17β-Estradiol Elevates cGMP and, via Plasma Membrane Recruitment of Protein Kinase GIα, Stimulates Ca2+ Efflux from Rat Hepatocytes*

Rapid non-genomic effects of 17β-estradiol, the principal circulating estrogen, have been observed in a wide variety of cell types. Here we investigate rapid signaling effects of 17β-estradiol in rat hepatocytes. We show that, above a threshold concentration of 1 nm, 17β-estradiol, but not 17α-estradiol, stimulates particulate guanylyl cyclase to elevate cGMP, which through activation and plasma membrane recruitment of protein kinase G isoform Iα, stimulates plasma membrane Ca2+-ATPase-mediated Ca2+ efflux from rat hepatocytes. These effects are extremely rapid in onset and are mimicked by a membrane-impermeant 17β-estradiol-BSA conjugate, suggesting that 17β-estradiol acts at the extracellular face of the plasma membrane. We also show that 17β-estradiol binds specifically to the intact hepatocyte plasma membrane through an interaction that is competed by an excess of atrial natriuretic peptide but also shows many similarities to the pharmacological characteristics of the putative γ-adrenergic receptor. We, therefore, propose that the observed rapid signaling effects of 17β-estradiol are mediated either through the guanylyl cyclase A receptor for atrial natriuretic peptide or through the γ-adrenergic receptor, which is either itself a transmembrane guanylyl cyclase or activates a transmembrane guanylyl cyclase through cross-talk signaling.

Direct measurements have shown that 17␤-estradiol elevates cGMP in PC12 pheochromocytoma cells (4), human umbilical vein endothelial cells (6), pancreatic ␣ (7) and ␤ cells (5), and human coronary smooth muscle cells (9). Moreover, several studies using protein kinase G (PKG) inhibitors have implicated cGMP in rapid effects of 17␤-estradiol (5,7,14,20). These include decreased contractility of isolated porcine coronary arterial rings (20), attenuation of acetylcholine-induced Ca 2ϩ transients in hypothalamic GT1-7 neuronal cells (14), inhibition of K ATP channel activity in pancreatic ␤ cells (5), increased frequency of [Ca 2ϩ ] c oscillations in pancreatic ␤ cells (5), and prevention of [Ca 2ϩ ] c oscillations in pancreatic ␣ cells (7). Cellular cGMP generation is controlled by two distinct classes of guanylyl cyclases that differ in their cellular location and activation by specific ligands; soluble guanylyl cyclase exists in the cytosol and is activated by nitric oxide (NO) (21), whereas particulate guanylyl cyclases are trans-plasma membrane proteins that are activated by agonist binding to their extracellular receptor portions (22). Seven mammalian particulate guanylyl cyclase isoforms have been identified: GC-A through to GC-G (23). GC-A and GC-B are receptors for atrial natriuretic peptide (ANP) and B-type natriuretic peptide, whereas GC-C mediates the effects of guanylin and uroguanylin (22). GC-D, -E, -F, and -G are orphan receptors; endogenous ligands have yet to be identified (23). GC-A (24) and GC-C (25) have been detected in rat hepatocytes. GC-C is developmentally regulated; it is synthesized by late fetal and early neonatal rat liver but is not expressed by adult rat liver except under conditions of liver regeneration (26).
The rapidity of the effects of 17␤-estradiol is consistent with it acting at the plasma membrane, and indeed, many of the reported rapid effects of 17␤-estradiol are mimicked by 17␤estradiol-rendered plasma membrane-impermeant by linkage to a large molecule e.g. BSA (6,(41)(42)(43)(44) or horseradish peroxidase (7,(45)(46). There is a large body of evidence in favor of the existence of plasma membrane receptors for 17␤-estradiol, and numerous such receptors have been proposed. These include classical ER␣ localized at the plasma membrane (47)(48)(49). Variant forms of ER␣, including a truncated form, ER46 (50,51), have been found associated with caveolae in the plasma membrane of some cells. Membrane estrogen receptors that are distinct from the classical estrogen receptors have also been described. These include an orphan G protein-coupled receptor GPR30 (52,53), ERX, a novel receptor found in post-natal neocortical and uterine plasma membrane (54), and a receptor that has been termed the "␥-adrenergic receptor" (5,7,55,56).
The pharmacology of the putative ␥-adrenergic receptor is distinct from any other estrogen receptors; intact cell competition binding studies revealed that the catecholestrogen 2-hydroxyestradiol, noradrenaline, adrenaline, and dopamine blocked the binding of the membrane-impermeant conjugate 17␤-estradiol-horseradish peroxidase to the plasma membrane of both pancreatic ␣ (7) and ␤ cells (55). In contrast, selective ␣-agonists, a ␤-antagonist, a D 2 dopamine receptor antagonist, and the classical ER antagonist ICI 182,780 were without effect. Nadal et al. (5,7,55,56) noted striking similarities to the so-called ␥-adrenergic receptor that was first described in vascular cells (57,58) and then characterized pharmacologically in presynaptic terminals of chick ciliary ganglion (59) and defined as a novel adrenergic receptor that is stimulated by noradrenaline but insensitive to ␣and ␤-adrenergic agonists and antagonists and activates particulate GC to elevate cGMP and activate PKG.
Here we investigate rapid effects of 17␤-estradiol on rat hepatocytes. We show that, above a threshold concentration of 1 nM, 17␤-estradiol stimulates particulate guanylyl cyclase to elevate cGMP, which through activation and plasma membrane recruitment of PKGI␣ stimulates PMCA-mediated Ca 2ϩ efflux from rat hepatocytes. These effects are extremely rapid, occurring within seconds, and are mimicked by a membrane-impermeant form of 17␤-estradiol, suggesting that 17␤-estradiol acts at the plasma membrane. We also show that 17␤-estradiol binds specifically to the intact hepatocyte plasma membrane through an interaction that is competed by an excess of ANP but also shows competitive binding characteristics that resemble those of the ␥-adrenergic receptor that have been proposed to mediate the effects of 17␤-estradiol in pancreatic ␣ and ␤ cells (5,7,55,56).
Water-soluble 17␤-estradiol was supplied as 17␤-estradiol encapsulated in 2-hydroxypropyl-␤-cyclodextrin (cyclodextrin vehicle), which releases free 17␤-estradiol in aqueous solution. This formulation has been used in numerous studies, e.g. Refs. 6, 60, and 61), as it negates the need for organic solvents and thereby more faithfully mimics in vivo conditions. Water-soluble 17␤-estradiol was, therefore, used extensively throughout the present study, except for the binding studies, for which it is not recommended. For binding studies, 17␤-estradiol was used from a 1000-fold concentrated stock solution dissolved in DMSO. Because 17␣-estradiol is not commercially available in cyclodextrinencapsulated form, it was also used from a 1000-fold concentrated DMSO-dissolved stock solution; in such experiments similarly DMSO-dissolved 17␤-estradiol also was used, enabling direct comparison of data.

