Nongenomic activation of the GC-A enzyme by resveratrol and estradiol downstream from membrane estrogen receptors in human coronary arterial cells
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Nutrition, Metabolism & Cardiovascular Diseases (2007) 17, 508e516
www.elsevier.com/locate/nmcd
Nongenomic activation of the GC-A enzyme
by resveratrol and estradiol downstream from
membrane estrogen receptors in human
coronary arterial cells
A.M. El-Mowafy a,*, M. Alkhalaf b, S.M. Jaffal b
a
Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
b
Department of Biochemistry, Faculty of Medicine, HSC, Kuwait University, Kuwait
Received 21 December 2005; received in revised form 6 April 2006; accepted 26 April 2006
KEYWORDS Abstract Background and aim: Resveratrol (RSVL), a polyphenolic phytoestrogen
Resveratrol; in grapes, confers multifaceted cardiovascular benefits. The cellular and molecular
Estradiol; basis of RSVL actions has been largely undefined until now.
Human coronary Methods and results: In human coronary smooth muscle cells (HCSMCs), RSVL mark-
artery; edly (3.2-fold) enhanced cGMP formation (t1/2: 6.3 min, EC50: 1.8 mM) and stimu-
cGMP; lated kinase-G activity (4-fold). By contrast, RSVL had no effect on cAMP or PKA
Kinase-G; activity in these cells. The RSVL-enhanced cGMP/kinase-G activity was not abro-
Nitric oxide synthase; gated by the nitric oxide synthase-inhibitor (L-NMMA, 10 mM), or the soluble guany-
Particulate guanylate lyl cyclase (sGC)-inhibitor (ODQ, 10 mM). In membrane preparations from HCSMCs,
cyclase; RSVL activated GC in the particulate-, but not in the soluble-membrane fraction.
Membrane estrogen- Similar effects were due to the specific particulate-GC-A agonist atrial natriuretic
receptor peptide (ANP, 0.1e1 mM). The combined effects of RSVL and ANP were competitive.
By contrast, the selective GC-B agonist (BNP) showed no response on cGMP,
whereas that for GC-C (guanylin) produced only slight increases in cGMP levels.
Estradiol (E2) mimicked the effects of RSVL on cGMP, but showed a 46% lower
maximal response. Combining E2 with RSVL showed a competitive, rather than an
additive, response. Further, cGMP formation by RSVL or E2 was significantly atten-
uated by the pure estrogen receptor blocker, ICI-182,780 (10 mM).
Conclusion: These findings are the first to link RSVL with pGC/kinase-G activation
downstream from membrane ERs in the vasculature, thus substantiating its coro-
nary protective effects, even in endothelium-disrupted coronary arteries.
ª 2006 Elsevier B.V. All rights reserved.
* Corresponding author. Tel./fax: þ20 50 224 7496.
E-mail address: aelmowafy@yahoo.com (A.M. El-Mowafy).
