Essential role of beta-adrenergic receptor kinase 1 in cardiac development and function.

The beta-adrenergic receptor kinase 1 (beta ARK1) is a member of the G protein-coupled receptor kinase (GRK) family that mediates the agonist-dependent phosphorylation and desensitization of G protein-coupled receptors. We have cloned and disrupted the beta ARK1 gene in mice by homologous recombination. No homozygote beta ARK1-/- embryos survive beyond gestational day 15.5. Prior to gestational day 15.5, beta ARK1-/- embryos display pronounced hypoplasia of the ventricular myocardium essentially identical to the "thin myocardium syndrome" observed upon gene inactivation of several transcription factors (RXR alpha, N-myc, TEF-1, WT-1). Lethality in beta ARK1-/- embryos is likely due to heart failure as they exhibit a > 70% decrease in cardiac ejection fraction determined by direct in utero intravital microscopy. These results along with the virtual absence of endogenous GRK activity in beta ARK1-/- embryos demonstrate that beta ARK1 appears to be the predominant GRK in early embryogenesis and that it plays a fundamental role in cardiac development.

The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.

Phosphorylation of G-protein-coupled receptors plays an important role in regulating their function. In this study the G-protein-coupled receptor phosphatase (GRP) capable of dephosphorylating G-protein-coupled receptor kinase-phosphorylated receptors is described. The GRP activity of bovine brain is a latent oligomeric form of protein phosphatase type 2A (PP-2A) exclusively associated with the particulate fraction. GRP activity is observed only when assayed in the presence of protamine or when phosphatase-containing fractions are subjected to freeze/thaw treatment under reducing conditions. Consistent with its identification as a member of the PP-2A family, the GRP is potently inhibited by okadaic acid but not by I-2, the specific inhibitor of protein phosphatase type 1. Solubilization of the membrane-associated GRP followed by gel filtration in the absence of detergent yields a 150-kDa peak of latent receptor phosphatase activity. Western blot analysis of this phosphatase reveals a likely subunit composition of AB alpha C. PP-2A of this subunit composition has previously been characterized as a soluble enzyme, yet negligible soluble GRP activity was observed. The subcellular distribution and substrate specificity of the GRP suggests significant differences between it and previously characterized forms of PP-2A.

An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G beta gamma subunits.

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.

Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate. The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain. We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. Gi alpha-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain.

Tyrosine phosphorylation of G protein alpha subunits by pp60c-src.

A number of lines of evidence suggest that cross-talk exists between the cellular signal transduction pathways involving tyrosine phosphorylation catalyzed by members of the pp60c-src kinase family and those mediated by guanine nucleotide regulatory proteins (G proteins). In this study, we explore the possibility that direct interactions between pp60c-src and G proteins may occur with functional consequences. Preparations of pp60c-src isolated by immunoprecipitation phosphorylate on tyrosine residues the purified G-protein alpha subunits (G alpha) of several heterotrimeric G proteins. Phosphorylation is highly dependent on G-protein conformation, and G alpha(GDP) uncomplexed by beta gamma subunits appears to be the preferred substrate. In functional studies, phosphorylation of stimulatory G alpha (G alpha s) modestly increases the rate of binding of guanosine 5'-[gamma-[35S]thio]triphosphate to Gs as well as the receptor-stimulated steady-state rate of GTP hydrolysis by Gs. Heterotrimeric G proteins may represent a previously unappreciated class of potential substrates for pp60c-src.

Chromosomal organization of adrenergic receptor genes.