Isolation of Hepatocytes
Single hepatocytes were isolated from 150 -250 g of female Wistar rats by collagenase digestion as described previously (62,63).

Measurement of cGMP
Isolated rat hepatocytes at a cell density of 1.5 ϫ 10 6 /ml were incubated in suspension in medium of composition 150 mM NaCl, 3.6 mM KCl, 1 mM MgCl 2 , 10 mM HEPES, 1.8 mM CaCl 2 , 11 mM glucose, pH 7.4, in a shaking water bath at 37°C. Agonists were added for the periods indicated. cGMP was measured by using the acetylation method in a cGMP enzyme immunoassay system (Biotrak cellular communication assays; Amersham Biosciences).

Measurement of Ca 2؉ Efflux from Hepatocytes
Isolated rat hepatocytes were counted using a hemocytometer and then stored in suspension in WME on ice. Before each assay ϳ10 6 cells were washed 3 times in ice-cold efflux medium composed of 150 mM NaCl, 3.6 mM KCl, 1 mM MgCl 2 , 10 mM HEPES, 11 mM glucose, pH 7.4. Ca 2ϩ efflux from the intact rat hepatocytes was monitored by measuring the extracellular concentration of Ca 2ϩ ([Ca 2ϩ ] o ) in the medium using fura-2 as extracellular Ca 2ϩ reporter. Thus, the cells were resuspended in 1 ml of efflux medium at 37°C to which the membrane-impermeant pentapotassium salt of fura-2 (7.5 M) was added. Fluorescence was measured at 37°C with constant stirring by using a PTI DeltaRam spectrofluorimeter (Photon Technologies International, Inc., Lawrenceville, NJ), exciting alternately at 340 and 380 nm and collecting emission at 510 nm. [Ca 2ϩ ] o was calculated by calibrating the photometric 340/380 nm ratio signal as described previously (33). ([Ca 2ϩ ] o ) never rose above 1.5 M, ensuring a true, unidirectional, Ca 2ϩ efflux measurement.

