Institute of Bioengineering, Miguel Hernández University, Campus of San Juan, Alicante 03550, Spain
Address all correspondence and requests for reprints to: Dr. Angel Nadal, Institute of Bioengineering and Department of Physiology, School of Medicine, Miguel Hernández University, San Juan Campus Carretera Alicante-Valencia Km 87. 03550, Alicante, Spain. E-mail: nadal{at}umh.es.
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ABSTRACT |
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INTRODUCTION |
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The hormones, insulin and glucagon, are released in a [Ca2+]i-dependent manner (1). ß-Cells respond to an increase of extracellular glucose by increasing insulin secretion. The stimulus-secretion coupling process involves glucose metabolism, which provokes the closure of ATP-dependent potassium channels (KATP), responsible for the resting membrane potential. As a result, the plasma membrane depolarizes, opening the voltage-dependent calcium channels and increasing [Ca2+]i (2, 3, 4). Glucose-induced Ca2+ signals in ß-cells within the islet of Langerhans are organized in a synchronous and homogeneous [Ca2+]i oscillatory pattern. This is a consequence of the bursting pattern of electrical activity characteristic of pancreatic ß-cells (5, 6, 7). As a result, pulsatile insulin secretion is triggered (8, 9, 10).
As opposed to ß-cells, -cells release glucagon at low glucose values (11, 12). These cells contain a specific set of ion channels, responsible for their electrical activity (13, 14). Consequently, [Ca2+]i oscillates at low glucose concentrations, both in cultured cells (15) and in intact islets (7). Due to the calcium influx, the exocytotic machinery is initiated and glucagon is released (16). When extracellular glucose concentration increases to the level needed for insulin release, [Ca2+]i oscillations and glucagon release decrease (7, 12).
Both insulin and glucagon secretion are modified by the presence of 17ß-E2 (17, 18, 19), but its mechanism of action is only partially known in ß-cells and is unknown in -cells. An estrogen membrane receptor has been visualized in pancreatic ß-cells, responsible for a rapid insulinotropic effect of 17ß-E2 (19). The estrogen binds to its membrane receptor and triggers cyclic GMP (cGMP) synthesis, which activates protein kinase G (PKG). The KATP channels close in a PKG-dependent manner, and therefore the plasma membrane depolarizes, enhancing [Ca2+]i signals (20). As a consequence, insulin secretion is increased (19).
The pancreatic islet of Langerhans is not the only system in which estrogens produce nongenomic actions through a plasma membrane receptor. There is a great variety of effects in different cell types. MAPK, cAMP, cGMP, and NO are some of the intracellular mechanisms involved in rapid actions of estrogens (21, 22, 23). Other hydrophobic hormones act through a similar nongenomic mechanism (24, 25).
Despite the long list of effects of E2 on the plasma membrane, the nature of the receptor involved remains elusive. Some evidence show that the intracellular ERs, ER and ERß, may behave as membrane receptors (26, 27). However, this hypothesis is not supported by other studies, which demonstrate the involvement of a different estrogen membrane receptor (28, 29, 30, 31, 32).
The ER that mediates the actions of 17ß-E2 in pancreatic ß-cells is unrelated to the classical ER and ERß and has the pharmacological profile of the so-called "
-adrenergic receptor" (31).
In the present work, we have studied the modulatory effect of 17ß-E2 on [Ca2+]i in - and ß-cells within intact islets of Langerhans. These actions are triggered after E2 binds to a common estrogen membrane receptor, which is different than the classical ERs, ER
and ERß. Following the Mannheim classification of nongenomic steroid actions (25), we have named it the nonclassical estrogen membrane receptor (ncmER).
