A Nonclassical Estrogen Membrane Receptor Triggers Rapid Differential Actions in the Endocrine Pancreas

Ana B. Ropero, Bernat Soria and Angel Nadal

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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucose homeostasis in blood is mainly maintained by insulin released from ß-cells and glucagon released from {alpha}-cells, both integrated within the pancreatic islet of Langerhans. The secretory processes in both types of cells are triggered by a rise in intracellular calcium concentration ([Ca2+]i). In this study, rapid effects of the natural hormone E2 on [Ca2+]i were studied in both types of cells within intact islets using laser scanning confocal microscopy. {alpha}- And ß-cells showed opposite [Ca2+]i responses when stimulated with physiological concentrations of 17ß-E2. Although the estrogen produced an increase in the frequency of glucose-induced [Ca2+]i oscillations in insulin-releasing ß-cells, it prevented the low glucose-induced [Ca2+]i oscillations in glucagon-releasing {alpha}-cells. The effects of 17ß-E2 on {alpha}-cells were mimicked by the cGMP permeable analog 8bromo-cGMP and blocked by the cGMP-dependent protein kinase (PKG) inhibitor KT5823. Evidence indicated that these were membrane actions mediated by a nonclassical ER. Both effects were rapid in onset and were reproduced by 17ß-E2 linked to horseradish peroxidase, a cell-impermeable molecule. Furthermore, these actions were not blocked by the specific ER blocker ICI 182,780. Competition studies performed with 17ß-E2 linked to horseradish peroxidase binding in {alpha}-cells supported the idea that the membrane receptor involved is neither ER{alpha} nor ERß. Additionally, the binding site was shared by the neurotransmitters epinephrine, norepinephrine, and dopamine and had the same pharmacological profile as the receptor previously described for ß-cells. Therefore, rapid estrogen actions in islet cells are initiated by a nonclassical estrogen membrane receptor.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PANCREATIC ISLETS OF Langerhans are composed of a heterogeneous population of cells: insulin-secreting ß-cells (65–90%), glucagon-releasing {alpha}-cells (15–20%), somatostatin-producing {delta}-cells (3–10%), and pancreatic polypeptide-producing cells (1%). Interaction between the different cell types within the islet is vital for an adequate control of insulin release. A diminished glucose-induced insulin secretion results in non-insulin-dependent diabetes mellitus, responsible for 75% of the diabetic syndromes.

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, {alpha}-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 {alpha}-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{alpha} 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{alpha} and ERß and has the pharmacological profile of the so-called "{gamma}-adrenergic receptor" (31).

In the present work, we have studied the modulatory effect of 17ß-E2 on [Ca2+]i in {alpha}- 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{alpha} and ERß. Following the Mannheim classification of nongenomic steroid actions (25), we have named it the nonclassical estrogen membrane receptor (ncmER).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
{alpha}- and ß-Cells [Ca2+]i Responses to 17ß-E2
To investigate the effect of 17ß-E2 on [Ca2+]i in individual {alpha}- and ß-cells within intact islets of Langerhans, isolated islets were loaded with the fluorescent calcium-sensitive dye Fluo-3, and changes in fluorescence were monitored using laser scanning confocal microscopy (33). Only the periphery of the islet was loaded in all cases as shown in Fig. 1AGo and described previously (7). Nonetheless, all cell types that form part of the islets were represented at the periphery and were easily distinguishable by their [Ca2+]i response to glucose. ß-Cells were characterized by a synchronous oscillatory pattern on [Ca2+]i in the presence of 7–16 mM glucose, whereas they remained silent in its absence. On the contrary, glucagon-containing {alpha}-cells presented an oscillatory [Ca2+]i pattern in the absence of glucose, which diminished as glucose concentration was increased (7, 34).