0939-4753/$ - see front matter ª 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.numecd.2006.04.008Nongenomic activation of the GC-A enzyme 509
Introduction Determination of cyclic nucleotide levels
The contention that natural edible components may cAMP and cGMP were determined by enzyme-
protect against diseases is currently best exempli- immunoassay according to the reported proce-
fied by resveratrol (trans-3,40 ,5-trihydroxystilbene, dures [16,17]. Human coronary smooth muscle
RSVL), a phytoalexin in grapes, berries and red wine cells, passages 3e8, were cultured in 48-well cul-
[1,2]. RSVL is believed to confer protection against ture dishes at equal densities, in an atmosphere
some cardiovascular diseases, a dogma commonly of 5% CO2/air, at 37 C. Media were exchanged
designated as ‘‘the French paradox of red wine’’ each 48 h. Experiments were run on 85e90% con-
[3]. These observations prompted an explosion of fluent cells. Confluent cells were incubated in
research that uncovered further biological effects low serum-media (0.5e1%) for 18 h. The media
for RSVL such as antineoplastic, antioxidant, anti- were removed and cells were washed three times
platelet, and anti-inflammatory actions [4e6]. Cor- with KRB-buffer (0.5 mL/well) containing 0.1% bo-
onary heart disease remains a primary contributor vine serum albumin. Cells were then preincubated
to morbidity and mortality in developed countries for 15 min at 37 C in a buffer containing 0.5 mM
[7]. The benefits of RSVL in such diseases have of IBMX, to inhibit phosphodiesterases. RSVL or
been linked to inhibition of platelet aggregation, solvent (ethanol) was then added. Solvent concen-
perturbation of prostanoid synthesis, and regula- tration was kept below 0.1%. Reactions were
tion of lipoprotein metabolism [8]. In the vascula- terminated after 10 min by removing the buffer
ture, RSVL also produced vasodilatory effects that and adding 0.5 mL of 0.1 N HCl for 30 min at
were attributed to endothelium-dependent release room temperature. cGMP and cAMP, extracted in
of nitric oxide (NO) [9]. Moreover, RSVL remarkably HCl, were measured by enzyme immunoassay using
relaxed endothelium-denuded vascular prepara- assay kits (Biomol) that included all reagents,
tions; however through largely unknown cellular antibodies and microtiter plates. Results were ex-
events [10]. In porcine coronaries, vasorelaxation pressed as fmol nucleotide/cell number. In gen-
by RSVL was attributed to its estrogen receptor eral, basal cAMP levels were 143e162 fmol/106
(ER) binding capacity [11]; however, RSVL antiproli- cells; 4e5-fold higher than those of cGMP
ferative effects in this preparation were indepen- (26e30 fmol/106).
dent from ERs [2]. Further, antioxidants such as
vitamin C and dithiothreitol have been shown to
stimulate guanylyl cyclase (GC) activity in some sys- Assay of guanylyl cyclase (GC) activity
tems [12,13]. Both kinase-G and kinase-A can confer in cellular fractions
protection in vascular smooth muscle through both
vasodilatory and antiproliferative effects [14]. Like- The following protocol was adapted from our
wise, estrogen receptor binding has been positively reported procedures [18]. Cells were washed
linked with vascular protection, as demonstrated with cold phosphate-buffered saline and then
with estradiol (E2) and RSVL [11,15]. Therefore, to with TriseHCl buffer 25 mM, pH 7.6, containing su-
verify the possible coronary protective effects of crose (250 mM). Cells were frozen at 80 C then
RSVL in humans, we investigated its effects on thawed on ice to break down the cell membranes.
kinase-G/kinase-A activity, and further attempted Cell debris was first removed by low-speed centri-
to delineate the molecular basis/trafficking path- fugation. The supernatant was then centrifuged at
ways entailing such RSVL coronary protection in 120,000 g (4 C) for 15 min. The produced super-
human coronary smooth muscle cells (HCSMCs). natant was collected as a soluble fraction and the
pellet was washed by dispersion into homogenizing
buffer and re-centrifugation. Pellet served as
the particulate membrane fraction. Guanylyl
Methods cyclase was determined in both soluble and partic-
ulate fractions in a total volume of 100 mL, at
Cell culture 37 C. The reaction mixture contained Tris (pH
7.6, 50 mM), IBMX (0.5 mM), GTP (1 mM), MgCl2
Human coronary artery smooth muscle cells were (4 mM), bovine serum albumin (0.1%), creatine
purchased from Clonetics/Cambrex Bio Science phosphate (25 mM), creatine kinase (55 U/ml),
(Walkersville, MD) and were grown in smooth membrane- or soluble-protein (5e10 mg), and the
muscle growth medium (SmGM-2) supplemented indicated concentrations of RSVL. Reactions were
with 5% fetal bovine serum. Passages 3e8 were terminated by immersion into boiling water
utilized for subsequent experiments. and centrifugation. The generated cGMP was510 A.M. El-Mowafy et al.
quantitated as mentioned above. Enzyme activity Statistical analyses
was expressed as pmol cGMP/min per mg tissue
protein. Statistical significance between two groups was
evaluated by Student’s t-test for unpaired data.