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RESULTS |
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These results demonstrate an opposite effect of E2 in - and ß-cells within the islet of Langerhans. On the one hand, 17ß-E2 increases the frequency of [Ca2+]i in ß-cells, producing an increase in insulin secretion (19). On the other hand, 17ß-E2 abolishes the low glucose-induced [Ca2+]i oscillations in
-cells and as a consequence, glucagon secretion will diminish (16).
cGMP Mediates 17ß-E2 Actions on -Cells
To examine the involvement of cGMP in the inhibition of low glucose-induced [Ca2+]i oscillations, 8-bromo-cGMP (8Br-cGMP) was tested in the virtual absence of glucose (0.5 mM). Application of 8Br-cGMP at concentrations as low as 10 µM reproduced the effect of 17ß-E2 on [Ca2+]i (Fig. 2A). The cGMP analog completely abolished low glucose-induced [Ca2+]i oscillations in 56% of the cells (32 of 57 cells tested), in 18% of the cells the frequency was decreased by 25 ± 5% (10 of 57 cells), and in 26% cGMP had no effect. In most cellular systems, including pancreatic ß-cells (20), cGMP activates the regulatory subunit of cGMP-dependent protein kinase (PKG). Therefore, if PKG were involved in 17ß-E2 action, then inhibition of the kinase would abolish the E2-induced blockade of [Ca2+]i oscillations. Application of a low dose of KT5823, a specific inhibitor of PKG, completely inhibited the effect of 17ß-E2 on [Ca2+]i signals in 55% of the cells tested (11 of 20 cells) (Fig. 2B
). These results are consistent with 17ß-E2 modulating [Ca2+]i signals by a PKG-dependent mechanism, although they do not exclude participation of other second messengers.
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In the experiment shown in Fig. 3, the mentioned antiestrogen did not modify the responses to 17ß-E2 in either ß- or
-cells. For that reason, we conclude that rapid [Ca2+]i regulation triggered by 17ß-E2 in both
- and ß-cells should be mediated by a nonclassical ER.
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Because the effects on [Ca2+]i were independent of the classical ER, we tested whether two classic antiestrogens, ICI 182,780 and tamoxifen, interact with the same binding site as E2 on the plasma membrane. Figure 6 shows that neither of these antiestrogens bound to the estrogen membrane receptor, either in
- or in ß-cells, because they did not compete with E-HRP. Furthermore, the H222 antibody raised against the ligand-binding domain of ER
did not compete with E-HRP. This indicates that the ligand-binding domain for this receptor should be different from that in ER
. Therefore, these results and those shown in Fig. 3
demonstrate that the membrane receptor involved in the rapid effect of 17ß-E2 in glucagon-containing
-cells must be different from the classical ER.
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To study the possible involvement of such a receptor in the effects described in -cells, we performed a competition assay with the neurotransmitters epinephrine, norepinephrine, and dopamine. Figure 7A
shows that the binding of E-HRP was blocked by the three catecholamines in
-cells as well as in ß-cells. The pharmacological profile was established by the experiment in Fig. 7B
. The binding of E-HRP was unaffected by the
- and the ß-adrenergic receptor ligands, clonidine and propranolol, and by the dopaminergic antagonist quinpirole.
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DISCUSSION |
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An increase in plasma levels of 17ß-E2 is achieved in proestrus and during pregnancy. In pregnant rats, plasma insulin is increased in response to increased levels of sex steroids (17, 36, 37). Moreover, 17ß-E2 at concentrations comparable to that of pregnancy enhances insulin secretion in perfused rat pancreas (38). E2 reverses the effects of menopause on glucose and insulin metabolism, resulting in an increase of pancreatic insulin secretion and a decrease in insulin resistance (39, 40).
Because glucagon inhibits insulin secretion, a decrease of glucagon release may contribute, in a paracrine manner, to the rapid insulinotropic effect manifested by 17ß-E2 via a plasma membrane receptor (19).