View larger version (27K):
[in this window]
[in a new window]
 
Figure 1. Effects of 1 nM 17ß-E2 on [Ca2+]i Oscillations in {alpha}- and ß-Cells Within the Islet of Langerhans

A, Image of a Fluo-3-loaded islet exposed to 0.5 mM glucose. Blue corresponds to low and red to high fluorescence intensity. The scale bar at the bottom right represents 20 µm. Location of five {alpha}-cells that oscillate at 0.5 mM glucose are labeled with numbers 1 to 5. B–D, Records of fluorescence vs. time from cells within the islet of Langerhans. The Ca2+-dependent fluorescence of Fluo-3 is expressed as %{Delta}F/F0 (see Materials and Methods). E2 or E-HRP was applied for the period indicated by the horizontal bar. B, Increase of the frequency of [Ca2+]i oscillations in a ß-cell induced by 1 nM 17ß-E2 in the presence of 8 mM glucose. C, Inhibition of [Ca2+]i oscillations elicited by 0.5 mM glucose in an {alpha}-cell by E2 (51%, 38 of 75 cells). The mean time for the abolishment of the oscillatory pattern was 10.1 ± 0.9 min. D, 1 nM E-HRP completely abolished [Ca2+]i oscillations in an {alpha}-cell (49%, 26 of 53 cells) in 10.5 ± 1.0 min. Note the high grade of mimetism between 17ß-E2 and E-HRP effects. Data are expressed as mean ± SEM.

 
When a physiological concentration of 17ß-E2, 1 nM, was applied to a ß-cell, an increase in the frequency of glucose-induced [Ca2+]i oscillations was recorded (Fig. 1BGo), as previously described (19, 20). Typical {alpha}-cells presented an oscillatory [Ca2+]i pattern in the absence of glucose, which was completely abolished by 17ß-E2 in 51% of the cells (38 of 75) in 10.1 ± 0.9 min (Fig. 1CGo). In 29% of the cells (22 cells of 75 tested), a decrease of 37 ± 4% in the frequency of the oscillations was observed after 10 min of E2 application. There was no effect in 20% of the cells (15 of 75) even after 25 min of E2 exposure. The rapid effect of E2 in {alpha}-cells should be triggered at the plasma membrane because it is rapid in onset and was reproduced by the membrane-impermeable E2 conjugated to horseradish peroxidase (E-HRP) (Fig. 1DGo). E-HRP, 1 nM, abolished [Ca2+]i oscillations in 49% of the {alpha}-cells (26 cells of 53 tested) in 10.5 ± 1.0 min and decreased the frequency of 31 ± 5% in 30% of the cells (16 of 53), with no effect in 21% of the cells (11 of 53).

These results demonstrate an opposite effect of E2 in {alpha}- 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 {alpha}-cells and as a consequence, glucagon secretion will diminish (16).

cGMP Mediates 17ß-E2 Actions on {alpha}-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. 2AGo). 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. 2BGo). 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.



View larger version (30K):
[in this window]
[in a new window]
 
Figure 2. Involvement of cGMP and PKG in 17ß-E2-Induced Abolishment of [Ca2+]i Oscillations in {alpha}-Cells

A, Application of 10 µM 8Br-cGMP completely abolishes [Ca2+]i oscillations induced by 0.5 mM glucose in {alpha}-cells in 56% of the cells tested. B, 1 µM KT5823, an inhibitor of PKG, fully blocked the effect of 1 nM 17ß-E2 on [Ca2+]i oscillations induced by 0.5 mM glucose in 55% of the {alpha}-cells tested.

 
Rapid 17ß-E2 Effects in both {alpha}- and ß-Cells Are Independent of Classical ER
Several reports have described rapid effects of 17ß-E2 mediated by either a classical intracellular ER expressed at the plasma membrane or a nonclassical estrogen membrane receptor. The existence of both receptors can be distinguished by their sensitivity to the antiestrogen ICI 182,780. The effects mediated by a classical membrane receptor are blocked by ICI 182,780, whereas nonclassical estrogen membrane receptors are insensitive to the antiestrogen (22, 23, 25).