Measurement of [Ca 2؉ ] c in Single Hepatocytes
Preparation of Single Cells for Microinjection-After harvesting, the hepatocytes were incubated at 37°C at low density (ϳ10 3 cells/ml) in 1.7% SeaPrep-agarose in WME. Single hepatocytes were transferred to 0.1-mm path length microslides containing 1.2% FMC SeaPlaque-agarose in WME, which was subsequently gelled at 4°C for 2 min. The cells were then held at 37°C under a layer of liquid paraffin.
Fura-2 Dextran Preparation-Fura-2 dextran (10 kDa) was dissolved in 150 mM KCl, 1 mM PIPES, pH 7.2, to make a 5 mM stock solution that was stored at Ϫ70°C. For microinjection, a small aliquot of 5 mM fura-2 dextran was held as a droplet under liquid paraffin.
Microinjection and Data Acquisition-Freshly pulled pipettes were filled with fura-2 dextran by dipping the tip for a few seconds in the fura-2 dextran droplet. Individual hepatocytes were injected to ϳ0.5% of the cell volume as described previously (64,65). The fura-2 dextran thus localized in the cytoplasmic compartment reports [Ca 2ϩ ] c only. Injected cells of healthy appearance were transferred within microslides to a heated (37°C) stainless steel perfusion chamber on the stage of an Olympus IX71 epifluorescence microscope. Cells were superfused with extracellular buffer to which ATP and 17␤-estradiol were added as indicated. Cells were alternately excited at 340and 380-nm wavelengths by means of a PTI DeltaRAM V high speed random access monchromator (Photon Technologies International Inc., Lawrenceville, NJ). The 510-nm emission was detected by a PTI Q-8 microscope photometer. [Ca 2ϩ ] c was calculated by calibrating the fura-2 signal in situ as described previously (66).
Immunolabeling-Hepatocytes were allowed to attach to glass coverslips for 24 h in WME supplemented with 100 IU/ml penicillin and 100 g/ml streptomycin at 37°C and then fixed in 4% paraformaldehyde. Nonspecific binding was prevented by blocking for 1 h at 25°C in phosphate-buffered saline (PBS) plus 0.01% Triton X-100 containing 5% normal rabbit serum. After three 5-min washes with PBS, cells were incubated at 4°C overnight with the corresponding primary antibody diluted (1:50) in PBS plus 0.01% Triton X-100. After antibody incubation, cells were washed three times with PBS and then incubated with Alexa Fluor 594-conjugated donkey anti-goat IgG (H ϩ L) for 2 h at 25°C. The secondary antibody was diluted 1:1000 in PBS plus 0.01% Triton X-100. Cells were then washed 3 times for 5 min in PBS, and coverslips were then mounted in Citifluor (glycerol/PBS solution) on glass slides. Cells were examined under oil immersion (ϫ63) using a Leica DMRE upright laser scanning confocal microscope with a TCS SP2 scan head.
Quantification of Fluorescence Localization-Raw confocal images were analyzed using MetaMorph imaging software (Universal Imaging, Marlow, Bucks, UK). A single horizontal transect was made through the widest portion of each cell. Regions of interest (2.5 m ϫ 2.5 m) were applied along the transect, and the average fluorescent staining intensity within each region was determined. For each cell, data were normalized by expressing staining intensity per region as a percentage of the average staining intensity across the entire transect. Subsequent analysis determined relative staining intensity at the cell periphery compared with average staining intensity across the entire transect. This was achieved by expressing the average staining intensity of the outer 2 regions of each side of the cell as a percentage of the average staining intensity across the entire transect. Similarly, relative staining intensity of the cell interior compared with average staining intensity across the entire transect was determined by expressing the average staining intensity of all except the outer 2 regions of each side of the cell as a percentage of the average staining intensity across the entire transect.

Assay for 17␤-Estradiol-BSA-FITC Binding
Hepatocytes were allowed to attach to glass coverslips for 24 h in WME supplemented with 100 IU/ml penicillin and 100 g/ml streptomycin at 37°C. The cells were then incubated with 1 M 17␤-estradiol-BSA-FITC for 30 min at 4°C and then fixed in 4% paraformaldehyde. After three 5-min washes in PBS, the coverslips were mounted on Citifluor on glass slides and then examined under oil immersion (ϫ63) using a Leica DMRE upright laser scanning confocal microscope with a TCS SP2 scan head.

Statistics
Differences between means were compared by Student's t test using a level of significance of p Ͻ 0.05. Data are expressed AUGUST Fig. 1A shows that 10 nM 17␤estradiol rapidly elevated cGMP levels in isolated rat hepatocytes; cGMP was significantly increased above basal levels within 30 s and remained significantly elevated for 10 min. No effect was seen with the cyclodextrin vehicle.