Comparison among multiple groups was conducted
Determination of PKG activity using the one-way analysis of variance (ANOVA)
test, followed by Tukey’s post hoc test to deter-
Incubation conditions were similar to those of mine significant differences among the means of
cGMP determination. Cells were lysed in a buffer the data groups. A probability of P < 0.05 was
containing (in mmol/L) TriseHCl (pH 7.4) 20, accepted as a significant difference.
EGTA 1, EDTA 1, and PMSF 1; 10 mg/mL leupeptin;
2 mg/mL aprotonin; and 0.1% Triton X-100. Cell
lysate was centrifuged at 13,000 g for 15 min at
4 C. The supernatant was used as a tissue extract Results
for determination of kinase activity [18]. Protein
concentrations were determined by the method Resveratrol (RSVL) appreciably (3.2-fold) enhanced
of Lowry. Kinase activity was determined by mea- cGMP formation in HCSMCs. This response occurred
suring 32Pi incorporation from [g-32P]ATP into the in a time- and concentration-dependent manner
serine residue of the synthetic peptide ‘‘Kemp- with a t1/2 value of 6.3 min and EC50 value of 1.8 mM
tide’’, containing a specifically designed sequence (Figs. 1A and 2). As can be seen in Fig. 2B, RSVL also
that governs high affinity to PKG. Reactions were elicited a concentration-dependent stimulation of
performed in a total volume of 50 mL that con- PKG (up to 4-fold). We also investigated the effects
tained (in mmol/L) TriseHCl (pH 7.5) 50, MgCl2 of RSVL on the cAMP-dependent kinase (PKA)
20, and MnCl2 10; 20 mL of tissue extract; because: (i) this cascade can produce similar pro-
100 mmol/L Kemptide; 100 mmol/L ATP, 0.5 mCi tective effects to that of the cGMP/kinase-G,
[g-32P]ATP (4 mCi/mmol); 0.1 mg/mL BSA; and (ii) RSVL was found to enhance cAMP formation in
the phosphatase inhibitors (in mmol/L): b-glycero- some systems [4], and (iii) cross-activation of
phosphate 50, sodium pyrophosphate 1, and sodium kinase-G by cAMP has been documented in vascular
vanadate 0.1. Reactions were carried out at 30 C smooth muscle [14,18]. Fig. 2 indicates that RSVL
for 10 min, and terminated by adding 20 mL of 20% had no effect on the AC/cAMP system. We also did
trichloroacetic acid (TCA) and ice cooling. After not detect any activation for PKA by RSVL (data
centrifugation, Kemptide-directed phosphorylation not presented).
was assessed by spotting 20 mL of each supernatant cGMP accumulation usually results from either
onto p-81 phosphocellulose paper discs. Discs were stimulation of GC activity, soluble or particulate
washed twice, each for 10 min with 1% phosphoric enzyme isoforms, or alternatively inhibition of
acid, followed by a similar washing with distilled cGMP-phosphodiesterases. Therefore, RSVL ef-
water. 32Pi incorporation was determined by liquid fects on cGMP were first challenged by specific
scintillation counting. Background for PKG activity inhibitors for soluble-GC (ODQ, 10 mM) and for NOS
was determined from parallel incubations contain- (L-NMMA, 10 mM). Relative to control levels, both
ing the selective kinase-G inhibitor KT5823 inhibitors did not alter RSVL’s capacity to enhance
(300 nmol/L) and was always less than 10% of total cGMP formation by RSVL (Fig. 3). These inhibitors,
Kemptide phosphorylation. Likewise, RSVL failed to however; reduced basal cGMP-, but not cAMP-,
elicit significant phosphorylation for Kemptide in levels; thus controlling for the activity and speci-
cultures of HCSMCs pretreated with this concentra- ficity of their actions (data not presented).