In the experiment shown in Fig. 3Go, the mentioned antiestrogen did not modify the responses to 17ß-E2 in either ß- or {alpha}-cells. For that reason, we conclude that rapid [Ca2+]i regulation triggered by 17ß-E2 in both {alpha}- and ß-cells should be mediated by a nonclassical ER.



View larger version (26K):
[in this window]
[in a new window]
 
Figure 3. 17ß-E2 Regulation of [Ca2+]i Is Unaffected by ICI 182,780

A, Effect of 1 µM ICI 182,780 in the action of 17ß-E2 on [Ca2+]i in a ß- and an {alpha}-cell (panel B). ICI was applied during the whole record and E2 during the time indicated by the horizontal bar. Records in the absence of ICI were made as control for the effect of E2 in islets from the same mouse.

 
Binding Studies Using E-HRP
We have used a binding assay method based on the interaction of E-HRP as a specific probe to detect E2 binding sites at the plasma membrane of nonpermeabilized cells (31). This assay has been combined with immunocytochemistry to specifically study the characteristics of the E2 binding site in {alpha}-cells. Figure 4AGo shows a cell stained with E-HRP and developed using Co-DAB (3,3'-diaminobenzidine in the presence of CoCl2). On completion of this assay, the cells were permeabilized (see Materials and Methods) to identify glucagon-containing {alpha}-cells by immunocytochemistry (Fig. 4BGo). The binding of E-HRP was blocked due to the competition with 17ß-E2 in a dose-dependent manner, which occurred in {alpha}- as well as in non-{alpha}-cells (Figs. 4CGo and 5Go).



View larger version (108K):
[in this window]
[in a new window]
 
Figure 4. E-HRP Binding at the Plasma Membrane of Glucagon-Containing {alpha}-Cells

E2 competition for the binding site of E-HRP at the plasma membrane of {alpha}-cells. A, Transmission image of a nonpermeabilized cell incubated with 100 nM E-HRP and developed with Co-DAB. B, Immunocytochemistry of the cell in panel A with an antiglucagon antibody and a secondary antibody conjugated to tetramethylrhodamine isothiocyanate. The cell was identified as a glucagon-containing {alpha}-cell. C, Competition of 30 µM 17ß-E2 for the binding site of E-HRP 100 nM. D, Identification of the cell in panel C as an {alpha}-cell by immunocytochemistry. Scale bars, 5 µm.

 


View larger version (18K):
[in this window]
[in a new window]
 
Figure 5. Dose-Dependent Competition of E-HRP Binding by 17ß-E2

Incubation of fixed nonpermeabilized islet cells in culture with 10 nM E-HRP and increased concentrations of 17ß-E2. {alpha}-Cells were identified as glucagon-containing cells, and the result was plotted separately ({blacksquare}) from the rest of the cells ({square}). As shown, there are no differences between the percentages of absorbed light in {alpha}-cells and non-{alpha}-cells (mostly ß-cells, see Results) in the same conditions. The results are expressed as the mean ± SEM of three different experiments with duplicate samples.

 
With this approach, we are able to study the properties of the E2 binding site on the {alpha}-cell membranes and also in non-{alpha}-cells. The population of non-{alpha}-cells will be mainly ß-cells, because somatostatin-secreting {delta}-cells and pancreatic polypeptide-containing PP-cells are normally less than 15% of the total population.

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 6Go shows that neither of these antiestrogens bound to the estrogen membrane receptor, either in {alpha}- or in ß-cells, because they did not compete with E-HRP. Furthermore, the H222 antibody raised against the ligand-binding domain of ER{alpha} did not compete with E-HRP. This indicates that the ligand-binding domain for this receptor should be different from that in ER{alpha}. Therefore, these results and those shown in Fig. 3Go demonstrate that the membrane receptor involved in the rapid effect of 17ß-E2 in glucagon-containing {alpha}-cells must be different from the classical ER.



