples were injected by a 717plus autosampler (Waters), with the injection volume being determined by the sample size. The UV detector was set on 280 nm, 0.4 AUFS. The chromatographic system was controlled by Millennium32 Chromatography Manager (Waters) version 3.2 software, and the chromatograms were integrated within Chromatograph software (Waters).
HPLC analysis was conducted in the following manner. The mobile phases were: buffer A: 100% H2O, buffer B: 100% methanol. Both buffers A and B were filtered through a 0.22 µm filter (Waters), and the temperature of the system was maintained at 25 C. The running conditions were: from 0 min, 60% buffer B; 15 min, 100% buffer B; at 20 min 100% buffer B; and at 25 min, 60% buffer B.
Our extraction experiments used either internal or external estradiol standards as a positive control. Extractions of seawater, from the mussels’ environment, as well as incubation water were used as negative controls.
RIA for 17ß-estradiol
To generate antiserum in rabbits, 6-keto-estadiol-17ß-6-oxime-BSA was used as the immunogen. The antiserum was titered so as to bind to 30–40% of the 125I-estradiol derivative in the absence of nonradioactive estradiol. The specificity of the antiserum was determined by ICN Pharmaceuticals, Inc. (Costa Mesa, CA). Seven standards were used at the following concentrations: 0, 10, 30, 100, 300, 100, and 3000 pg/ml, and the assay was carried out in the following steps: 1) 100 µl of the estradiol standards were added as controls, and each of our samples was added to the coated tubes; 2) 1.0 ml 125I-estradiol was then added to all tubes; 3) the tubes were thoroughly vortexed and incubated at 37 C for 90 min; 4) decanted tubes were then counted in a 
-counter calibrated for 125I; and 5) the results were calculated using the formula supplied by ICN Pharmaceuticals, Inc.
Mass spectrometry
Mass spectrometry analysis has been shown to be an effective way of demonstrating the presence of estrogen in different samples (10, 16, 17). A commercial Q-TOF-MS system (Micromass, Manchester, UK) was used to test the fractions from the HPLC. One microliter methanol/water/NH4OH (50:49.7:0.3, vol/vol/vol) containing the sample was loaded in a gold-coated capillary (Micromass F-type needle). The sample was sprayed at a flow rate of 30 nl/min, giving an extended analysis time during which we acquired an MS spectrum. The source temperature was 100 C, capillary voltage was -2660 V, cone voltage was -8 V for MS and -50 V for MS/MS, and mass accuracy was 5 ppm (about 0.005 Da).
NO determination
For the NO determination, M. edulis pedal ganglia (10 per treatment) were dissected from animals maintained in laboratory conditions and stored on ice (13). The ganglia were subsequently washed and placed in sterile, filtered seawater as previously described (14).
NO release from the pedal ganglia was directly measured using an NO-specific amperometric probe (30 µm, 0.5 mm, World Precision Instruments, Sarasota, FL) in a manner described by Stefano et al. (18) and Magazine et al. (19). Briefly, the ganglia were placed in a superfusion chamber with 1 ml seawater along with a micromanipulator (World Precision Instruments), which was attached to the stage of an inverted microscope (Diaphot, Nikon, Melville, NY) that was used to position the amperometric probe 15 µm above the ganglia surface. Dissected ganglia were washed before measurement to remove any materials that might interfere with NO release. The system was calibrated daily with the nitrosothiol donor S-nitroso-N-acetyl-DL-penicillamine, resulting in the liberation of a known quantity of NO (World Precision Instruments). Baseline levels were determined by evaluation of NO release in sterile, 0.2 µm filtered seawater. The amperometric probe was allowed to equilibrate for at least 12 h before being transferred to the chamber containing the ganglia, and manipulation of the tissue was performed with only glass instruments. Each experiment was repeated four times along with a control from the same tissue source (vehicle alone) so as to exclude experimental drift in NO release unrelated to the study drugs.
