Nongenomic activation of the GC-A enzyme by resveratrol and estradiol downstream from membrane estrogen receptors in human coronary arterial cells

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 b

Department of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt 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 Resveratrol; Estradiol; Human coronary artery; cGMP; Kinase-G; Nitric oxide synthase; Particulate guanylate cyclase; Membrane estrogenreceptor

Abstract Background and aim: Resveratrol (RSVL), a polyphenolic phytoestrogen in grapes, confers multifaceted cardiovascular benefits. The cellular and molecular basis of RSVL actions has been largely undefined until now. Methods and results: In human coronary smooth muscle cells (HCSMCs), RSVL markedly (3.2-fold) enhanced cGMP formation (t1/2: 6.3 min, EC50: 1.8 mM) and stimulated kinase-G activity (4-fold). By contrast, RSVL had no effect on cAMP or PKA activity in these cells. The RSVL-enhanced cGMP/kinase-G activity was not abrogated by the nitric oxide synthase-inhibitor (L-NMMA, 10 mM), or the soluble guanylyl cyclase (sGC)-inhibitor (ODQ, 10 mM). In membrane preparations from HCSMCs, RSVL activated GC in the particulate-, but not in the soluble-membrane fraction. Similar effects were due to the specific particulate-GC-A agonist atrial natriuretic 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 attenuated 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 coronary 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: [email protected] (A.M. El-Mowafy). 0939-4753/$ - see front matter ª 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.numecd.2006.04.008

Nongenomic activation of the GC-A enzyme

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Introduction

Determination of cyclic nucleotide levels

The contention that natural edible components may protect against diseases is currently best exemplified by resveratrol (trans-3,40 ,5-trihydroxystilbene, RSVL), a phytoalexin in grapes, berries and red wine [1,2]. RSVL is believed to confer protection against some cardiovascular diseases, a dogma commonly designated as ‘‘the French paradox of red wine’’ [3]. These observations prompted an explosion of research that uncovered further biological effects for RSVL such as antineoplastic, antioxidant, antiplatelet, and anti-inflammatory actions [4e6]. Coronary heart disease remains a primary contributor to morbidity and mortality in developed countries [7]. The benefits of RSVL in such diseases have been linked to inhibition of platelet aggregation, perturbation of prostanoid synthesis, and regulation of lipoprotein metabolism [8]. In the vasculature, RSVL also produced vasodilatory effects that were attributed to endothelium-dependent release of nitric oxide (NO) [9]. Moreover, RSVL remarkably relaxed endothelium-denuded vascular preparations; however through largely unknown cellular events [10]. In porcine coronaries, vasorelaxation by RSVL was attributed to its estrogen receptor (ER) binding capacity [11]; however, RSVL antiproliferative effects in this preparation were independent from ERs [2]. Further, antioxidants such as vitamin C and dithiothreitol have been shown to stimulate guanylyl cyclase (GC) activity in some systems [12,13]. Both kinase-G and kinase-A can confer protection in vascular smooth muscle through both vasodilatory and antiproliferative effects [14]. Likewise, estrogen receptor binding has been positively linked with vascular protection, as demonstrated with estradiol (E2) and RSVL [11,15]. Therefore, to verify the possible coronary protective effects of RSVL in humans, we investigated its effects on kinase-G/kinase-A activity, and further attempted to delineate the molecular basis/trafficking pathways entailing such RSVL coronary protection in human coronary smooth muscle cells (HCSMCs).

cAMP and cGMP were determined by enzymeimmunoassay according to the reported procedures [16,17]. Human coronary smooth muscle cells, passages 3e8, were cultured in 48-well culture dishes at equal densities, in an atmosphere of 5% CO2/air, at 37  C. Media were exchanged each 48 h. Experiments were run on 85e90% confluent cells. Confluent cells were incubated in low serum-media (0.5e1%) for 18 h. The media were removed and cells were washed three times with KRB-buffer (0.5 mL/well) containing 0.1% bovine serum albumin. Cells were then preincubated for 15 min at 37  C in a buffer containing 0.5 mM of IBMX, to inhibit phosphodiesterases. RSVL or solvent (ethanol) was then added. Solvent concentration was kept below 0.1%. Reactions were terminated after 10 min by removing the buffer and adding 0.5 mL of 0.1 N HCl for 30 min at room temperature. cGMP and cAMP, extracted in HCl, were measured by enzyme immunoassay using assay kits (Biomol) that included all reagents, antibodies and microtiter plates. Results were expressed as fmol nucleotide/cell number. In general, basal cAMP levels were 143e162 fmol/106 cells; 4e5-fold higher than those of cGMP (26e30 fmol/106).