View larger version (18K):
[in this window]
[in a new window]
 
Figure 6. No Competition with Antiestrogens for the E2 Binding Site at the Plasma Membrane of {alpha}-Cells

Binding assays with 100 nM E-HRP and competition with 17ß-E2 (E2), the antiestrogens ICI 182,780 (ICI) and tamoxifen (T), and the antibody H222 against the ligand binding domain of ER{alpha} (H222). All of them were applied at 30 µM and H222 at 1:100 dilution. Neither ICI182,780 nor tamoxifen competed for E-HRP binding, whereas E2 decreased light absorption up to 80%. Neither antiestrogen affected either {alpha}- ({blacksquare}) or ß-cell ({square}) binding of E-HRP. Background binding obtained with 100 nM peroxidase has been subtracted. Dimethylsulfoxide was used as a vehicle for insoluble reactives, and it was present both in control and peroxidase samples. Data are from two duplicate experiments, expressed as mean ± SEM.

 
17ß-E2 and Catecholamines Share a Common Membrane Receptor
The results presented up to now strongly suggest the presence of a similar estrogen membrane receptor in {alpha}- and ß-cells. We have recently described an estrogen membrane receptor in pancreatic ß-cells that binds 17ß-E2, catecholestrogens, and, remarkably, catecholamines (31). This receptor differs pharmacologically from the dopamine and the {alpha}- or the ß-adrenergic receptors, and it was previously defined as the {gamma}-adrenergic receptor (35).

To study the possible involvement of such a receptor in the effects described in {alpha}-cells, we performed a competition assay with the neurotransmitters epinephrine, norepinephrine, and dopamine. Figure 7AGo shows that the binding of E-HRP was blocked by the three catecholamines in {alpha}-cells as well as in ß-cells. The pharmacological profile was established by the experiment in Fig. 7BGo. The binding of E-HRP was unaffected by the {alpha}- and the ß-adrenergic receptor ligands, clonidine and propranolol, and by the dopaminergic antagonist quinpirole.



View larger version (21K):
[in this window]
[in a new window]
 
Figure 7. Pharmacological Profile of the Nonclassical Estrogen Membrane Receptor

A, Competition of E-HRP binding by the catecholamines dopamine (Dop), norepinephrine (NE), and epinephrine (E) in {alpha}-cells ({blacksquare}). Similar results were obtained in ß-cells ({square}). B, Lack of competition with ligands for the {alpha}-adrenergic receptor (clonidine, Clo), ß-adrenergic receptor (propranolol, Prop), and dopamine receptor (quinpirole, Quin) in both {alpha}- and ß-cells. All reactives were used at 30 µM. Background binding obtained with 100 nM peroxidase has been subtracted. Data are from at least three duplicate experiments, expressed as mean ± SEM.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This report demonstrates that physiological concentrations of the gonadal hormone 17ß-E2 inhibits the [Ca2+]i oscillatory pattern induced by low glucose concentrations in pancreatic {alpha}-cells. Because glucagon secretion depends on an increase in [Ca2+]i (1), a diminished glucagon secretion is to be expected. Such an effect is in agreement with previous results that described an inhibitory role of 17ß-E2 in {alpha}-cells using long-term exposure. After E2 exposure for a period of 4–8 h, a dose-dependent inhibitory effect on the arginine-stimulated glucagon secretion was manifested (18).

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 {alpha}-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 {alpha}-cells using immunostaining.