To evaluate NO release, ganglia were exposed to various concentrations of 17ß-estradiol (17ß-E2). If an antagonist or NOS inhibitor was used, it was administered 5 min before that of the various estrogens. The NOS inhibitor, N-
-nitro-L-arginine methyl ester (L-NAME; Sigma, St. Louis, MO), was used in these studies. From the concentration curves of the estrogen agonist-stimulated NO release, the EC50 has been determined, and from antagonist concentration curves (10-13 to 10-6 M) against 10-9 M 17ß-E2 conjugated to BSA (E2-BSA), the IC50 of stimulated NO release occurs was also determined.
Data acquisition was by the computer-interfaced DUO-18 software (World Precision Instruments). The experimental values were then transferred to Sigma-Plot and -Stat (Jandel, San Rafael, CA) for graphic representation and evaluation. The differences between the data before and after treatment were tested for normal distribution. All data were normally distributed and subsequently evaluated by t test for paired samples. Data gatherers were unaware of the experimental treatments.
Ligands
Ganglia were stimulated with various concentrations of 17ß-E2 (10-11 to 10-6 M) or E2-BSA (10-11 to 10-6 M of 17ß-E2; Sigma). They were also stimulated with 17ß-E2 (10-7 M; n = 4) or tamoxifen (10-7 M; Sigma); estrogen receptor antagonist (n = 4) or tamoxifen (10-7 M) plus 17ß-E2 (10-7 M; n = 4); or tamoxifen (10-7 M) plus 17ß-E2 covalently conjugated to BSA (E2-BSA; 10-11 to 10-7 M of 17ß-E2) or E2-BSA (10-9 M; n = 4). Tamoxifen was added to the milieu 5 min before 17ß-E2 or E2-BSA. ICI 182,780 was used at a concentration of 10-7 M (Tocris Cookson, Inc., Ellisville, MO).
To demonstrate that there was no dissociation between 17ß-E2 and BSA, a RIA kit optimized for the direct quantitative determination of very low concentrations of free 17ß-E2 (ICN) was used. 17ß-E2 was measured in the cytosolic fraction of pedal ganglia (5 ganglia per milliliter) treated with 10-9 and 10-8 M E2-BSA. After washing the cells, they were put through a freeze-thaw cycle (cells were frozen in PBS for five min at -70 C and thawed in a 37 C water bath for 1 min). This process was repeated five times. The supernatants were harvested after centrifugation for 10 min at 12,000 x g in a refrigerated centrifuge and the pellet containing cell debris discarded. The cytosolic material was then evaluated for free E2. E2 was not detected in the cytosol, demonstrating that this material initiated its action via cell surface ER. The assay sensitivity was 0.2 pg/ml.
ER expression/Western blot analysis
Forty pedal ganglia excised from M. edulis were washed twice with cold PBS. Membrane and cytosolic proteins were separated and extracted from the tissues using the MEM-PER mammalian membrane protein extraction kit (Pierce Chemical Co., Rockford, IL), according to the manufacturer’s instructions. Protein concentration was determined by a protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). Cytosolic and membrane proteins (10 µg) were boiled for 5 min in a 2x loading buffer and then separated by electrophoresis on a 10% SDS-PAGE gel. The proteins were transferred onto a nitrocellulose membrane (Pierce Chemical Co.) and incubated overnight at 4 C in 5% blocking buffer [5% wt/vol nonfat dry milk in Tris-buffered saline/Tween 20 (TBS-T) buffer, pH 7.6]. The membrane was washed twice in TBS-T buffer (pH 7.6), and incubated for 2 h at room temperature in 5% blocking buffer containing a rabbit polyclonal antibody specific for ER-ß (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:5000. After incubation with the primary antibody, the membrane was washed three times, 10 min each wash. The blot was then incubated for 1 h in blocking buffer containing antirabbit IgG peroxidase-conjugated secondary antibody (Sigma) at a dilution of 1:50,000. A 10-min wash was done followed by two 15-min washes and then incubated for 5 min in 5 ml SuperSignal West substrate solution (Pierce Chemical Co.). The blot was then exposed to film (Biomax ML, Kodak, Rochester, NY). In the competition studies, 5-fold excess of blocking peptide was combined with the ER-ß polyclonal antibody and then incubated overnight at 4 C. This mixture was then used in the Western blot.