Methods Cell culture Human coronary artery smooth muscle cells were purchased from Clonetics/Cambrex Bio Science (Walkersville, MD) and were grown in smooth muscle growth medium (SmGM-2) supplemented with 5% fetal bovine serum. Passages 3e8 were utilized for subsequent experiments.

Assay of guanylyl cyclase (GC) activity in cellular fractions The following protocol was adapted from our reported procedures [18]. Cells were washed with cold phosphate-buffered saline and then with TriseHCl buffer 25 mM, pH 7.6, containing sucrose (250 mM). Cells were frozen at 80  C then thawed on ice to break down the cell membranes. Cell debris was first removed by low-speed centrifugation. The supernatant was then centrifuged at 120,000  g (4  C) for 15 min. The produced supernatant 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 cyclase was determined in both soluble and particulate fractions in a total volume of 100 mL, at 37  C. The reaction mixture contained Tris (pH 7.6, 50 mM), IBMX (0.5 mM), GTP (1 mM), MgCl2 (4 mM), bovine serum albumin (0.1%), creatine phosphate (25 mM), creatine kinase (55 U/ml), membrane- or soluble-protein (5e10 mg), and the indicated concentrations of RSVL. Reactions were terminated by immersion into boiling water and centrifugation. The generated cGMP was

510 quantitated as mentioned above. Enzyme activity was expressed as pmol cGMP/min per mg tissue protein.

Determination of PKG activity Incubation conditions were similar to those of cGMP determination. Cells were lysed in a buffer containing (in mmol/L) TriseHCl (pH 7.4) 20, 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 for determination of kinase activity [18]. Protein concentrations were determined by the method of Lowry. Kinase activity was determined by measuring 32Pi incorporation from [g-32P]ATP into the serine residue of the synthetic peptide ‘‘Kemptide’’, containing a specifically designed sequence that governs high affinity to PKG. Reactions were performed in a total volume of 50 mL that contained (in mmol/L) TriseHCl (pH 7.5) 50, MgCl2 20, and MnCl2 10; 20 mL of tissue extract; 100 mmol/L Kemptide; 100 mmol/L ATP, 0.5 mCi [g-32P]ATP (4 mCi/mmol); 0.1 mg/mL BSA; and the phosphatase inhibitors (in mmol/L): b-glycerophosphate 50, sodium pyrophosphate 1, and sodium vanadate 0.1. Reactions were carried out at 30  C for 10 min, and terminated by adding 20 mL of 20% trichloroacetic acid (TCA) and ice cooling. After centrifugation, Kemptide-directed phosphorylation was assessed by spotting 20 mL of each supernatant onto p-81 phosphocellulose paper discs. Discs were washed twice, each for 10 min with 1% phosphoric acid, followed by a similar washing with distilled water. 32Pi incorporation was determined by liquid scintillation counting. Background for PKG activity was determined from parallel incubations containing the selective kinase-G inhibitor KT5823 (300 nmol/L) and was always less than 10% of total Kemptide phosphorylation. Likewise, RSVL failed to elicit significant phosphorylation for Kemptide in cultures of HCSMCs pretreated with this concentration of KT5823.

Chemicals Kits for cGMP were purchased from Biomol. Kemptide, KT-5823, ODQ, and L-NMMA were purchased from Calbiochem. P-81 phosphocellulose paper discs were obtained from Gibco. [g-32P]ATP was purchased from Amersham. Resveratrol, ICI182,780, 17-b-estradiol, guanylin, ANP, and BNP were purchased from Sigma.

A.M. El-Mowafy et al.