Basis for the Opposite Effect of 17ß-E2 in {alpha}- 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 {alpha}-cells, where the existence of KATP channels has recently been described (14). The KATP channel has the same function in both {alpha}- 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 {alpha}- and ß-cells (14). The model predicts that action potential firing in {alpha}-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 {alpha}- 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 {alpha}-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{alpha}, has been described (26, 41). As a result, the membrane receptor involved should be very similar in structure to ER{alpha}. Recently, it has been shown that both ER{alpha} and ERß can be expressed on the plasma membrane, but only at a 2–3% 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{alpha}q and G{alpha}s. These experiments have been performed in Chinese hamster ovary cells transiently transfected with cDNA for both ER{alpha} 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{alpha} 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{alpha} 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{alpha} 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{alpha} 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 {alpha}- 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{alpha}. 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 {alpha}-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 {gamma}-adrenergic receptor, which is characterized by its equal activation with the neurotransmitters, epinephrine, norepinephrine and dopamine. It is affected neither by the {alpha}- 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 {alpha}- and ß-cells unrelated to the classical ER{alpha} and ERß is strong evidence in favor of a new class of estrogen membrane receptor.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
ICI 182,780 was a kind gift of Astra USA, Inc.-Zeneca Pharmaceuticals (Bristol, UK). Fluo-3 AM was obtained from Molecular Probes, Inc. (Leiden, The Netherlands). Propranolol, Clonidine, and Quinpirole were obtained from Tocris Neuramin (Bristol, UK). KT 5823 was from Alexis (Laufelfingen, Switzerland). Other substances were obtained from Sigma (Madrid, Spain).

Measuring [Ca2+]i in {alpha} and ß-Cells Within Intact Islets of Langerhans
Swiss albino OF1 male mice (8–10 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 ({Delta}F) is expressed as a percentage of the basal fluorescence (F0) observed in absence of stimulus. {alpha} 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. 4Go). 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 {alpha}-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. 6Go and 7Go for clarity.


    ACKNOWLEDGMENTS
 
The authors thank Drs. Esther Fuentes and Cristina Ripoll for their help in several aspects of the project and E. Pérez García, A. Pérez Vegara, and N. Illera (Miguel Hernandez University, Alicante, Spain) for technical assistance. H222 antibody was a kind gift of Professor G. Greene (University of Chicago, Chicago, IL).


    FOOTNOTES
 
This work was supported by Grants from European Union-Comisión Interministerial de Ciencia y Tecnología (IFD97-1064-103-02) and Fundación Navarro Trípodi. A.B.R. is a recipient of a research scholarship from de Ministerio de Educación, Cultura y Deporte.