For ER
analysis, a Western blot was performed using a rabbit polyclonal antibody specific for ER (Santa Cruz Biotechnology). Ten micrograms cytosolic and membrane proteins were boiled for 5 min in a 2x loading buffer and then separated by electrophoresis on a 10% SDS-PAGE gel. The proteins were transferred onto a nitrocellulose membrane (Pierce Chemical Co.) and incubated overnight at 4 C in 5% blocking buffer (5% wt/vol nonfat dry milk in TBS-T buffer, pH 7.6). The membrane was washed twice in TBS-T buffer (pH 7.6) and incubated for 2 h at room temperature in 5% blocking buffer containing a rabbit polyclonal antibody specific for ER (Santa Cruz Biotechnology) at a dilution of 1:2000. After incubation with the primary antibody, the membrane was washed three times, 10 min each wash. The blot was then incubated for 1 h in blocking buffer containing antirabbit IgG peroxidase-conjugated secondary antibody (Sigma) at a dilution of 1:50,000. A 10-min wash was done followed by two 15-min washes and then incubated for 5 min in 5 ml SuperSignal West substrate solution (Pierce Chemical Co.). The blot was then exposed to film (Biomax ML, Kodak). In the competition studies, 5-fold excess of blocking peptide was combined with the ER polyclonal antibody and then incubated overnight at 4 C. This mixture was then used in the Western blot.
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Results |
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NO release was measured in real time using a NO-specific amperometric probe following the addition of 17ß-E2 to pedal ganglia (Fig. 1
). Normally pedal ganglia release low levels of cNOS-derived NO (0- to 1-nM range; Refs. 19 and 22). Increasing concentrations of 17ß-E2 (10-11 to 10-6 M) resulted in a dose-dependent increase in NO release with a maximal effect observed at 10-7 M (27-nM peak value; Fig. 1). This peak was observed during the 1-min period subsequent to the addition of 17ß-E2 (Fig. 1A, inset). Addition of 10-10 M 17ß-E2 failed to stimulate a significant increase in NO release (Fig. 1). Tamoxifen (10-7 M), an ER antagonist, inhibited 17ß-E2-stimulated ganglionic NO release (P < 0.005; Figs. 1 and 2), whereas ICI 182,780, another antagonist, did not. L-NAME (10-4 M), an NOS inhibitor, also inhibited the NO-stimulating activities of 17ß-E2 (Fig. 2). Addition of various concentrations of 17-E2 did not stimulate any NO release from the pedal ganglia (Fig. 1B). The results from these experiments demonstrate that the marine mollusk M. edulis possesses a 17ß-E2 signaling system, similar to the system in vertebrates, which acts via the release of NO.
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and 2). The E2-BSA-stimulated NO release is dose dependent, with a maximal effect at the same concentration (10-7 M) as 17ß-E2 (Fig. 2). Stimulation with 10-11 M E2-BSA failed to stimulate a significant increase in NO release. The EC50 for E2-BSA-stimulated NO release is approximately 7 x 10-9 M. Thus, our data support the hypothesis that E2-BSA acts at the cell surface and demonstrates that E2-BSA is as potent as 17ß-E2 in stimulating NO release (Figs. 1 and 2).
To ascertain whether the ganglia demonstrate ERß immunoreactivity that localizes to the membrane components of the ganglia, we performed Western blot analysis on both the membrane and cytosolic fractions with a rabbit polyclonal ERß antibody. The results show a protein of about 55 kDa, which corresponds to the expected size of the ERß protein in both the membrane and cytosolic fractions (Fig. 3, lane 3, membrane fraction; lane 4, cytosolic fraction). Furthermore, a band of similar size was observed in both membrane and cytosolic fractions of the positive control, human leukocytes, which are known to express a cell-surface ERß receptor (23) (Fig. 3, lane 5, membranes; lane 6, cytosol). No ERß immunoreactivity was observed in mantle tissue of M. edulis (Fig. 3, lanes 1 and 2) or Cos-7 cells (lanes 7 and 8) that do not express ERß. Blocking peptide controls for ERß (Santa Cruz Biotechnology) blocked the antibody, and no band was observed in the ERß-positive tissues (data not shown). Furthermore, cytosolic and membrane protein from pedal ganglia, mantle tissue, and human leukocytes was analyzed by Western blot analysis with an ER polyclonal antibody. A band at about 67 kDa corresponding to ER was observed in the cytosolic protein of human leukocytes but not in pedal ganglia or mantle tissue (data not shown).