tion of KT5823. Because RSVL effects on cGMP were generally
determined in the presence of the broad-spectrum
PDE-inhibitor, 3-isobutyl-5-methylxanthine (IBMX,
Chemicals 0.5 mM), an inhibitory effect for RSVL on this en-
zyme can be ruled out. Accordingly, a possibility
Kits for cGMP were purchased from Biomol. Kemp- remained that RSVL could activate pGC. To clarify
tide, KT-5823, ODQ, and L-NMMA were purchased this assumption, the enzymatic activity of GC was
from Calbiochem. P-81 phosphocellulose paper determined in both soluble- and particulate-
discs were obtained from Gibco. [g-32P]ATP was membrane fractions from HCSMCs. Fig. 4A shows
purchased from Amersham. Resveratrol, ICI- that RSVL stimulated GC activity in the particu-
182,780, 17-b-estradiol, guanylin, ANP, and BNP late- but not in the soluble-fraction, indicating
were purchased from Sigma. the activation of membrane-bound GC isoform.Nongenomic activation of the GC-A enzyme 511
350 350
A
Cyclic nucleotide level (% of Control)
cGMP
300 cAMP 300
cGMP (% of control)
250
250
200
200
150
150
100
100
50
-9 -8 -7 -6 -5 -4
0.0 2.5 5.0 7.5 10.0 12.5 15.0
Log Resveratrol (M) Time of resveratrol treatment (min)
Figure 2 Time course for RSVL (10 mM)-induced cGMP
1.4
B formation. Incubations were performed in the presence
* of IBMX (0.5 mM). Basal cGMP level was 29 5 fmol/106
cells. Data are means SEM of 6 experiments.
PKG Activity (pmol/min/mg protein)
1.2
1.0 of this response from cytosolic signaling, at least
to a large extent.
To confirm these results and further identify the
0.8 * pGC-isoform(s) that signal(s) cGMP formation for
0.6
* 300
Pretreatment
None
0.4
LNMMA
250
ODQ
0.2
cGMP (% of Control)
200
0.0
0 1 10 100
Resveratrol ( M) 150
Figure 1 (A) Short-term (15 min) effects of various re-
sveratrol (RSVL) concentrations on cGMP and cAMP levels 100
in human coronary smooth muscle cells (HCSMCs). Data
were obtained in the presence of IBMX (0.5 mM). Basal
levels were 26 4 and 156 11 fmol/106 cells; respec- 50
tively. Data are means SEM of 5e7 experiments. (B)
Concentration-dependent stimulation of kinase-G by
RSVL in HCSMCs. Data are means SEM of 7 experi- 0
ments. *Significantly higher than untreated cells. 1 10
Resveratrol (µM)
Figure 3 Effect of the NOS-inhibitor (L-NMMA, 10 mM),
By contrast, the NO donor (SNAP, 10 mM) did not
or the soluble-GC inhibitor (ODQ, 10 mM) on RSVL (1e
alter GC activity in the particulate fraction, but
10 mM)-induced cGMP formation in HCSMCs. Incubation
activated this enzyme in the soluble fraction, in with enzyme inhibitors lasted for 20 min in the presence
a concentration-dependent manner (Fig. 4B). of IBMX (0.5 mM) before RSVL was added for 15 addi-
These results attest to the purity of prepared tional minutes. Incubations were performed in the pres-
membrane fractions, to the specificity of RSVL’s ence of IBMX (0.5 mM). Data are means SEM of 5e6
stimulatory effects on pGC, and to the dissociation experiments. *Significantly lower than untreated cells.512 A.M. El-Mowafy et al.
600
A
Particulate fraction
* 550
0.6
Guanylyl cyclase (pmol/min/mg)
Soluble fraction 500
cGMP (% of control)
450
* *
400
0.4 350 * *
300
250
200
0.2
150
100
0 -9 -8 -7 -6 -5
0.0 Log Resveratrol (M)
0 1 10
Resveratrol (µM) Figure 5 Effect of pretreatment with RSVL (1 nMe
10 mM) on ANP (0.1 mM)-induced cGMP formation in
* HCSMCs. Incubation with RSVL continued for 15 min in
1.0
the presence of IBMX (0.5 mM) before ANP was added.