17␤-Estradiol Elevates Hepatocyte cGMP through Activation of Particulate Guanylyl Cyclase-
Investigation of the concentration dependence of the effect of 17␤-estradiol (Fig. 1B) showed that a significant increase in cGMP levels was seen above a threshold concentration of 1 nM 17␤-estradiol and that the optimum concentration of 17␤-estradiol was 10 nM. Interestingly, the magnitude of the increase in cGMP levels was reduced at concentrations of 17␤-estradiol above 10 nM. This could be due to a nonspecific effect of 17␤estradiol on the plasma membrane; high concentrations of steroid hormones can interact non-specifically with cell membranes (67). Cytotoxicity arising from high concentrations of 17␤-estradiol has been associated with a decrease in membrane fluidity (68). Fig. 1C shows that 10 nM 17␤-estradiol-BSA also elevated hepatocyte cGMP to a level similar to that seen with 17␤-estradiol, whereas, in contrast, 17␣-estradiol, the stereoisomer of 17␤-estradiol, did not elevate cGMP.
Regarding the mechanism through which 17␤-estradiol elevates hepatocyte cGMP, three possibilities exist; that is, soluble guanylyl cyclase activation, particulate guanylyl cyclase activation, or cGMP phosphodiesterase (PDE) inhibition. Because soluble guanylyl cyclase can remain plasma membrane-associated (in caveolae) (69), soluble versus particulate guanylyl cyclase involvement cannot be distinguished using the traditional approach of comparing effects on isolated plasma membrane preparations versus intact cells. Instead, to investigate whether 17␤-estradiol elevates cGMP through stimulation of soluble or particulate guanylyl cyclase, NS2028, a potent and specific inhibitor of soluble guanylyl cyclase (70), was used.
Agonists were added to hepatocytes in the presence of 10 M NS2028 hepatocytes (after 5 min of preincubation with NS2028 alone). The specificity and efficacy of this inhibitor is demonstrated in Fig. 2A; NS2028 prevented any significant elevation of cGMP upon the addition of sodium nitroprusside, an NO donor that stimulates soluble guanylyl cyclase (21,32,33). In contrast, ANP, a known activator of particulate guanylyl cyclase (22,24,32), was still able to elevate cGMP to similar levels in the presence or absence of NS2028. Fig. 2A shows that inhibition of soluble guanylyl cyclase with NS2028 did not prevent 17␤estradiol from elevating cGMP. This suggests that soluble guanylyl cyclase stimulation is not the mechanism responsible for the 17␤-estradiol-mediated elevation of cGMP in hepatocytes.
Intracellular cGMP can also be elevated through inhibition of cyclic nucleotide PDEs. PDEs 3B, 7B, 9A, and 11A have been identified in hepatocytes (71)(72)(73). IBMX is generally used as a universal nonspecific inhibitor of cyclic nucleotide PDEs; however, it has been shown to be ineffective at inhibiting PDE9 (72), a cGMP-specific PDE. PDE9A has the highest affinity for cGMP and has been found in nearly every tissue, including the liver (72,73). For this reason, a combination of IBMX (300 M) and zaprinast (300 M), an inhibitor of PDE9 and PDE5 (72), was used to inhibit hepatocyte PDEs. Agonists were, thus, added in the presence of IBMX and zaprinast (after a 10-min preincubation of hepatocytes with IBMX and zaprinast alone).
Preincubation of cells with IBMX and zaprinast significantly elevated cGMP levels above basal, control levels (Fig. 2B). The addition of 17␤-estradiol elevated cGMP to levels significantly greater than those seen in cells preincubated with the PDE inhibitors alone. These data, therefore, suggest that 17␤-estradiol elevates cGMP through a mechanism distinct from either stimulation of soluble guanylyl cyclase or inhibition of cyclic nucleotide phosphodiesterases. We, therefore, propose that 17␤-estradiol acts by stimulating particulate guanylyl cyclase.