The adrenergic receptors (ARs) (subtypes alpha 1, alpha 2, beta 1, and beta 2) are a prototypic family of guanine nucleotide binding regulatory protein-coupled receptors that mediate the physiological effects of the hormone epinephrine and the neurotransmitter norepinephrine. We have previously assigned the genes for beta 2- and alpha 2-AR to human chromosomes 5 and 10, respectively. By Southern analysis of somatic cell hybrids and in situ chromosomal hybridization, we have now mapped the alpha 1-AR gene to chromosome 5q32----q34, the same position as beta 2-AR, and the beta 1-AR gene to chromosome 10q24----q26, the region where alpha 2-AR is located. In mouse, both alpha 2- and beta 1-AR genes were assigned to chromosome 19, and the alpha 1-AR locus was localized to chromosome 11. Pulsed field gel electrophoresis has shown that the alpha 1- and beta 2-AR genes in humans are within 300 kilobases (kb) and the distance between the alpha 2- and beta 1-AR genes is less than 225 kb. The proximity of these two pairs of AR genes and the sequence similarity that exists among all the ARs strongly suggest that they are evolutionarily related. Moreover, they likely arose from a common ancestral receptor gene and subsequently diverged through gene duplication and chromosomal duplication to perform their distinctive roles in mediating the physiological effects of catecholamines. The AR genes thus provide a paradigm for understanding the evolution of such structurally conserved yet functionally divergent families of receptor molecules.

Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors.

beta-Adrenergic receptor kinase (beta-AR kinase) is a cytosolic enzyme that phosphorylates the beta-adrenergic receptor only when it is occupied by an agonist [Benovic, J. Strasser, R. H., Caron, M. G. & Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. USA 83, 2797-2801.] It may be crucially involved in the processes that lead to homologous or agonist-specific desensitization of the receptor. Stimulation of DDT1MF-2 hamster smooth muscle cells or S49 mouse lymphoma cells with a beta-agonist leads to translocation of 80-90% of the beta-AR kinase activity from the cytosol to the plasma membrane. The translocation process is quite rapid, is concurrent with receptor phosphorylation, and precedes receptor desensitization and sequestration. It is also transient, since much of the activity returns to the cytosol as the receptors become sequestered. Stimulation of beta-AR kinase translocation is a receptor-mediated event, since the beta-antagonist propranolol blocks the effect of agonist. In the kin- mutant of the S49 cells (lacks cAMP-dependent protein kinase), prostaglandin E1, which provokes homologous desensitization of its own receptor, is at least as effective as isoproterenol in promoting beta-AR kinase translocation to the plasma membrane. However, in the DDT1MF-2 cells, which contain alpha 1-adrenergic receptors coupled to phosphatidylinositol turnover, the alpha 1-agonist phenylephrine is ineffective. These results suggest that the first step in homologous desensitization of the beta-adrenergic receptor may be an agonist-promoted translocation of beta-AR kinase from cytosol to plasma membrane and that beta-AR kinase may represent a more general adenylate cyclase-coupled receptor kinase that participates in regulating the function of many such receptors.

Adjunctive β2-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with Pompe disease.

Enzyme or gene replacement therapy with acid α-glucosidase (GAA) has achieved only partial efficacy in Pompe disease. We evaluated the effect of adjunctive clenbuterol treatment on cation-independent mannose-6-phosphate receptor (CI-MPR)-mediated uptake and intracellular trafficking of GAA during muscle-specific GAA expression with an adeno-associated virus (AAV) vector in GAA-knockout (KO) mice. Clenbuterol, which increases expression of CI-MPR in muscle, was administered with the AAV vector. This combination therapy increased latency during rotarod and wirehang testing at 12 wk, in comparison with vector alone. The mean urinary glucose tetrasaccharide (Glc4), a urinary biomarker, was lower in GAA-KO mice following combination therapy, compared with vector alone. Similarly, glycogen content was lower in cardiac and skeletal muscle following 12 wk of combination therapy in heart, quadriceps, diaphragm, and soleus, compared with vector alone. These data suggested that clenbuterol treatment enhanced trafficking of GAA to lysosomes, given that GAA was expressed within myofibers. The integral role of CI-MPR was demonstrated by the lack of effectiveness from clenbuterol in GAA-KO mice that lacked CI-MPR in muscle, where it failed to reverse the high glycogen content of the heart and diaphragm or impaired wirehang performance. However, the glycogen content of skeletal muscle was reduced by the addition of clenbuterol in the absence of CI-MPR, as was lysosomal vacuolation, which correlated with increased AKT signaling. In summary, β2-agonist treatment enhanced CI-MPR-mediated uptake and trafficking of GAA in mice with Pompe disease, and a similarly enhanced benefit might be expected in other lysosomal storage disorders.