B Data are means SEM of 4e6 experiments. *Significantly
Particulate fraction lower than ANP-treated cells.
Soluble fraction
Guanylyl cyclase (pmol/min/mg)
0.8
predominant pGC-isoform in HCSMCs, that is also
* targeted by RSVL to enhance cGMP formation in
0.6
these cells.
Furthermore, RSVL is known as a phytoestrogen
that can bind to and modulate the estrogen ma-
0.4 chinery [6,15]. Hence, we first checked whether
0.2 400
RSVL
Guanylin + RSVL
350
0.0
0 1 10
SNAP (µM) 300
cGMP (% of control)
Figure 4 Differential short-term (15 min) effects of
RSVL (A), and SNAP (B), on guanylyl cyclase activity in 250
soluble- and particulate-membrane fractions from
HCSMCs. Data are means SEM of 6e7 experiments.
200
*Significantly higher than basal enzyme activity.
150
RSVL, competition studies were performed using
RSVL and selective agonists for the three main
pGC-isoforms. The selective GC-A activator, ANP, 100
elicited a 5.5-fold increase in cGMP levels, a re-
sponse that was attenuated by RSVL in a concen- Control -9 -8 -7 -6 -5 -4 -3
tration-dependent manner (Fig. 5). Conversely, Resveratrol (log M)
the selective GC-B agonist, CNP had no significant
Figure 6 The combined effects of RSVL (1 nM-10 mM)
effect on cGMP levels in HCSMCs (data not shown). and guanylin (0.1 mM) on cGMP formation in HCSMCs. In-
Meanwhile, the selective GC-C agonist, guanylin, cubation with RSVL continued for 15 min in the presence
elicited only a slight (40e45%) increase in cGMP of IBMX (0.5 mM) before guanylin was added. Basal cGMP
levels that was virtually additive with RSVL effects level was 31 5 fmol/106 cells. Data are means SEM of
(Fig. 6). These observations imply that GC-A is the 4e6 experiments.Nongenomic activation of the GC-A enzyme 513
estradiol (E2) has similar stimulatory effects on Pretreatment
pGC. Fig. 7 demonstrates that E2 produced rapid, 300
None
concentration-dependent increases in cGMP with (1µM ICI-182,780)
(10µM ICI-182,780)
an EC50 value of 1 mM; a comparable value to that
250
of RSVL (1.8 mM). These effects for E2 were, like-
wise, insensitive to the s-GC inhibitor (ODQ 10 mM;
cGMP (% of Control)
data not shown), but were blunted by the ER- 200
blocker ICI-182,780 (Fig. 7). The maximal response *
to E2, however; was remarkably lower than that of
RSVL (230% vs. 335%). Further, competition studies 150 *
between E2 and RSVL (Fig. 7) suggest that the ef-
fects of these ligands are not additive, but rather
100
competitive; thus indicating that they compete for
the same effector(s). In this same vein, the effects
of RSVL on cGMP were partially, but significantly, 50
inhibited by the estrogen-receptor blocker,
ICI-182,780 (10 mM) (Fig. 8).
0
1 10
Resveratrol (µM)
Discussion
Figure 8 Effect of ICI-180,780 (1, 10 mM) on RSVL (1,
Resveratrol (RSVL) confers a plethora of beneficial 10 mM)-induced cGMP formation in HCSMCs. Incubation
biological responses against cancer and cardiovas- with ICI-182,780 continued for 20 min in the presence
cular disease; thereby becoming a main target in of IBMX (0.5 mM) before RSVL was added for 15 min.
Data are means SEM of 4e5 experiments.
recent experimental and clinical research [20].