17␤-Estradiol Recruits PKGI␣ to the Hepatocyte Plasma
Membrane-PKG is the major mediator of cGMP signaling. Two mammalian isotypes of PKGI have been described; that is, type I, consisting of an ␣ and a ␤ isoform, splice variants of a single gene, and type II (reviewed in Ref. 74). PKGI␣ and PKGI␤ arise from alternative splicing of exons encoding the N-terminal domain of PKGI. These two different PKGI isoforms differ from PKGII and from one another in terms of sensitivity to cGMP, tissue distribution, and substrate specificity, interacting with different proteins through their distinct N termini (74).
In confirmation of our previous findings (33,34), immunoreactivity of the commercially available antibodies cGKI␣ (N-16) and cGKI␤ (L-16) (visualized using Alexa 594, red sec-ondary) demonstrated expression of PKGI␣ and PKGI␤ in isolated rat hepatocytes (Fig. 3). No staining was observed using primary antibodies pre-absorbed with corresponding blocking peptides or using the Alexa 594 secondary antibody alone (results not shown). In unstimulated rat hepatocytes, the pattern of staining of both PKGI␣ (Fig. 3, Ai and C) and PKGI␤ (Fig. 3, Bi and D) was punctate and distributed diffusely throughout the cytosol, lacking clear nuclear or plasma membrane localization. Stimulation of hepatocytes with 17␤-estradiol (10 nM) for 5 min increased the intensity of plasma membrane-localized immunostaining of PKGI␣ (Fig. 3, Aii and C), but, in contrast, the pattern of localization of PKGI␤ was unaffected by stimulation with 17␤-estradiol (Fig. 3, Bii and D). Fluorescence intensity scans of confocal images revealed significantly increased PKGI␣ immunostaining at the cell periphery and decreased PKGI␣ immunostaining in the cell interior after treatment of hepatocytes with 17␤-estradiol (Fig. 3C). In contrast, the distribution of PKGI␤ immunostaining was not significantly altered (Fig. 3D). These observations suggest a selective translocation of PKGI␣ to the plasma membrane upon stimulation with 17␤-estradiol.
PKGI␣ is a homodimer, with each subunit containing an N-terminal dimerization domain, a regulatory domain, and a catalytic domain. The regulatory domain contains two tandem cGMP binding sites, occupation of which releases inhibition of the catalytic domain by the autoinhibitory site on the N-terminal domain. The catalytic domain when, thus, exposed phosphorylates serine/threonine residues in target proteins (74). To determine whether the activation of PKGI␣ is required for its translocation to the plasma membrane, two distinct membrane-permeant inhibitors of PKG were used: Rp-8-pCPT-cGMPS and DT-3. Rp-8-pCPT-cGMPS inhibits all three PKG isoforms (75) by blocking cGMP binding to the regulatory domain, thereby preventing exposure of the catalytic domain. DT-3 acts through a distinct mechanism to that of Rp-8-pCPT-cGMPS; the PKGI-inhibitor peptide portion of DT-3, W45, prevents binding and, hence, phosphorylation of substrates at the PKGI catalytic domain (76). DT-3 inhibits isoforms I␣ and the I␤ at low M concentrations, whereas PKGII is relatively (ϳ100-fold less) insensitive to this inhibitor (77).
Plasma membrane recruitment of PKGI␣ (Fig. 3, Av and C) was also seen upon treatment of hepatocytes with 17␤-estradiol-BSA (10 nM). In contrast, 17␣-estradiol did not affect the pattern of localization of PKGI␣ (Fig. 3, Avi and C).
17␤-Estradiol Stimulates PMCA-mediated Ca 2ϩ Efflux from Hepatocytes-Consistent with our previous findings (32)(33)(34)78), a basal rate of Ca 2ϩ efflux was recorded from intact rat hepatocytes in the absence of agonist (Fig. 4, A and G) and was blocked by caloxin 1b1, a specific PMCA inhibitor (79) (Fig. 4, B and G), confirming that the basal hepatocyte Ca 2ϩ efflux recorded here is predominantly due to PMCA activity (33, 78). Specific inhibitors were applied to intact rat hepatocytes to investigate the involvement of soluble guanylyl cyclase and cyclic nucleotide phosphodiesterases in the elevation of cGMP by 17␤-estradiol. Cells were incubated at 37°C. Total cGMP levels were measured by enzyme immunoassay. All data shown are the mean values of at least six experiments from at least three independent hepatocyte preparations. A, 17␤-estradiol does not stimulate soluble guanylyl cyclase. Cells were incubated with either no treatment (control) or soluble guanylyl cyclase inhibitor NS2028 (10 M) for 5 min. 10 nM water-soluble 17␤-estradiol (E2), 200 nM ANP, or 100 nM sodium nitroprusside (SNP) was then added, and cells were incubated for a further 5 min. *, significantly different from control; **, significantly different from NS2028 alone. B, 17␤-estradiol does not elevate cGMP through inhibition of cyclic nucleotide phosphodiesterases. Cells were incubated with either no treatment (control) or 300 M IBMX plus 300 M zaprinast (zap) for 10 min. 10 nM water-soluble 17␤-estradiol (E2) or no treatment were then added, and cells were incubated for a further 5 min. *, significantly different from control; **, significantly different from control plus IBMX and zaprinast.