Statistical analyses Statistical significance between two groups was evaluated by Student’s t-test for unpaired data. Comparison among multiple groups was conducted using the one-way analysis of variance (ANOVA) test, followed by Tukey’s post hoc test to determine significant differences among the means of the data groups. A probability of P < 0.05 was accepted as a significant difference.

Results Resveratrol (RSVL) appreciably (3.2-fold) enhanced cGMP formation in HCSMCs. This response occurred in a time- and concentration-dependent manner with a t1/2 value of 6.3 min and EC50 value of 1.8 mM (Figs. 1A and 2). As can be seen in Fig. 2B, RSVL also elicited a concentration-dependent stimulation of PKG (up to 4-fold). We also investigated the effects of RSVL on the cAMP-dependent kinase (PKA) because: (i) this cascade can produce similar protective effects to that of the cGMP/kinase-G, (ii) RSVL was found to enhance cAMP formation in some systems [4], and (iii) cross-activation of kinase-G by cAMP has been documented in vascular smooth muscle [14,18]. Fig. 2 indicates that RSVL had no effect on the AC/cAMP system. We also did not detect any activation for PKA by RSVL (data not presented). cGMP accumulation usually results from either stimulation of GC activity, soluble or particulate enzyme isoforms, or alternatively inhibition of cGMP-phosphodiesterases. Therefore, RSVL effects on cGMP were first challenged by specific inhibitors for soluble-GC (ODQ, 10 mM) and for NOS (L-NMMA, 10 mM). Relative to control levels, both inhibitors did not alter RSVL’s capacity to enhance cGMP formation by RSVL (Fig. 3). These inhibitors, however; reduced basal cGMP-, but not cAMP-, levels; thus controlling for the activity and specificity of their actions (data not presented). Because RSVL effects on cGMP were generally determined in the presence of the broad-spectrum PDE-inhibitor, 3-isobutyl-5-methylxanthine (IBMX, 0.5 mM), an inhibitory effect for RSVL on this enzyme can be ruled out. Accordingly, a possibility remained that RSVL could activate pGC. To clarify this assumption, the enzymatic activity of GC was determined in both soluble- and particulatemembrane fractions from HCSMCs. Fig. 4A shows that RSVL stimulated GC activity in the particulate- but not in the soluble-fraction, indicating the activation of membrane-bound GC isoform.

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Figure 2 Time course for RSVL (10 mM)-induced cGMP 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.

of this response from cytosolic signaling, at least to a large extent. To confirm these results and further identify the pGC-isoform(s) that signal(s) cGMP formation for

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Figure 1 (A) Short-term (15 min) effects of various resveratrol (RSVL) concentrations on cGMP and cAMP levels 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; respectively. Data are means  SEM of 5e7 experiments. (B) Concentration-dependent stimulation of kinase-G by RSVL in HCSMCs. Data are means  SEM of 7 experiments. *Significantly higher than untreated cells.

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By contrast, the NO donor (SNAP, 10 mM) did not alter GC activity in the particulate fraction, but activated this enzyme in the soluble fraction, in a concentration-dependent manner (Fig. 4B). These results attest to the purity of prepared membrane fractions, to the specificity of RSVL’s stimulatory effects on pGC, and to the dissociation

Figure 3 Effect of the NOS-inhibitor (L-NMMA, 10 mM), or the soluble-GC inhibitor (ODQ, 10 mM) on RSVL (1e 10 mM)-induced cGMP formation in HCSMCs. Incubation with enzyme inhibitors lasted for 20 min in the presence of IBMX (0.5 mM) before RSVL was added for 15 additional minutes. Incubations were performed in the presence of IBMX (0.5 mM). Data are means  SEM of 5e6 experiments. *Significantly lower than untreated cells.

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predominant pGC-isoform in HCSMCs, that is also targeted by RSVL to enhance cGMP formation in these cells. Furthermore, RSVL is known as a phytoestrogen that can bind to and modulate the estrogen machinery [6,15]. Hence, we first checked whether

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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 the presence of IBMX (0.5 mM) before ANP was added. Data are means  SEM of 4e6 experiments. *Significantly lower than ANP-treated cells.