Differential expression of components of the cardiomyocyte adrenomedullin/intermedin receptor system following blood pressure reduction in nitric oxide-deficient hypertension.

Adrenomedullin (AM) and intermedin (IMD; adrenomedulln-2) are vasodilator peptides related to calcitonin gene-related peptide (CGRP). The actions of these peptides are mediated by the calcitonin receptor-like receptor (CLR) in association with one of three receptor activity-modifying proteins. CGRP is selective for CLR/receptor activity modifying protein (RAMP)1, AM for CLR/RAMP2 and -3, and IMD acts at both CGRP and AM receptors. In a model of pressure overload induced by inhibition of nitric-oxide synthase, up-regulation of AM was observed previously in cardiomyocytes demonstrating a hypertrophic phenotype. The current objective was to examine the effects of blood pressure reduction on cardiomyocyte expression of AM and IMD and their receptor components. Nomega-nitro-L-arginine methyl ester (L-NAME) (35 mg/kg/day) was administered to rats for 8 weeks, with or without concurrent administration of hydralazine (50 mg/kg/day) and hydrochlorothiazide (7.5 mg/kg/day). In left ventricular cardiomyocytes from L-NAME-treated rats, increases (-fold) in mRNA expression were 1.6 (preproAM), 8.4 (preproIMD), 3.4 (CLR), 4.1 (RAMP1), 2.8 (RAMP2), and 4.4 (RAMP3). Hydralazine/hydrochlorothiazide normalized systolic blood pressure (BP) and abolished mRNA up-regulation of hypertrophic markers sk-alpha-actin and BNP and of preproAM, CLR, RAMP2, and RAMP3 but did not normalize cardiomyocyte width nor preproIMD or RAMP1 mRNA expression. The robust increase in IMD expression indicates an important role for this peptide in the cardiac pathology of this model but, unlike AM, IMD is not associated with pressure overload upon the myocardium. The concordance of IMD and RAMP1 up-regulation indicates a CGRP-type receptor action; considering also a lack of response to BP reduction, IMD may, like CGRP, have an anti-ischemic function.

Role of conformational constraints of position 7 of the disulphide bridge of h-alpha-CGRP derivatives in their agonist versus antagonist properties

Previous structure-activity studies have shown that the disulphide bridge of calcitonin gene-related peptide (CGRP) is important for the highly potent, CGRP receptor-mediated effects of this peptide. In this study penicillamine (Pen) was substituted for one or both of the cysteinyl residues to determine conformational and topographical properties of the disulphide bridge favourable for binding to CGRP receptors and/or receptor activation. Pen constrains the conformational flexibility of disulphide bridges in other peptides. Binding affinities were measured using a radioligand binding assay with membranes prepared from pig coronary arteries and I-125-h-alpha-CGRP. Functional effects were characterized using a previously reported pig coronary artery relaxation bioassay. The binding affinity of [Pen(2)]h-alpha-CGRP was not significantly different from that of h-alpha-CGRP. All other analogues showed reduced affinity for CGRP receptors. [Pen(2)]h-alpha-CGRP also caused relaxation of coronary arteries. The remaining analogues either caused relaxation with significantly reduced potency or failed to relax the arteries at concentrations up to 1 x 10(-5) M. All analogues that did not relax coronary arteries contained a D-Pen in position 7 and inhibited CGRP-induced relaxation. [D-Pen(2,7)]h-alpha- CGRP was the most potent antagonist with a K-B value of 630 nM. This affinity is similar to that of the classical CGRP receptor antagonist, h-alpha-CGRP(8-37), on these arteries (K-B, 212 nM). These studies show that modifying the topography of the disulphide bridge can cause large and variable effects on ligand binding and activation of CGRP receptors. The contribution of position 7 to the conformation and topography of the disulphide bridge of h-alpha-CGRP is crucial to the future design of agonists of CGRP receptors. Furthermore, position 7 is important for the development of new CGRP receptor antagonists with structures based on the whole sequence of h-alpha-CGRP.