Cardiovascular disease is the leading cause of
death in many societies all over the world [21]. some protective actions [23]. However, the molec-
The cardiovascular benefits of RSVL include inhibi- ular underpinnings of such vascular effects remain
tion of LDL-oxidation and protection against ische- largely elusive [3,22]. In particular, the mecha-
mia/reperfusion-induced myocardial damage [22]. nisms whereby RSVL can dilate endothelium-
At the vascular level, RSVL appears to also exert denuded vessels have been more speculative
than certain; and appear to be inconsistent among
blood vessels [9,10]. When characterized, these
350
E2 signaling mechanisms can provide convincing clues
E2 + RSVL 1 µM for the protective effects of RSVL in the
E2 + ICI-182,780 vasculature.
300
The present study demonstrates the capacity of
RSVL to stimulate the GC/cGMP/kinase-G cascade
cGMP (% of control)
250 in an endothelium-free system; i.e., the human
coronary smooth muscle cells (HCSMCs). This
signaling cascade is known to culminate into both
200
vasodilatory and antiatherogenic effects in smooth
muscles [24]. At the molecular level, cGMP dilates
150
blood vessels through reduction of intracellular
calcium, inhibition of myosin-light-chain phosphor-
ylation, or stimulation of potassium efflux and
100 membrane repolarization [18,25]. On the other
hand, cGMP elicits cytostatic actions in smooth
Control -9 -8 -7 -6 -5 -4 -3 muscles by enhancing apoptosis, inhibiting mito-
Estradiol (E2) (log M) genic enzymes such as PI3K and MAPKs, and/or in-
terfering with the cell-cycle machinery [2,23,24].
Figure 7 The effects of RSVL (1 mM) and ICI-182,780
(10 M) on estradiol (E2, 1 nM-200 mM)-induced cGMP for- In HCSMCs, our present observations showed that
mation in HCSMCs. Incubation with E2 or ICI-182,780 the stimulatory effects of RSVL on cGMP are not
continued for 20 min in the presence of IBMX (0.5 mM) mediated by sGC or via inhibition of phosphodies-
before RSVL or E2 were added for 15 min. Data are terases, as confirmed by the use of subcellular
means SEM of 5e6 experiments. fractions and specific inhibitors for sGC and PDE514 A.M. El-Mowafy et al. enzymes. Instead, these effects for RSVL involved Moreover, we were able to demonstrate stimula- the activation of membrane-bound GC isoform tion of pGC by RSVL in a microsomal-membrane (pGC). This response occurred in both time- and preparation, implying that the enhanced cGMP re- concentration-related fashions. The time-course sponse occurs mostly at the cell-membrane level. of this response (10 min for maximal cGMP forma- Further, we showed that effects for RSVL and the tion/kinase-G activation) rules out the involve- GC-A agonist, ANP, were competitive rather than ment of genomic mechanisms. This notion was additive. On the other hand, E2 reproduced these also confirmed by using conventional inhibitors rapid GC-A stimulatory effects of RSVL in HCSMCs. for transcription and translation (data not pre- Both responses to RSVL and E2 were sensitive to sented). The estimated EC50 value of this re- the pure ER-blocker, ICI-182,780, supporting the sponse, 1.8 mM, is congruent to those found for mediation by membrane ERs. Lines of evidence inhibition of MAPKs (2 mM) [2], and for relaxation that RSVL and E2 share common ERs and mem- of vascular beds (0.5e10 mM) [10,13]. Indeed, brane-bound GC-A enzyme have currently evolved pGC has been a major player in maintaining cardio- from competition experiments on cGMP formation, vascular hemodynamic mechanisms and integrity. which revealed that these ligands act ‘‘competi- Not surprisingly, lower pGC activity was observed tively’’ on one and the same effector. in vascular preparations from hypertensive animals Despite these similarities between RSVL and E2 [26], whereas treatments with exogenous ANP trig- actions, some mechanistic differences have been gered both vasodilation and cytostatic responses observed in their present cGMP response. First, the [24,27]. These views are supported by the re- maximal cGMP stimulatory response for RSVL was ported ability of RSVL to improve the vascular me- appreciably (46%) higher than that of E2, which chanical properties in hypertensive animals [28]. was likewise spotted between E2 and tamoxifen in Unlike sGC, the membrane-bound pGC enzyme another cell system [32]. We propose that this is a receptor-linked enzyme that exists in at least higher intrinsic activity for RSVL could be the con- seven isoforms in mammalian tissues (GC-A sequence of its nature as a mixed ER-agonist/ through -G) [24,29]. Albeit being primarily acti- antagonist, as compared to the pure agonistic vated by the endogenous ligands ‘‘natriuretic profile of E2. Our dynamic simulation studies for peptides’’, recent observations showed that pGC the mechanism of interaction of these ligands could also be stimulated by exogenous agents, with ER-a may well support this assumption [33]. like vitamin-C and muscarinic agonists, in diverse Because also these ligands appear to chiefly target systems [2,30]. The exact scenario underlying a membrane receptor, their differential lipid this process has not been defined. However, an im- solubility and cellular penetrability can be an portant regulatory mechanism for pGC is its sus- additional element. Lastly, as the identity, struc- ceptibility for desensitization by a PKC-triggered tural details, and ligand-binding characteristics dephosphorylation [19]. get unraveled, more of these differential ligand Because RSVL is a phytoestrogen with a capacity responses can be better explained. In the present to bind to estrogen receptors (ERs), numerous work, the partial, but significant attenuation of cardiovascular studies have investigated the link RSVL response by the pure ER blocker, ICI- between ER-binding and cardiovascular protection 182,780, suggests that RSVL may trigger additional by RSVL [1,6]. In this context, in porcine coronary signaling pathways to enhance GC-A activity. This arteries, we reported that RSVL rapidly inhibited view is supported by the current finding that this MAPKs through an ER-independent mechanism ER-blocker completely blunted the E2 response; [2]. However, the vasorelaxant effects of RSVL, needless to say, RSVL, away from ERs, can modu- also in porcine coronaries, were ascribed to modu- late a variety of cellular trafficking pathways lation of potassium current, through an ER- [1e3]. For instance, the remarkable RSVL antioxi- mediated pathway [12]. Interestingly, in arterial dant activity has been associated with many bio- cells isolated from hypertensive animals, the ER- logical effects; including cGMP formation [12]. blocker (ICI-182,780) only partly reversed the Thus, collectively, it appears that the signaling long-term inhibitory effects of RSVL on atheroscle- pathway that couples RSVL to GC-A activation is rosis, DNA synthesis and prolyl hydroxylase activity not necessarily a simple/direct one. Lastly, in this [31]. Our present finding that activation of GC by context, it also remains to be investigated whether RSVL in HCSMCs was both rapid (minutes) and me- RSVL can produce additional effects by binding to diated by the GC-A isoform agrees with the study such membrane ERs. of Chen and colleagues that showed an agonistic Indeed, functional evidence has been accumu- effect for the ER-ligand tamoxifen in the porcine lated that both long-term (genomic)- and short- kidney proximal tubular LLC-PK1 cells [32]. term-effects can be mediated by E2 binding to
Nongenomic activation of the GC-A enzyme 515
membrane ERs [34]. However, a paucity of informa- membrane-bound GC-A isoform downstream from
tion is available on the mechanisms whereby E2 membrane estrogen receptors, and is functional in
generates its non-genomic effects; and many postu- the absence of vascular endothelium.
lations have been driven therein. Classical ER-a
receptors were reported to signal non-genomic
activation of NO synthase and mitogen-activated Acknowledgement
protein kinase [35,36]. Also, E2 has been shown to
employ another type of cell-surface receptor, This study was supported by Kuwait University
GPR30da G-protein-coupled receptor homologdin grant PT 01/01 to A.M.El-M. and M.A.
order to activate the ERK [37]. Therefore, all these
previous observations are in line with our present
findings that RSVL binds to membrane ERs to en-
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