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). This experiment was repeated three times for each tissue (50 animals/group) with similar results.
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Following the HPLC, RIA was performed on each of the HPLC fractions. The fractions containing estradiol showed positive readings at the same concentrations, compared with our HPLC results (Fig. 5). As predicted, the fractions that did not correspond to the estradiol standards did not show any positive readings. There were no positive readings from the HPLC fractions of seawater and incubation water (Fig. 5). These results substantiate that estradiol is both present in the extracted ganglia and endogenously produced.
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); incubation of ganglia with the HPLC fraction resulted in the immediate release of NO (27 ± 3.28 nM). This release was subsequently inhibited by the antagonist tamoxifen (10-7 M), which further attests to the existence of an ER in these tissues (see Fig. 2).
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), was virtually identical with the corresponding value for the authentic standard (Fig. 7B) and the theoretical value (271.1698 Da). Fragmentation analysis showed estradiol from the ganglia has the identical fragments (Fig. 7C), compared with the standard (Fig. 7D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-estradiol) rapidly stimulate NO release from M. edulis pedal ganglia. Our results suggest that rapid estrogen-mediated NO release is mediated by a specific ERß on the cell surface. The cell-surface localization of the ER is supported by the following observations. First, our data show that NO release is stimulated by E2-BSA (which does not enter the cell). Second, our data show that E2-BSA itself does not localize to the cytosol. Third, Western blot analysis shows that membrane fractions of M. edulis ganglia express an ERß-like protein of the expected size of 55 kDa but not ER. This protein is also expressed in human leukocytes that have been shown to express ERß on the cell surface (23). Furthermore, 17ß-E2- and E2-BSA-stimulated NO release are inhibited by L-NAME, a NOS inhibitor, indicating that the effect of the agonists on NO release is mediated by coupling the membrane ER to cNOS. Western blot analysis of M. edulis pedal ganglia demonstrates the expression of an ERß-like protein, suggesting that ERß is mediating estradiol’s action at the plasma membrane. Interestingly, a rabbit polyclonal anti-ERß antibody was able to identify a specific protein in M. edulis pedal ganglia that was also expressed in human leukocytes but not in M. edulis mantle tissue. This suggests that, in addition to the conservation of this signaling pathway, the molecules involved in the pathway, including the receptor itself, have retained a high degree of similarity.
In addition, using HPLC, we purified and with RIA identified material with estrogen-like activity when applied to pedal ganglia. Lastly, when this material is subjected to mass spectroscopy determination, it was confirmed to be 17ß-E2, conclusively demonstrating its presence in the ganglia. These data suggest that we have identified the same signaling pathway in the marine mollusk that had previously been identified in human tissue, meaning that this signaling system has been conserved over 500 million years of evolution.
This report complements other studies demonstrating that estrogen signaling may be present in invertebrates. In 1996 D’Aniello et al. (3) provided indirect biochemical evidence for the concurrence of sex steroid hormones and their binding proteins in Octopus vulgaris. Di Cosmo et al. (1) found sex steroid hormone fluctuations and morphological changes of the reproductive system in the female O. vulgaris throughout its annual cycle. In their study, estrogen was identified indirectly via RIA. In a more recent report (2), they identified, characterized, and immunolocalized an estradiol-17ß receptor in the reproductive system of the female O. vulgaris.
In unpublished data from our laboratory, we have also identified an estrogen-like molecule via HPLC and RIA in M. edulis gonadal tissue. The current finding of this molecule in the animals’ hemolymph increases the potential signaling mechanisms that may be involved. This molecule may exert both close and distant signaling in the organism’s reproductive structures and neural tissues, i.e. of the hormonal type.