17␤-Estradiol Stimulates Hepatocyte Ca 2؉ Efflux
The addition of 10 nM 17␤-estradiol stimulated an approximate 1.6-fold increase in the rate of Ca 2ϩ efflux (Fig. 4, C and  G). The increase was extremely rapid, occurring within ϳ2-4 s. No effect on the rate of Ca 2ϩ efflux was seen upon the addition of the equivalent concentration of the cyclodextrin vehicle (Fig.  4G). Caloxin 1b1 prevented any stimulation of Ca 2ϩ efflux by 17␤-estradiol (Fig. 4, D and G), indicating that the 17␤-estradiol-stimulated Ca 2ϩ efflux is PMCA-mediated. Fig. 4, E and G, show that an equivalent concentration of 17␤-estradiol-BSA also stimulates Ca 2ϩ efflux from hepatocytes, with an approximate 1.5-fold increase, similar to the increase seen with 17␤estradiol. The stereoisomer 17␣-estradiol had no effect on the rate of Ca 2ϩ efflux from hepatocytes (Fig. 4, F and G).
The relationship between hepatocyte Ca 2ϩ efflux stimulation and concentration of 17␤-estradiol was investigated (Fig.  4H). The optimum concentration was 10 nM, although a significant increase in the rate of Ca 2ϩ efflux was seen at and above a threshold concentration of 1 nM. Consistent with the effect on cGMP elevation (Fig. 1B), the magnitude of the Ca 2ϩ efflux stimulation was reduced at higher concentrations of 17␤-estradiol (Fig. 4H).  (80). Fig. 5A shows that 10 nM 17␤-estradiol applied alone did not alter resting [Ca 2ϩ ] c levels in single rat hepatocytes. Single rat hepatocytes responded to 25 M ATP by the elevation of [Ca 2ϩ ] c (Fig. 5, B, D, and  E). Co-application of 10 nM 17␤-estradiol decreased both the amplitude and duration of the ATP-induced [Ca 2ϩ ] c rise (Fig. 5C, D, -Fig. 6, A and C, shows that soluble guanylate cyclase inhibitor NS2028 did not prevent 17␤-estradiol from stimulating hepatocyte Ca 2ϩ efflux. In confirmation of our previous findings, both the membrane-permeant cGMP analogue 8-Br-cGMP (Fig.  6, B and C) and ANP-mediated particulate guanylyl cyclase activation (Fig. 6C) stimulated Ca 2ϩ efflux from rat hepatocytes. However, sodium nitroprusside-mediated soluble guanylyl cyclase activation did not stimulate Ca 2ϩ efflux (Fig. 6C) (32)(33)(34).

17␤-Estradiol Does Not Elevate [Ca 2ϩ ] c but Attenuates ATP-induced Elevations in [Ca 2ϩ ] c in Sin
Stimulation of Ca 2ϩ Efflux by 17␤-Estradiol Is Mediated by PKG-To investigate a possible link between elevation of cGMP, activation of PKG, and Ca 2ϩ efflux stimulation by 17␤estradiol, the two distinct membrane-permeant PKG inhibitors, Rp-8-pCPT-cGMPS and DT-3, were used.
17␤-Estradiol Binds Specifically at the Plasma Membrane of Intact Rat Hepatocytes-To investigate whether 17␤-estradiol binds at the plasma membrane of intact rat hepatocytes, the membrane-impermeant, fluorescently labeled conjugate 17␤-estradiol-BSA-FITC was used. Fig. 8C shows  AUGUST 27, 2010 • VOLUME 285 • NUMBER 35 that 17␤-estradiol-BSA-FITC binds to the plasma membrane of intact hepatocytes that had been cultured for 24 h. Similar plasma membrane binding of 17␤-estradiol-BSA-FITC was also observed in hepatocytes cultured for 6 h (results not shown). An excess of either 17␤-estradiol (Fig. 8E) or 17␤-estradiol-BSA (Fig. 8G) prevented 17␤-estradiol-BSA-FITC binding to the plasma membrane. In contrast, an excess of 17␣-estradiol did not compete with the binding of 17␤-estradiol-BSA-FITC to the plasma membrane (Fig. 8I). An excess of guanylyl cyclase A receptor agonist ANP was, however, effective in preventing plasma membrane binding of 17␤-estradiol-BSA-FITC (Fig. 8K).