The mechanism of action of estrogens in -cells is still greatly unknown; indeed this is the first report describing a rapid effect of 17ß-E2 in this particular type of cell. The results presented in this work demonstrate that the receptor responsible is located at the plasma membrane. E-HRP, which is membrane impermeable, mimics the effect of 17ß-E2 at the same concentration. The existence of a membrane receptor is confirmed by visualization using E-HRP and by transmission laser scanning microscopy in unequivocally identified
-cells using immunostaining.
Basis for the Opposite Effect of 17ß-E2 in - and ß-Cells
The rapid effects of 17ß-E2 and E-HRP on [Ca2+]i are mimicked by 8Br-cGMP and blocked in more than 50% of the cases with the PKG inhibitor KT5823. These experiments indicate the involvement of cGMP in the 17ß-E2 response. We have recently demonstrated in ß-cells (20) that physiological levels of 17ß-E2, as those used here, decrease KATP channel activity via a cGMP-dependent phosphorylation process. This produces a depolarization of the membrane and the subsequent opening of voltage-gated calcium channels, causing a potentiation of glucose-induced [Ca2+]i oscillations. A similar mechanism may operate in pancreatic -cells, where the existence of KATP channels has recently been described (14). The KATP channel has the same function in both
- and ß-cells, and depolarization of the membrane as a result of glucose metabolism is observed in both types of cells. The opposite effect of glucose is explained as a consequence of the different set of ion channels presented by
- and ß-cells (14). The model predicts that action potential firing in
-cells is only possible in a narrow window of KATP channel activity. Within this window the membrane potential can pass from being positive enough to open pacemaker ion channels (likely T-type Ca2+ channels) and at the same time being sufficiently negative to prevent voltage-dependent inactivation of the membrane conductances involved in action potential generation. The effect of 17ß-E2 observed here is similar to that of glucose (7), in both
- and ß-cells: E2 will increase cGMP, inducing PKG activation and the subsequent decrease of KATP activity. The latter will depolarize the membrane in both types of cells: in ß-cells depolarization will lead to [Ca2+]i potentiation as explained previously, whereas in
-cells the depolarization will inactivate the membrane conductances, inducing the abolishment of [Ca2+]i oscillations. In this manner a common second messenger will cause opposite effects by using the same molecular mechanism. The fact that there exists a common second messenger in both types of cells also suggests that the ER involved would be the same.
Characteristics of the Membrane ER Involved
Two different hypotheses have been established for steroid receptors with ligand specificity involved: a classical steroid nuclear receptor and a nonclassical steroid receptor (25). The existence of proteins on the plasma membrane that cross-react with antibodies raised against different domains of the classical ER, ER, has been described (26, 41). As a result, the membrane receptor involved should be very similar in structure to ER
. Recently, it has been shown that both ER
and ERß can be expressed on the plasma membrane, but only at a 23% density, compared with the nuclear receptor density. Once these receptors have been activated by 17ß-E2, they stimulate adenylate cyclase as well as IP3 formation via a previous activation of G proteins, G
q and G
s. These experiments have been performed in Chinese hamster ovary cells transiently transfected with cDNA for both ER
and ERß. This study shows that the membrane and the nuclear protein are from the same transcript and have the same molecular weight (27).
Other evidence of the involvement of the classical ERs in rapid signaling is based on the inhibition of the E2 effect by antiestrogens. ICI 182,780 is a specific antiestrogen for ER and ß that inhibits the activation of the nitric oxide intracellular pathway by E2 (42, 43). It also blocks the MAPKs pathway activated by E2 in endothelial cells (44). Moreover, the E2 activation of endothelial nitric oxide synthase is enhanced by the overexpression of ER
in endothelial cells and inhibited by ICI (45).
The existence of the estrogen membrane receptor with a molecular structure related to a classical nuclear receptor does not exclude the existence of a specific nonclassical ER. Mounting evidence has appeared in the last years supporting this hypothesis (46). A strong indication is the fact that 17ß-E2 affects neuronal excitability in ER knockout mice. The possibility that ERß is responsible for this 17ß-E2 action is ruled out by the absence of effect of the specific antiestrogen ICI 182780 (28). Likewise, 17ß-E2 produces rapid effects in cell lines that lack both ER
and ERß (47).