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Figure 4 Differential short-term (15 min) effects of RSVL (A), and SNAP (B), on guanylyl cyclase activity in soluble- and particulate-membrane fractions from HCSMCs. Data are means  SEM of 6e7 experiments. *Significantly higher than basal enzyme activity.

RSVL, competition studies were performed using RSVL and selective agonists for the three main pGC-isoforms. The selective GC-A activator, ANP, elicited a 5.5-fold increase in cGMP levels, a response that was attenuated by RSVL in a concentration-dependent manner (Fig. 5). Conversely, the selective GC-B agonist, CNP had no significant effect on cGMP levels in HCSMCs (data not shown). Meanwhile, the selective GC-C agonist, guanylin, elicited only a slight (40e45%) increase in cGMP levels that was virtually additive with RSVL effects (Fig. 6). These observations imply that GC-A is the

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Figure 6 The combined effects of RSVL (1 nM-10 mM) and guanylin (0.1 mM) on cGMP formation in HCSMCs. Incubation with RSVL continued for 15 min in the presence of IBMX (0.5 mM) before guanylin was added. Basal cGMP level was 31  5 fmol/106 cells. Data are means  SEM of 4e6 experiments.

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estradiol (E2) has similar stimulatory effects on pGC. Fig. 7 demonstrates that E2 produced rapid, concentration-dependent increases in cGMP with an EC50 value of 1 mM; a comparable value to that of RSVL (1.8 mM). These effects for E2 were, likewise, insensitive to the s-GC inhibitor (ODQ 10 mM; data not shown), but were blunted by the ERblocker ICI-182,780 (Fig. 7). The maximal response to E2, however; was remarkably lower than that of RSVL (230% vs. 335%). Further, competition studies between E2 and RSVL (Fig. 7) suggest that the effects of these ligands are not additive, but rather 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, inhibited by the estrogen-receptor blocker, ICI-182,780 (10 mM) (Fig. 8).

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Discussion Resveratrol (RSVL) confers a plethora of beneficial biological responses against cancer and cardiovascular disease; thereby becoming a main target in recent experimental and clinical research [20]. Cardiovascular disease is the leading cause of death in many societies all over the world [21]. The cardiovascular benefits of RSVL include inhibition of LDL-oxidation and protection against ischemia/reperfusion-induced myocardial damage [22]. At the vascular level, RSVL appears to also exert 350

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Figure 7 The effects of RSVL (1 mM) and ICI-182,780 (10 M) on estradiol (E2, 1 nM-200 mM)-induced cGMP formation in HCSMCs. Incubation with E2 or ICI-182,780 continued for 20 min in the presence of IBMX (0.5 mM) before RSVL or E2 were added for 15 min. Data are means  SEM of 5e6 experiments.

Figure 8 Effect of ICI-180,780 (1, 10 mM) on RSVL (1, 10 mM)-induced cGMP formation in HCSMCs. Incubation with ICI-182,780 continued for 20 min in the presence of IBMX (0.5 mM) before RSVL was added for 15 min. Data are means  SEM of 4e5 experiments.

some protective actions [23]. However, the molecular underpinnings of such vascular effects remain largely elusive [3,22]. In particular, the mechanisms whereby RSVL can dilate endotheliumdenuded vessels have been more speculative than certain; and appear to be inconsistent among blood vessels [9,10]. When characterized, these signaling mechanisms can provide convincing clues for the protective effects of RSVL in the vasculature. The present study demonstrates the capacity of RSVL to stimulate the GC/cGMP/kinase-G cascade in an endothelium-free system; i.e., the human coronary smooth muscle cells (HCSMCs). This signaling cascade is known to culminate into both vasodilatory and antiatherogenic effects in smooth muscles [24]. At the molecular level, cGMP dilates blood vessels through reduction of intracellular calcium, inhibition of myosin-light-chain phosphorylation, or stimulation of potassium efflux and membrane repolarization [18,25]. On the other hand, cGMP elicits cytostatic actions in smooth muscles by enhancing apoptosis, inhibiting mitogenic enzymes such as PI3K and MAPKs, and/or interfering with the cell-cycle machinery [2,23,24]. In HCSMCs, our present observations showed that the stimulatory effects of RSVL on cGMP are not mediated by sGC or via inhibition of phosphodiesterases, as confirmed by the use of subcellular fractions and specific inhibitors for sGC and PDE