17␤-Estradiol Stimulates Hepatocyte Ca 2؉ Efflux
Two classical estrogen receptors have been described: ER␣ and ER␤ (2). A previous study has shown that ER␣, but not ER␤, is present in rat liver (81). Fig. 8M shows that 17␤estradiol-BSA-FITC stained both the plasma membrane and the cytosol of digitonin-permeabilized hepatocytes. The cytosolic staining is consistent with 17␤-estradiol-BSA-FITC binding to classical 17␤-estradiol receptors, which have an intracellular location. Indeed, when the cells were preincubated with ICI 182,780, an antagonist of the classical ERs (82), the cytosolic staining was greatly reduced (Fig. 8, O  and Q). In marked contrast, however, neither 50 nor 100 M ICI 182,780 appeared to affect the binding of 17␤-estradiol-BSA-FITC at the plasma membrane of either permeabilized (Fig. 8, O and Q) or intact hepatocytes (Fig. 8, S and U). These findings suggest that 17␤-estradiol binding to the hepatocyte plasma membrane is not mediated by classical ER␣ or ER␤. Fig. 8W shows that G-1, a specific agonist (83) for the GPR30 receptor (52, 53), did not compete with 17␤-estra-

DISCUSSION
We have shown here that 17␤-estradiol stimulates particulate guanylyl cyclase to rapidly elevate rat hepatocyte cGMP and promote plasma membrane localization of PKGI␣. We also show that 17␤-estradiol, above a similar threshold concentration of 1 nM, stimulates PMCA-dependent Ca 2ϩ efflux from rat hepatocytes through a PKGImediated mechanism. Our study is the first to observe particulate guanylyl cyclase activation and Ca 2ϩ efflux stimulation by 17␤-estradiol in one cell type and to demonstrate that they are linked by the activation and translocation of PKGI␣ to the plasma membrane.
Our model proposes that a spatially and functionally distinct pool of cGMP close to the plasma membrane (30,84), generated by activation of particulate guanylyl cyclase but not soluble guanylyl cyclase, recruits activated PKGI␣ into its vicinity. PKGI␣ thus localized at the plasma membrane activates the PMCA (85, 86) thereby stimulating plasma membrane Ca 2ϩ efflux (33,34). PKGI␣, but not PKGI␤, stimulates PMCA partially purified from aortic smooth muscle cells (86), which like rat hepatocytes (87), predominantly express PMCA isotype I.
How PKGI␣ is recruited to the hepatocyte plasma membrane is not known. However, we propose the involvement of an interaction between PKGI␣ and PKG-anchoring proteins. In addition to holding the homodimers together and controlling activation/inhibition of the catalytic domain, the N-terminal domain of PKGI␣ has a further important function in that it mediates binding to PKG-anchoring proteins, which target PKGI␣ to different intracellular locations (74). Such PKG-anchoring proteins include regulator of G protein signaling 2 (RGS-2) and the myosin-binding subunit of phosphatase I. Indeed, studies in both primary mouse vascular smooth muscle cells (88) and isolated mouse cardiomyocytes (89) have shown that activated PKGI␣ phosphorylates and binds to RGS-2, resulting in translocation of the PKGI␣-RGS-2 complex to the plasma membrane. In both cell types, consistent with our data, PKG inhibition prevents PKGI␣ plasma membrane recruitment by preventing PKGI␣-mediated phosphorylation of the N terminus of RGS2 (88,89).
We have previously presented a novel mechanism through which ANP protects hepatocytes by particulate guanylyl cyclase-mediated Ca 2ϩ efflux stimulation, thereby attenuating harmful elevations in [Ca 2ϩ ] c (33). 17␤-estradiol also protects liver and isolated hepatocytes under pathophysiological conditions associated with a rise in [Ca 2ϩ ] c (90 -93) through an unknown mechanism that appears to be unrelated to classical ER activation (91,92). Our finding that 17␤-estradiol can also, like ANP, stimulate Ca 2ϩ efflux through particulate guanylyl cyclase activation and attenuate [Ca 2ϩ ] c elevations suggests that such a cytoprotective mechanism may underlie 17␤-estradiol-mediated hepatoprotection and may, thus, prove to be of more widespread physiological significance.
Our data show that the plasma membrane-impermeant conjugate 17␤-estradiol-BSA closely mimicked all of the effects of free 17␤-estradiol, elevating cGMP, promoting PKGI␣ plasma membrane localization, and stimulating Ca 2ϩ efflux. We, therefore, propose that the rapid effects of 17␤-estradiol on hepatocyte cGMP, PKGI␣ plasma membrane recruitment, and Ca 2ϩ efflux observed here are mediated by 17␤-estradiol acting at the extracellular face of the plasma membrane.