The absence of effect of the antiestrogens tamoxifen and ICI 182,780 in 17ß-E2-induced rapid effects also supports the existence of a nonclassical ER. 17ß-E2 acutely regulates Cl- secretion in distal colonic epithelium in a tamoxifen-independent manner (48). In addition, ICI 182,780 does not affect rapid 17ß-E2 actions in dopaminergic neurons (49) and activation of MAPK in neuroblastoma cells (50) and hypothalamus (51). Very recently, new effects via a membrane ER unrelated to the classical have been demonstrated in pancreatic ß-cells (31) and in mouse IC-21 macrophages (32). In these two studies antibodies against classical nuclear ER failed to stain the plasma membrane.
In pancreatic ß-cells, we have demonstrated that neither of the two classical nuclear ERs are present at the membrane, and therefore they could not be responsible for the rapid actions of 17ß-E2 (31). In addition, we demonstrate in the present paper that regulation of [Ca2+]i by 17ß-E2 is not modified by the antiestrogen ICI 182,780 in either - or ß-cells. Moreover, no competition for E-HRP binding is manifested by the antiestrogens tamoxifen and ICI 182,780, as well as the antibody H222 against the ligand-binding domain of ER
. Involvement of ERß at the membrane is also unlikely based on the pharmacological profile of the receptor described here, although no antibody against the ligand binding domain of ERß is available to perform a competition study to fully rule out this possibility.
The binding studies presented in this paper demonstrate that the membrane receptor acting in -cells has the same pharmacological profile as that described in ß-cells (31), i.e. it has a binding site shared by the hormone 17ß-E2 and the neurotransmitters, dopamine, epinephrine, and norepinephrine. It is, nonetheless, neither a classical adrenergic receptor nor a dopaminergic receptor, because their classical ligands are without effect on E-HRP binding. This pharmacological profile is the same manifested by the
-adrenergic receptor, which is characterized by its equal activation with the neurotransmitters, epinephrine, norepinephrine and dopamine. It is affected neither by the
- nor the ß-adrenergic receptor antagonist and produces activation of protein kinase G via a nitric oxide-independent guanylyl cyclase (35, 52, 53, 54, 55).
The presence of a membrane receptor both in - and ß-cells unrelated to the classical ER
and ERß is strong evidence in favor of a new class of estrogen membrane receptor.
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MATERIALS AND METHODS |
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Measuring [Ca2+]i in and ß-Cells Within Intact Islets of Langerhans
Swiss albino OF1 male mice (810 wk old) were killed by cervical dislocation according to national guidelines. Pancreatic islets of Langerhans were isolated by collagenase digestion as previously described (56) and loaded with 5 µM Fluo-3 AM for at least 1 h at room temperature. Loaded islets were kept in a medium containing (mM): 115 NaCl, 25 NaHCO3, 5 KCl, 1.1 MgCl2, 1.2 NaH2PO4, 2.5 CaCl2, and 2.5 HEPES; plus 1% albumin and 5 mM D-glucose, continuously gassed with a mixture of 95% O2 and 5% CO2, pH 7.35. Islets were perfused with a modified Ringer solution containing (mM): 120 NaCl, 5 KCl, 25 NaHCO3, 1.1 MgCl2, and 2.5 CaCl2, pH 7.35, when gassed with 95% O2 and 5% CO2.
Calcium records in individual cells were obtained by imaging [Ca2+]i under a LSM 510 confocal microscope (Carl Zeiss, Jena, Germany) using a Carl Zeiss x40 oil immersion lens, numerical aperture 1.3. Images were collected at 2-sec intervals, and fluorescent signals from individual cells were measured in function of time using the Carl Zeiss LSM software package. Experiments were performed at 34 C. Results were plotted using a commercially available software (Sigmaplot, Jandel Scientific, San Rafael, CA) where the change in fluorescence (F) is expressed as a percentage of the basal fluorescence (F0) observed in absence of stimulus.