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Johansson H, Gylfe E, Hellman B 1987 The actions of arginine and glucose on glucagon secretion are mediated by opposite effects on cytoplasmic Ca2+. Biochem Biophys Res Commun 147:309–314[Medline]
  2. Prentki M, Matschinsky FM 1987 Ca2+, cAMP, and phospholipid-derived messengers in coupling mechanisms of insulin secretion. Physiol Rev 67:1185–1248[Free Full Text]
  3. Ashcroft FM, Rorsman P 1989 Electrophysiology of the pancreatic ß-cell. Prog Biophys Mol Biol 54:87–143[CrossRef][Medline]
  4. Valdeolmillos M, Nadal A, Contreras D, Soria B 1992 The relationship between glucose-induced K+ATP channel closure and the rise in [Ca2+]i in single mouse pancreatic ß-cells. J Physiol (Lond) 455:173–186[Abstract]
  5. Santos RM, Rosario LM, Nadal A, Garcia-Sancho J, Soria B, Valdeolmillos M 1991 Widespread synchronous [Ca2+]i oscillations due to bursting electrical activity in single pancreatic islets. Pflugers Arch 418:417–422[Medline]
  6. Valdeolmillos M, Nadal A, Soria B, Garcia-Sancho J 1993 Fluorescence digital image analysis of glucose-induced [Ca2+]i oscillations in mouse pancreatic islets of Langerhans. Diabetes 42:1210–1214[Abstract]
  7. Nadal A, Quesada I, Soria B 1999 Homologous and heterologous asynchronicity between identified {alpha}-, ß- and {delta}-cells within intact islets of Langerhans in the mouse. J Physiol (Lond) 517:85–93[Abstract/Free Full Text]
  8. Rosario LM, Atwater I, Scott AM 1986 Pulsatile insulin release and electrical activity from single ob/ob mouse islets of Langerhans. Adv Exp Med Biol 211:413–425[Medline]
  9. Martin F, Reig JA, Soria B 1995 Secretagogue-induced [Ca2+]i changes in single rat pancreatic islets and correlation with simultaneously measured insulin release. J Mol Endocrinol 15:177–185[Abstract]
  10. Barbosa RM, Silva AM, Tome AR, Stamford JA, Santos RM, Rosario LM 1998 Control of pulsatile 5-HT/insulin secretion from single mouse pancreatic islets by intracellular calcium dynamics. J Physiol (Lond) 510:135–143[Abstract/Free Full Text]
  11. Dunbar JC, Walsh MF 1982 Glucagon and insulin secretion by dispersed islet cells: possible paracrine relationships. Horm Res 16:257–267[Medline]
  12. Opara EC, Atwater I, Go VL 1988 Characterization and control of pulsatile secretion of insulin and glucagon. Pancreas 3:484–487[Medline]
  13. Rorsman P, Hellman B 1988 Voltage-activated currents in guinea pig pancreatic {alpha} 2 cells. Evidence for Ca2+-dependent action potentials. J Gen Physiol 91:223–242[Abstract]
  14. Gopel SO, Kanno T, Barg S, Weng X, Gromada J, Rorsman P 2000 Regulation of glucagon release in mouse-cells by KATP channels and inactivation of TTX-sensitive Na+ channels. J Physiol 528:509–520[Abstract/Free Full Text]
  15. Berts A, Ball A, Gylfe E, Hellman B 1996 Suppression of Ca2+ oscillations in glucagon-producing {alpha}2-cells by insulin/glucose and amino acids. Biochim Biophys Acta 1310:212–216[CrossRef][Medline]
  16. Gromada J, Bokvist K, Ding WG, Barg S, Buschard K, Renstrom E, Rorsman P 1997 Adrenaline stimulates glucagon secretion in pancreatic A-cells by increasing the Ca2+ current and the number of granules close to the L-type Ca2+ channels. J Gen Physiol 110:217–228[Abstract/Free Full Text]
  17. SutterDub MT 1976 Preliminary report: effects of female sex hormones on insulin secretion by the perfused rat pancreas. J Physiol (Paris) 72:795–800[Medline]
  18. Faure A, Haouari M, Sutter BC 1988 Short term and direct influence of oestradiol on glucagon secretion stimulated by arginine. Diabet Metab 14:452–454[Medline]
  19. Nadal A, Rovira JM, Laribi O, Leon-quinto T, Andreu E, Ripoll C, Soria B 1998 Rapid insulinotropic effect of 17ß-estradiol via a plasma membrane receptor. FASEB J 12:1341–1348[Abstract/Free Full Text]