The nature of the plasma membrane interaction was investigated by both competition binding and functional (Ca 2ϩ efflux) studies using agonists and antagonists of characterized plasma membrane receptors for 17␤-estradiol. Our observations that the classical ER antagonist ICI 182,780 (82) prevented intracellular 17␤-estradiol-BSA-FITC binding in permeabilized hepatocytes but not 17␤-estradiol-BSA-FITC binding at the plasma membrane of either intact or permeabilized hepatocytes and did not prevent 17␤-estradiol-stimulated Ca 2ϩ efflux indicate that neither classical receptor, ER␣ or ER␤, mediates either hepatocyte plasma membrane 17␤-estradiol binding or the rapid signaling effects observed here. Likewise, because the GPR30 agonist G-1 (83) did not compete with 17␤-estradiol-BSA-FITC for binding to intact hepatocytes nor mimic the ability of 17␤-estradiol to stimulate Ca 2ϩ efflux, we have discounted the receptor GPR30 (52,53). This conclusion is further supported by the absence of effect of ICI 182,780 on Ca 2ϩ efflux; ICI 182,780 is, in addition to its action as a classical ER antagonist, a potent GPR30 agonist (52). Furthermore, our observations that 17␣-estradiol is without effect on plasma membrane binding (consistent with the findings of an early study (94)) of 17␤-estradiol-BSA-FITC, cGMP, PKGI␣ translocation or Ca 2ϩ efflux rules out receptor ERX, which is preferentially activated by 17␣-estradiol (61).
Our finding that an excess of ANP competes with 17␤-estradiol-BSA-FITC for hepatocyte plasma membrane binding raises the possibility that 17␤-estradiol binds directly to the extracellular receptor portion of the GC-A receptor for ANP, thus stimulating the intrinsic particulate guanylyl cyclase activity of this receptor and thereby promoting plasma membrane recruitment of PKGI␣ and PMCA stimulation (33,34). However, there are other feasible explanations of this observation. First, an excess of ANP may simply block binding of 17␤-estradiol to its distinct plasma membrane receptor. Second, the GC-A receptor and the plasma membrane 17␤-estradiol receptor may reside in close proximity such that binding of ANP to the GC-A receptor blocks binding of 17␤-estradiol to its distinct receptor. Therefore, it remains possible that 17␤-estradiol acts through a distinct plasma membrane receptor.
Our observation that dopamine and noradrenaline compete with 17␤-estradiol-BSA-FITC binding to the plasma membrane of intact rat hepatocytes, whereas a specific ␣ 2adrenergic agonist, a ␤-adrenergic antagonist and D 2 dopamine receptor antagonist are without effect, is consistent with the properties of the putative ␥-adrenergic receptor characterized pharmacologically in chick ciliary ganglion (59). The ␥adrenergic receptor was proposed by Nadal et al. (5,7,55,56) to mediate the rapid signaling effects of 17␤-estradiol in ␣ and ␤ pancreatic cells, which resemble, in terms of both particulate guanylyl cyclase stimulation and insensitivity to ICI 182,780, the plasma membrane-initiated signaling events we have observed in hepatocytes.
Despite compelling pharmacological evidence for its existence (57)(58)(59), the molecular identity of the ␥-adrenergic receptor remains to be elucidated. Perhaps the ␥-adrenergic receptor is one of the orphan particulate guanylyl cyclase isoforms (22)(23) for which 17␤-estradiol may be revealed to be the endogenous ligand. However, sensitivity of 17␤-estradiol signaling to pertussis toxin in pancreatic ␣ cells suggests that this putative receptor is a G protein-coupled receptor (46). Pharmacological similarities have been noted to a recently identified cloned and characterized Drosophila melanogaster G protein-coupled receptor, DmDopEcR (95), that mediates the effects of ecdysteroids and catecholamines. It is an intriguing possibility that the ␥-adrenergic receptor may be a G protein-coupled receptor that activates a transmembrane guanylyl cyclase, perhaps the GC-A receptor, by cross-talk signaling mediated by G protein activation. There are, indeed, several reports of coupling of G protein-coupled receptors to transmembrane guanylyl cyclases (96 -97).
In summary, we have shown here that 17␤-estradiol, acting at the extracellular face of the plasma membrane, stimulates particulate guanylyl cyclase, rapidly elevating cGMP, which through activation and plasma membrane recruitment of PKGI␣, stimulates PMCA-mediated Ca 2ϩ efflux from hepatocytes. We have also shown that 17␤-estradiol binds specifically at the hepatocyte plasma membrane through an interaction

17␤-Estradiol Stimulates Hepatocyte Ca 2؉ Efflux
that is competed by an excess of ANP but that also shows many similarities to the pharmacological characteristics of the putative ␥-adrenergic receptor. We, therefore, propose that either the observed rapid signaling effects of 17␤-estradiol are mediated through the GC-A receptor or they are mediated by the ␥-adrenergic receptor, which is either itself a transmembrane guanylyl cyclase or activates a transmembrane guanylyl cyclase perhaps the GC-A receptor through cross-talk signaling.