And ß-cells within the islets were identified by their [Ca2+]i oscillatory pattern in 0.5 mM and 8 mM glucose, respectively (7, 34).
Cell Isolation and Culture
Islets isolated as above were dispersed into single cells and cultured as previously described (4). Briefly, islets were disaggregated into single cells with trypsin. Cells were then centrifuged, resuspended in cultured medium RPMI 1640 supplemented with 10% FCS, 200 U ml-1 penicillin, 0.2 mg ml-1 streptomycin, 2 mM L-glutamine and 11 mM glucose, and plated on poly-L-lysine-coated glass coverslips. Cells were kept at 37 C in a humidified atmosphere of 95% O2 and 5% CO2 for 24 h.
E2-Peroxidase Binding Assay
After 24 h in culture, the cells were washed and fixed in 4% (wt/vol) fresh paraformaldehyde for 30 sec, which does not permeabilize the cells (19), and exposed overnight to 4.5 µg/ml E-HRP at 4 C (0.45 µg/ml for Fig. 4). The competition for the E2 binding site was tested using various reactives while adding E-HRP simultaneously. Cells were then washed and E-HRP binding was developed using 3,3'-diaminobenzidine tetrahydrochloride in the presence of urea hydrogen peroxide and CoCl2 for 30 min (Sigma FAST 3, 3'-diaminobenzidine tetrahydrochloride with metal enhancer tablet set, Co-DAB). After the development with Co-DAB, the cells were washed three times with PBS.
Immunocytochemistry
To identify -cells, immunocytochemistry was performed using an antiglucagon antibody. After the staining with E-HRP and Co-DAB, the cells were permeabilized with 1% Triton for 2 min. They were then incubated with the primary antibody (1:500 dilution) for 2 h, after which the secondary antibody antimouse IgG-tetramethylrhodamine isothiocyanate (1:125 dilution) was added for 1 h, both at room temperature. There was no labeling with the secondary antibody alone.
Binding Studies Using E-HRP and Transmission Laser Scanning Microscopy
The reaction between HRP and Co-DAB produces a dark precipitate that absorbs light, permitting us to visualize it using transmission laser scanning microscopy (57). The use of a reactive that competes for the E2 binding site will decrease the amount of precipitate. To identify those reactives that bind to the same site as 17ß-E2, the light absorbed by the precipitate was measured, as previously reported (31). Nonetheless, we should note that quantifying the amount of precipitate simply by measuring the light absorbed may be an unreliable procedure if changes in the amount of absorbed light are small. This method is of interest for comparative purpose, and it works extremely well for conditions in which the difference in the amount of precipitate is large. For that reason, detailed concentration-response profiles have been omitted. Transmission photographs were taken with a Carl Zeiss LSM 510 laser scanning confocal microscope, at 543 nm. In this work, the light intensity was taken from the whole area of the cell using Carl Zeiss LSM 510 software, rather than along a line as we have previously reported (31). A similar area was chosen to measure the background light. The background light and the microscope settings were constant throughout the different experiments. The quantity of E-HRP bound is expressed as the percentage of absorbed light with respect to the control condition. The lower the percentage of absorbed light, the higher the competition for an E-HRP binding site. To obtain an appropriate staining background, incubation and developmental procedure with peroxidase were performed in identical conditions. This background light has been subtracted in Figs. 6 and 7
for clarity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: 8Br-cGMP, 8-Bromo-cGMP; Co-DAB, 3,3'-diaminobenzidine in the presence of CoCl2; E-HRP, E2 conjugated to horseradish peroxidase; KATP, ATP-dependent potassium channel; PKG, protein kinase G.
Received for publication May 23, 2001. Accepted for publication November 7, 2001.
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REFERENCES |
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