514 enzymes. Instead, these effects for RSVL involved the activation of membrane-bound GC isoform (pGC). This response occurred in both time- and concentration-related fashions. The time-course of this response (10 min for maximal cGMP formation/kinase-G activation) rules out the involvement of genomic mechanisms. This notion was also confirmed by using conventional inhibitors for transcription and translation (data not presented). The estimated EC50 value of this response, 1.8 mM, is congruent to those found for inhibition of MAPKs (2 mM) [2], and for relaxation of vascular beds (0.5e10 mM) [10,13]. Indeed, pGC has been a major player in maintaining cardiovascular hemodynamic mechanisms and integrity. Not surprisingly, lower pGC activity was observed in vascular preparations from hypertensive animals [26], whereas treatments with exogenous ANP triggered both vasodilation and cytostatic responses [24,27]. These views are supported by the reported ability of RSVL to improve the vascular mechanical properties in hypertensive animals [28]. Unlike sGC, the membrane-bound pGC enzyme is a receptor-linked enzyme that exists in at least seven isoforms in mammalian tissues (GC-A through -G) [24,29]. Albeit being primarily activated by the endogenous ligands ‘‘natriuretic peptides’’, recent observations showed that pGC could also be stimulated by exogenous agents, like vitamin-C and muscarinic agonists, in diverse systems [2,30]. The exact scenario underlying this process has not been defined. However, an important regulatory mechanism for pGC is its susceptibility for desensitization by a PKC-triggered dephosphorylation [19]. Because RSVL is a phytoestrogen with a capacity to bind to estrogen receptors (ERs), numerous cardiovascular studies have investigated the link between ER-binding and cardiovascular protection by RSVL [1,6]. In this context, in porcine coronary arteries, we reported that RSVL rapidly inhibited MAPKs through an ER-independent mechanism [2]. However, the vasorelaxant effects of RSVL, also in porcine coronaries, were ascribed to modulation of potassium current, through an ERmediated pathway [12]. Interestingly, in arterial cells isolated from hypertensive animals, the ERblocker (ICI-182,780) only partly reversed the long-term inhibitory effects of RSVL on atherosclerosis, DNA synthesis and prolyl hydroxylase activity [31]. Our present finding that activation of GC by RSVL in HCSMCs was both rapid (minutes) and mediated by the GC-A isoform agrees with the study of Chen and colleagues that showed an agonistic effect for the ER-ligand tamoxifen in the porcine kidney proximal tubular LLC-PK1 cells [32].

A.M. El-Mowafy et al. Moreover, we were able to demonstrate stimulation of pGC by RSVL in a microsomal-membrane preparation, implying that the enhanced cGMP response occurs mostly at the cell-membrane level. Further, we showed that effects for RSVL and the GC-A agonist, ANP, were competitive rather than additive. On the other hand, E2 reproduced these rapid GC-A stimulatory effects of RSVL in HCSMCs. Both responses to RSVL and E2 were sensitive to the pure ER-blocker, ICI-182,780, supporting the mediation by membrane ERs. Lines of evidence that RSVL and E2 share common ERs and membrane-bound GC-A enzyme have currently evolved from competition experiments on cGMP formation, which revealed that these ligands act ‘‘competitively’’ on one and the same effector. Despite these similarities between RSVL and E2 actions, some mechanistic differences have been observed in their present cGMP response. First, the maximal cGMP stimulatory response for RSVL was appreciably (46%) higher than that of E2, which was likewise spotted between E2 and tamoxifen in another cell system [32]. We propose that this higher intrinsic activity for RSVL could be the consequence of its nature as a mixed ER-agonist/ antagonist, as compared to the pure agonistic profile of E2. Our dynamic simulation studies for the mechanism of interaction of these ligands with ER-a may well support this assumption [33]. Because also these ligands appear to chiefly target a membrane receptor, their differential lipid solubility and cellular penetrability can be an additional element. Lastly, as the identity, structural details, and ligand-binding characteristics get unraveled, more of these differential ligand responses can be better explained. In the present work, the partial, but significant attenuation of RSVL response by the pure ER blocker, ICI182,780, suggests that RSVL may trigger additional signaling pathways to enhance GC-A activity. This view is supported by the current finding that this ER-blocker completely blunted the E2 response; needless to say, RSVL, away from ERs, can modulate a variety of cellular trafficking pathways [1e3]. For instance, the remarkable RSVL antioxidant activity has been associated with many biological effects; including cGMP formation [12]. Thus, collectively, it appears that the signaling pathway that couples RSVL to GC-A activation is not necessarily a simple/direct one. Lastly, in this context, it also remains to be investigated whether RSVL can produce additional effects by binding to such membrane ERs. Indeed, functional evidence has been accumulated that both long-term (genomic)- and shortterm-effects can be mediated by E2 binding to