  20. Ropero AB, Fuentes E, Rovira JM, Ripoll C, Soria B, Nadal A 1999 Non-genomic actions of 17ß-oestradiol in mouse pancreatic ß-cells are mediated by a cGMP-dependent protein kinase. J Physiol (Lond) 521:397–407[Abstract/Free Full Text]
  21. Kelly MJ, Wagner EJ 1999 Estrogen modulation of G-protein-coupled receptors. Trends Endocrinol Metab 10:369–374[CrossRef][Medline]
  22. Kelly MJ, Levin ER 2001 Rapid actions of plasma membrane estrogen receptors. Trends Endocrinol Metab 12:152–156[CrossRef][Medline]
  23. Nadal A, Diaz M, Valverde MA 2001 The estrogen trinity: membrane, cytosolic and nuclear effects. News Physiol Sci 16:251–255[Abstract/Free Full Text]
  24. Wehling M 1997 Specific, nongenomic actions of steroid hormones. Annu Rev Physiol 59:365–393[CrossRef][Medline]
  25. Falkenstein E, Tillmann HC, Christ M, Feuring M, Wehling M 2000 Multiple actions of steroid hormones—a focus on rapid, nongenomic effects. Pharmacol Rev 52:513–556[Abstract/Free Full Text]
  26. Watson CS, Campbell CH, Gametchu B 1999 Membrane oestrogen receptors on rat pituitary tumour cells: immuno-identification and responses to oestradiol and xenoestrogens. Exp Physiol 84:1013–1022[Abstract]
  27. Razandi M, Pedram A, Greene GL, Levin ER 1999 Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ER{alpha} and ERß expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319[Abstract/Free Full Text]
  28. Gu Q, Korach KS, Moss RL 1999 Rapid action of 17ß-estradiol on kainate-induced currents in hippocampal neurons lacking intracellular estrogen receptors. Endocrinology 140:660–666[Abstract/Free Full Text]
  29. Das SK, Taylor JA, Korach KS, Paria BC, Dey SK, Lubahn DB 1997 Estrogenic responses in estrogen receptor-{alpha} deficient mice reveal a distinct estrogen signaling pathway. Proc Natl Acad Sci USA 94:12786–12791[Abstract/Free Full Text]
  30. Toran-Allerand CD 2000 Novel sites and mechanisms of oestrogen action in the brain. In: Chadwick DJ, Goode JA, eds. Neuronal and cognitive effects of oestrogens. Chichester, West Sussex, UK: John Wiley & Sons; 56–69; discussion 69–73
  31. Nadal A, Ropero AB, Laribi O, Maillet M, Fuentes E, Soria B 2000 Nongenomic actions of estrogens and xenoestrogens by binding at a plasma membrane receptor unrelated to estrogen receptor {alpha} and estrogen receptor ß. Proc Natl Acad Sci USA 97:11603–11608[Abstract/Free Full Text]
  32. Benten WP, Stephan C, Lieberherr M, Wunderlich F 2001 Estradiol signaling via sequestrable surface receptors. Endocrinology 142:1669–1677[Abstract/Free Full Text]
  33. Nadal A, Soria B 2000 Imaging intracellular calcium in living tissue by laser-scanning confocal microscopy. In: Pochet R, ed. Calcium: the molecular basis of calcium action in biology and medicine. Amsterdam: Kluwer Academic Publishers; 661–671
  34. Quesada I, Nadal A, Soria B 1999 Different effects of tolbutamide and diazoxide in {alpha}, ß-, and {delta}-cells within intact islets of Langerhans. Diabetes 48:2390–2397[Abstract]
  35. Hirst GD, Neild TO, Silverberg GD 1982 Noradrenaline receptors on the rat basilar artery. J Physiol (Lond) 328:351–360[Abstract]
  36. Malaisse WJ, Malaisse-Lagae F, Picard C, Flament- Durand J 1969 Effects of pregnancy and chorionic growth hormone upon insulin secretion. Endocrinology 84:41–44[Medline]
  37. Costrini NV, Kalkhoff RK 1971 Relative effects of pregnancy, estradiol, and progesterone on plasma insulin and pancreatic islet insulin secretion. J Clin Invest 50:992–999[Medline]
  38. Sutter-Dub MT 1979 Effects of pregnancy and progesterone and/or oestradiol on the insulin secretion and pancreatic insulin content in the perfused rat pancreas. Diabete Metab 5:47–56[Medline]