In the human vascular system, we have demonstrated rapid estrogen signaling via cell surface ERs that are coupled to cNOS-derived NO release (4, 5, 6, 7). This NO release occurs within seconds of 17ß-E2 or E2-BSA application. Given the fact that E2-BSA does not enter the cytosol, we concluded, as in the current report, that this signaling is mediated by a cell surface receptor. Our studies, as well as those from other laboratories (24, 25, 26), demonstrate that estrogen signaling involves a rapid nongenomic component as well as a genomic component. The identification of this membrane-signaling pathway in M. edulis suggests that the multifaceted nature and complexity of estrogen signaling processes first evolved in simpler animals.
The significance of NO coupling in the rapid estrogen signaling mechanism can be, in part, deduced from previous studies involving opiate alkaloid and endocannabinoid signaling in M. edulis nervous tissues. NO was found to mediate neurotransmitter release and microglial egress from traumatized pedal ganglia (20, 21, 27, 28, 29, 30). Thus, estrogen signaling may influence these processes as well.
Furthermore, if there is redundancy in the signaling pathway, as suggested by endocannbinoid- and opiate alkaloid-coupled NO release, why would there be a need for estrogen signaling? In answer to this question, we speculated that each signaling system performs this common function, i.e. cNOS derived NO release, under different circumstances. Morphine levels, given the long latency before increases in its levels are detected, rise after trauma/inflammation to down-regulate these processes in neural and immune tissues (31, 32, 33). Anandamide, part of the ubiquitous arachidonate and eicosanoid signaling processes, serves to maintain tonal NO in vascular tissues (34). Estrogen, because of the fact that neither testosterone nor progesterone exerts this NO-generating action (7), provides an extra degree of functional down-regulation in female organisms. This most probably is due to both the immune, neural, and vascular trauma associated with cyclic reproduction activities when a high degree of vascular, neural, and immune activities are occurring (4, 5). Given the high degree of proliferative growth capacity during estrogen peak levels in this cycle, NO may function to enhance down-regulation of the immune system that allows for these changes. Clearly, as demonstrated by Di Cosmo et al. (1, 2) and D’Aniello et al. (3), estrogen processes appear to be involved in O. vulgaris’ reproductive cycles because they are presumed to be involved in those of M. edulis. Thus, the need for enhanced NO release, via estrogen signaling, at critical reproductive time points was initiated in invertebrates.
In conclusion, we demonstrate the presence of 17ß-E2 and an ERß receptor in invertebrate ganglia that is coupled to NO release. Estrogen is present in the animals’ gonads and hemolymph, suggesting a hormonal as well as reproductive function. Our data further suggest that in pedal ganglia 17ß-E2 can act through an ERß receptor that is expressed on the cell surface, suggesting immediate communication that is not genomic in nature. Finally, estrogen processes appear to have been developed much earlier in evolution than previously thought.
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Acknowledgments |
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Footnotes |
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Abbreviations: cNOS, Constitutive nitric oxide synthase; E2-BSA, 17ß-estradiol conjugated to BSA; ER, estrogen receptor; L-NAME, N--nitro-L-arginine methyl ester; NO, nitric oxide; TBS-T, Tris-buffered saline/Tween 20.
Received October 25, 2002.
Accepted for publication December 19, 2002.
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opioid receptor on human and invertebrate immune cells. Proc Natl Acad Sci USA 89:9316–9320 and ERß expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319This article has been cited by other articles:
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S. Gingerich and T. L. Krukoff Estrogen Modulates Endothelial and Neuronal Nitric Oxide Synthase Expression via an Estrogen Receptor {beta}-Dependent Mechanism in Hypothalamic Slice Cultures Endocrinology, July 1, 2005; 146(7): 2933 - 2941. [Abstract] [Full Text] [PDF]
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G. A. LeBlanc, M. P. Gooding, and R. M. Sternberg Testosterone-Fatty Acid Esterification: A Unique Target for the Endocrine Toxicity of Tributyltin to Gastropods Integr. Comp. Biol., January 1, 2005; 45(1): 81 - 87. [Abstract] [Full Text] [PDF]
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Y. Xia and T. L. Krukoff Estrogen Induces Nitric Oxide Production via Activation of Constitutive Nitric Oxide Synthases in Human Neuroblastoma Cells Endocrinology, October 1, 2004; 145(10): 4550 - 4557. [Abstract] [Full Text] [PDF]
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