Nongenomic activation of the GC-A enzyme membrane ERs [34]. However, a paucity of information is available on the mechanisms whereby E2 generates its non-genomic effects; and many postulations have been driven therein. Classical ER-a receptors were reported to signal non-genomic activation of NO synthase and mitogen-activated protein kinase [35,36]. Also, E2 has been shown to employ another type of cell-surface receptor, GPR30da G-protein-coupled receptor homologdin 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 enhance cGMP formation through a membrane-bound GC enzyme. Because of its reported multifaceted health benefits, it has been appealing to correlate RSVL’s therapeutic effects with its plasma levels and consumption of red wine. For instance, the study by Goldberg and colleagues has argued against a possible therapeutic plasma level for RSVL following normal consumption of red wine [38]. Therefore, it appears important that red wine should not be considered as the only source or the ‘‘gold standard’’ of dietary RSVL. In this vein, an elegant study by Bertelli and co-workers revealed that ‘‘diets’’ rich in the phytoestrogens, RSVL and quercetin, such as fruits and vegetables, can evidently provide adequate plasma levels to trigger antihypertensive effects and cardiovascular benefits in humans [39], or protection/palliation against stroke and hypertension in genetic animal models of such diseases [28]. On the other hand, many commercial drug-preparations with extracted RSVL as the sole or component of their active ingredients are currently sold in drugstores. These, if optimally dosed, can confer better plasma RSVL levels, while also avoiding the biological hazards/glitches posed by the alcohol in wine. Therefore, not surprisingly, in their commentary review Corder and colleagues have concluded that red wine consumption should not be encouraged until a clear understanding of the relation between diet and health is generated [40]. With these concerns in mind, more experimental studies are also accumulating every day to further support the cardiovascular benefits of RSVL. Of these, RSVL was proven effective in models of preconditioning and stunning [7], and also showed promise as an antagonist of the aryl hydrocarbon (AhR) receptors [41], a fundamental trigger of cardiovascular diseases caused by xenobiotics and tobacco smoking. Taken together, the present study reveals that in human coronary arterial smooth muscle, RSVL and E2 rapidly stimulate the kinase-G system to elicit antiproliferative and vasodilatory signals in human coronary artery. This process is mediated by the

515 membrane-bound GC-A isoform downstream from membrane estrogen receptors, and is functional in the absence of vascular endothelium.

Acknowledgement This study was supported by Kuwait University grant PT 01/01 to A.M.El-M. and M.A.