  39. Stevenson JC, Crook D, Godsland IF, Collins P, Whitehead MI 1994 Hormone replacement therapy and the cardiovascular system. Nonlipid effects. Drugs 47(Suppl 2):35–41
  40. Brussaard HE, Gevers Leuven JA, Frolich M, Kluft C, Krans HM 1997 Short-term oestrogen replacement therapy improves insulin resistance, lipids and fibrinolysis in postmenopausal women with NIDDM. Diabetologia 40:843–849[CrossRef][Medline]
  41. Pappas TC, Gametchu B, Watson CS 1995 Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB J 9:404–410[Abstract/Free Full Text]
  42. Kim HP, Lee JY, Jeong JK, Bae SW, Lee HK, Jo I 1999 Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor {alpha} localized in caveolae. Biochem Biophys Res Commun 263:257–262[CrossRef][Medline]
  43. Haynes MP, Sinha D, Russell KS, Collinge M, Fulton D, Morales-Ruiz M, Sessa WC, Bender JR 2000 Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells. Circ Res 87:677–682[Abstract/Free Full Text]
  44. Russell KS, Haynes MP, Sinha D, Clerisme E, Bender JR 2000 Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling. Proc Natl Acad Sci USA 97:5930–5935[Abstract/Free Full Text]
  45. Chen Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn ME, Shaul PW 1999 Estrogen receptor {alpha} mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 103:401–406[Abstract/Free Full Text]
  46. Nadal A, Ropero AB, Fuentes E, Soria B 2001 The plasma membrane estrogen receptor: nuclear or unclear? Trends Pharmacol Sci 22:597–599[CrossRef][Medline]
  47. Filardo EJ, Quinn JA, Bland KI, Frackelton Jr AR 2000 Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 14:1649–1460[Abstract/Free Full Text]
  48. Condliffe SB, Doolan CM, Harvey BJ 2001 17ß-Oestradiol acutely regulates Cl- secretion in rat distal colonic epithelium. J Physiol 530:47–54[Abstract/Free Full Text]
  49. Beyer C, Karolczak M 2000 Estrogenic stimulation of neurite growth in midbrain dopaminergic neurons depends on cAMP/protein kinase A signalling. J Neurosci Res 59:107–116[CrossRef][Medline]
  50. Watters JJ, Campbell JS, Cunningham MJ, Krebs EG, Dorsa DM 1997 Rapid membrane effects of steroids in neuroblastoma cells: effects of estrogen on mitogen activated protein kinase signalling cascade and c-fos immediate early gene transcription. Endocrinology 138:4030–4033[Abstract/Free Full Text]
  51. Kuroki Y, Fukushima K, Kanda Y, Mizuno K, Watanabe Y 2000 Putative membrane-bound estrogen receptors possibly stimulate mitogen-activated protein kinase in the rat hippocampus. Eur J Pharmacol 400:205–209[CrossRef][Medline]
  52. Hirst GD, Neild TO 1980 Evidence for two populations of excitatory receptors for noradrenaline on arteriolar smooth muscle. Nature 283:767–768[Medline]
  53. Benham CD, Tsien RW 1988 Noradrenaline modulation of calcium channels in single smooth muscle cells from rabbit ear artery. J Physiol (Lond) 404:767–784[Abstract]
  54. Yawo H 1996 Noradrenaline modulates transmitter release by enhancing the Ca2+ sensitivity of exocytosis in the chick ciliary presynaptic terminal. J Physiol (Lond) 493:385–391[Abstract]
  55. Yawo H 1999 Involvement of cGMP-dependent protein kinase in adrenergic potentiation of transmitter release from the calyx-type presynaptic terminal. J Neurosci 19:5293–5300[Abstract/Free Full Text]
  56. Nadal A, Valdeolmillos M, Soria B 1994 Metabolic regulation of intracellular calcium concentration in mouse pancreatic islets of Langerhans. Am J Physiol 267:E769–E774
  57. Halbhuber KJ, Krieg R, Konig K 1998 Laser scanning microscopy in enzyme histochemistry. Visualization of cerium-based and DAB-based primary reaction products of phosphatases, oxidases and peroxidases by reflectance and transmission laser scanning microscopy. Cell Mol Biol (Noisy-le-grand) 44:807–826