Structure-activity studies on position 14 of human alpha-calcitonin gene-related peptide

A structure-activity study was performed to examine the role of position 14 of human alpha-calcitonin gene-related peptide (h-alpha-CGRP) in activating the CGRP receptor. Interestingly, position 14 of h-alpha-CGRP contains a glycyl residue and is part of an alpha-helix spanning residues 8-18. Analogues [Ala(14)]-h-alpha-CGRP, [Aib(14)]-h-alpha-CGRP, [Asp(14)]-h-alpha-CGRP, [Asn(14)]-h-alpha-CGRP, and [Pro(14)]-h-alpha-CGRP were synthesized by solid phase peptide methodology and purified by RP-HPLC. Secondary structure was measured by circular dichroism spectroscopy. Agonist activities were determined as the analogues' ability to stimulate amylase secretion from guinea pig pancreatic acini and to relax precontracted porcine coronary arteries. Analogues [Ala(1)4]-h-alpha-CGRP, [Aib(14)]-h-alpha-CGRP, [Asp(14)]-h-alpha-CGRP, and [Asn(14)]-h-alpha-CGRP, all containing residues with a high helical propensity in position 14, were potent full agonists compared to h-alpha-CGRP in both tissues. Interestingly, replacement of Gly(14) of h-alpha-CGRP with these residues did not substantially increase the helical content of these analogues. [Pro(14)]-h-alpha-CGRP, predictably, has significantly lower helical content and is a 20-fold less potent agonist on coronary artery, known to contain CGRP-1 receptor subtypes, and an antagonist on pancreatic acini, known to contain CGRP-2 receptor subtypes. In conclusion, the residue in position 14 plays a structural role in stabilizing the alpha-helix spanning residues 8-18. The alpha-helix is crucial for maintaining highly potent agonist effects of h-alpha-CGRP at CGRP receptors. The wide variety of functional groups that can be tolerated in position 14 with no substantial modification of agonist effects suggests the residue in this position is not in contact with the CGRP receptor. [Pro(14)]-h-alpha-CGRP may be a useful pharmacological tool to distinguish between CGRP-1 and CGRP-2 receptor subtypes.

Influence of atenolol and nifedipine on nitric oxide deficient cardiomyocyte hypertrophy and expression of the cardio-endocrine peptide intermedin and its receptor components.

BACKGROUND/AIMS: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial ischemia, oxidative stress and hypertrophy; expression of adrenomedullin (AM) and intermedin (IMD) and their receptor activity modifying proteins (RAMPs 1-3) is augmented in cardiomyocytes, indicating that the myocardial AM/ IMD system may be activated in response to pressure loading and ischemic insult. The aim was to examine effects on (i) parameters of cardiomyocyte hypertrophy and on (ii) expression of AM and IMD and their receptor components in NO-deficient cardiomyocytes of an intervention chosen specifically for ability to alleviate pressure loading and ischemic injury concurrently. METHODS: The NO synthesis inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME, 35 mg.kg(-1).day(-1)) was given to rats for 8 weeks, with/ without concurrent administration of beta-adrenoceptor antagonist, atenolol (25 mg.kg(-1).day(-1)) / calcium channel blocker, nifedipine (20mg.kg(-1).day(-1)). RESULTS: In L-NAME treated rats, atenolol / nifedipine abolished increases in systolic blood pressure and plasma AM and IMD levels and in left ventricular cardiomyocytes: (i) normalized increased cell width and mRNA expression of hypertrophic (sk-alpha-actin) and cardio-endocrine (ANP, BNP, ET) genes; (ii) normalized augmented membrane protein oxidation; (iii) normalized mRNA expression of AM, IMD, RAMP1, RAMP2 and RAMP3. CONCLUSIONS: normalization of blood pressure and membrane oxidant status together with prevention of hypertrophy and normalization of the augmented expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes by atenolol / nifedipine supports involvement of both pressure loading and ischemic insult in stimulating cardiomyocyte hypertrophy and induction of these counter-regulatory peptides and their receptor components. Attenuation of augmented expression of IMD in this model cannot however be explained simply by prevention of cardiomyocyte hypertrophy.