References [1] Fremont L. Biological effects of resveratrol. Life Sci January 14, 2000;66(8):663e73. [2] El-Mowafy AM, White RE. Resveratrol inhibits MAPK activity nuclear translocation in coronary artery smooth muscle reversal of Endothelin-1 stimulatory effects. FEBS Lett 1999; 451(1):63e7. [3] Kopp P. Resveratrol a phytoestrogen found in red wine A possible explanation for the conundrum of the ‘French paradox’? Eur J Endocrinol 1998;138(6):619e20. [4] El-Mowafy AM, Alkhalaf M. Resveratrol activates adenylylcyclase in human breast cancer cells: a novel estrogen receptor-independent cytostatic mechanism. Carcinogenesis 2003;24(5):869e73. [5] Gentilli M, Mazoit JX, Bouaziz H, Fletcher D, Casper RF, Benhamou D, et al. Resveratrol decreases hyperalgesia induced by carrageenan in the rat hind paw. Life Sci 2001;68(11):1317e21. [6] Basly JP, Marre-Fournier F, LeBail JC, Habrioux G, Chulia AJ. Estrogenic/antiestrogenic scavenging properties of (E)-(Z)-resveratrol. Life Sci 2000;66(9):769e77. [7] de Lorgeril M, Salen P, Guiraud A, Boucher F, de Leiris J. Resveratrol and non-ethanolic components of wine in experimental cardiology. Nutr Metab Cardiovasc Dis 2003; 13(2):100e3. [8] Soleas GJ, Diam EP, Goldberg DM. Wine as a biological fluid history production role in disease prevention. J Clin Lab Anal 1997;11(5):287e313. [9] Chen CK, Pace-Asciak CR. Vasorelaxing activity of resveratrol and quercetin in isolated rat aorta. Gen Pharmacol 1996;27(2):363e6. [10] Naderali EK, Smith SL, Doyle PJ, Williams G. The mechanism of resveratrol-induced vasorelaxation differs in the mesenteric resistance arteries of lean obese rats. Clin Sci (Lond) 2001;100(1):55e60. [11] Jager U, Nguoyen-Doung. Relaxant effects of trans-resveratrol on isolated porcine coronary arteries. Drug Res 1999; 49(1):207e11. [12] Chen ZJ, Che D, Chang CH. Antioxidants, vitamin C and dithiothreitol activate membrane-bound guanylate cyclase in PC12 cells. J Pharm Pharmacol 2001;53(2):243e7. [13] Chen ZJ, Vetter M, Chang GD, Liu S, Chang CH. Quercetin a phytoestrogen dietary flavonoid activates different membrane-bound guanylate cyclase isoforms in LLC-PK1 PC12 cells. J Pharm Pharmacol 2003;55(3):353e8. [14] Pelligrino DA, Wang Q. Cyclic nucleotide crosstalk and the regulation of cerebral vasodilation. Prog Neurobiol 1998; 56(1):1e18. [15] Abou-Zeid LA, El-Mowafy AM. Differential recognition of resveratrol isomers by human estrogen receptor-a: a molecular dynamics evaluation of stereoselective ligand binding. Chirality 2004;16(3):190e5.

516 [16] El-Mowafy AM, Abou-Mohamed GA. Non-epithelial ETA receptors activate adenylyl cyclase in rat trachea Molecular mechanisms Physiological implications. Life Sci 1997; 61(15):1529e38. [17] El-Mowafy AM, Biggs DF. ETB receptor activates adenylyl cyclase via a c-PLA2-dependent mechanism: a novel counterregulatory mechanism of ET-Induced contraction in airway smooth muscle. Biochem Biophys Res Commun 2001; 286(2):388e93. [18] White RE, Kryman J, El-Mowafy AM, Han G, Carrier GO. cAMP-dependent vasodilators cross-activate the cGMPdependent protein kinase to stimulate BKCa channel activity in coronary artery smooth muscle cells. Circ Res 2000;86:897e905. [19] Potter LR, Hunter T. Activation of protein kinase C stimulates the dephosphorylation of natriuretic peptide receptor-B at a single serine residue a possible mechanism of heterologous desensitization. J Biol Chem 2000;275(40): 31099e106. [20] Pervaiz S. Resveratrol: from grapevines to mammalian biology Effect of dietary patterns on ambulatory blood pressure: results from the Dietary Approaches to Stop Hypertension (DASH), Trial DASH Collaborative Research Group. FASEB J 2003;17(14):1975e85. [21] Moore TJ, Vollmer WM, Appel LJ, Sacks FM, Svetkey LP, Vogt TM, et al. Effect of dietary patterns on ambulatory blood pressure: results from the Dietary Approaches to Stop Hypertension (DASH) Trial. DASH Collaborative Research Group. Hypertension 1999;34(3):472e7. [22] Wu JM, Wang ZR, Hsieh TC, Bruder JL, Zou JG, Huang YZ. Mechanism of cardioprotection by resveratrol, a phenolic antioxidant present in red wine. Int J Mol Med 2001;8(1): 3e17. [23] Zou J, Huang Y, Chen Q, Wang N, Cao K, Hsieh TC, et al. Suppression of mitogenesis and regulation of cell cycle traverse by resveratrol in cultured smooth muscle cells. Intl J Oncol 1999;15(4):647e51. [24] Lucas KA, Pitari GM, Kazerounian S, et al. Guanylyl cyclases and signaling by cyclic GMP. Pharmacol Rev 2000; 52(3):375e414. [25] Soderling SH, Beavo JA. Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions. Curr Opin Cell Biol 2000;12(2):174e9. [26] Lee J, Kang DG, Kook H, Kim IK, Oh BK. Differentiallyaltered vascular guanylate cyclase isoforms in experimental hypertensive rats. J Korean Med Sci 1999;14:386e92. [27] Arjona AA, Hsu CA, Wrenn DS, Hill NS. Effects of natriuretic peptides on vascular smooth-muscle cells derived from different vascular beds. Gen Pharmacol 1997;28(3):387e92. [28] Mizutani K, Ikeda K, Kawai Y, Yamori Y. Extract of wine phenolics improves aortic biomechanical properties in stroke-prone spontaneously hypertensive rats (SHRSP). J Nutr Sci Vitaminol 1999;45(1):95e106.