Transient receptor potential melastatin 8 (TRPM8) channel involvement in the regulation of vascular tone

The transient receptor potential melastatin 8 (TRPM8) channel has been characterized as a cold and menthol receptor expressed in a subpopulation of sensory neurons but was recently identified in other tissues, including the respiratory tract, urinary system, and vasculature. Thus TRPM8 may play multiple functional roles, likely to be in a tissue- and activation state-dependent manner. We examined the TRPM8 channel presence in large arteries from rats and the functional consequences of their activation. We also aimed to examine whether these channels contribute to control of conscious human skin blood flow. TRPM8 mRNA and protein were detected in rat tail, femoral and mesenteric arteries, and thoracic aorta. This was confirmed in single isolated vascular myocytes by immunocytochemistry. Isometric contraction studies on endothelium-denuded relaxed rat vessels found small contractions on application of the TRPM8-specific agonist menthol (300 microM). However, both menthol and another agonist icilin (50 microM) caused relaxation of vessels precontracted with KCl (60 mM) or the alpha-adrenoceptor agonist phenylephrine (2 microM) and a reduction in sympathetic nerve-mediated contraction. These effects were antagonized by bromoenol lactone treatment, suggesting the involvement of Ca(2+)-independent phospholipase A(2) activation in TRPM8-mediated vasodilatation. In thoracic aorta with intact endothelium, menthol-induced inhibition of KCl-induced contraction was enhanced. This was unaltered by preincubation with either N(omega)-nitro-l-arginine methyl ester (l-NAME; 100 nM), a nitric oxide synthase inhibitor, or the ACh receptor antagonist atropine (1 microM). Application of menthol (3% solution, topical application) to skin caused increased blood flow in conscious humans, as measured by laser Doppler fluximetry. Vasodilatation was markedly reduced or abolished by prior application of l-NAME (passive application, 10 mM) or atropine (iontophoretic application, 100 nM, 30 s at 70 microA). We conclude that TRPM8 channels are present in rat artery vascular smooth muscle and on activation cause vasoconstriction or vasodilatation, dependent on previous vasomotor tone. TRPM8 channels may also contribute to human cutaneous vasculature control, likely with the involvement of additional neuronal mechanisms.

Src and ADAM-17-Mediated Shedding of Transforming Growth Factor-alpha Is a Mechanism of Acute Resistance to TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-alpha (TGF-alpha) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-alpha was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-alpha also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-alpha, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL. [Cancer Res 2008;68(20):8312-21]

Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase

Oestrogen produces diverse biological effects through binding to the oestrogen receptor (ER)(1). The ER is a steroid hormone nuclear receptor, which, when bound to oestrogen, modulates the transcriptional activity of target genes(2). Controversy exists, however, concerning whether ER has a role outside the nucleus(3), particularly in mediating the cardiovascular protective effects of oestrogen(4). Here we show that the ER isoform, ER alpha, binds in a ligand-dependent manner to the p85 alpha regulatory subunit of phosphatidylinositol-3-OH kinase (PI(3)K). Stimulation with oestrogen increases ER alpha-associated PI(3)K activity, leading to the activation of protein kinase B/Akt and endothelial nitric oxide synthase (eNOS). Recruitment and activation of PI(3)K by ligand-bound ERa are independent of gene transcription, do not involve phosphotyrosine adapter molecules or src-homology domains of p85 alpha, and extend to other steroid hormone receptors. Mice treated with oestrogen show increased eNOS activity and decreased vascular leukocyte accumulation after ischaemia and reperfusion injury. This vascular protective effect of oestrogen was abolished in the presence of PI(3)K or eNOS inhibitors. Our findings define a physiologically important non-nuclear oestrogen-signalling pathway involving the direct interaction of ERa with PI(3)K.