A.M. El-Mowafy et al. [29] Hamad AM, Knox AJ. Mechanisms involved in desensitization of particulate guanylyl cyclase in human airway smooth muscle the role of protein kinase C. Biochem Biophys Res Commun 1999;266(1):152e5. [30] Alfonzo MJ, de Becemberg IL, de Villaroel SS, de Herrera VN, Misle AJ, de Alfonzo RG. Two opposite signal transducing mechanisms regulate a G-protein-coupled guanylyl cyclase. Arch Biochem Biophys 1998;1350(1): 19e25. [31] Mizutani K, Ikeda K, Yamori Y. Resveratrol inhibits AGEsinduced proliferation and collagen synthesis activity in vascular smooth muscle cells from stroke-prone spontaneously hypertensive rats. Biochem Biophys Res Commun 2000;274(1):61e7. [32] Chen Z-J, Vetter M, Geen-Dong Chang G, Liu S, Ding Y, Chang C-H. Non-genomic effects of tamoxifen on the activation of membrane-bound guanylate cyclase GC-A. J Pharm Pharmacol 2003;55(11):1539e45. [33] El-Mowafy AM, Abou-Zeid LA, Edafiogho I. Recognition of Resveratrol by the human estrogen receptor-a: a molecular modeling approach to understand its biological actions. Med Princ Pract 2002;11:86e92. [34] Kelley MJ, Levin ER. Rapid actions of plasma membrane estrogen receptors. Trends Endocrinol Metab 2001;12: 152e6. [35] Chen Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn ME, Shaul PW. Estrogen receptor-mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 1999;103:401e6. [36] Watters JJ, Campbell JS, Cunningham MJ, Krebs EG, Dorsa DM. Rapid membrane effects of steroids in neuroblastoma cells: effects of estrogen on mitogen activated protein tyrosine kinase signaling cascade and c-fos immediate early gene transcription. Endocrinology 1997;138: 4030e3. [37] Filardo EJ, Quinn JA, Bland KI, Frackelton AR. Estrogeninduced 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 2000; 14:1649e60. [38] Goldberg DM, Yan J, Soleas GJ. Absorption of three winerelated polyphenols in three different matrices by healthy subjects. Clin Biochem 2003;36(1):79e87. [39] Bertelli A, Bertelli AA, Gozzini A, Giovannini L. Plasma tissue resveratrol concentrations pharmacological activity. Drugs Exp Clin Res 1998;24(3):133e8. [40] Corder R, Crozier A, Kroon PA. Drinking your health? It’s too early to say. Nature 2003;426(6963):119. [41] Savouret JF, Berdeaux A, Casper RF. The aryl hydrocarbon receptor and its xenobiotic ligands: a fundamental trigger for cardiovascular diseases. Nutr Metab Cardiovasc Dis 2003;13(2):104e13.