Abstract-Nitric oxide (NO) is a powerful angiogenic mediator acting downstream of vascular endothelial growth factor (VEGF). Both the endothelial NO synthase (eNOS) and the VEGFR-2 receptor colocalize in caveolae. Because the structural protein of these signaling platforms, caveolin, also represses eNOS activity, changes in its abundance are likely to influence the angiogenic process in various ways. In this study, we used mice deficient for the caveolin-1 gene (Cav Ϫ/Ϫ ) to examine the impact of caveolae suppression in a model of adaptive angiogenesis obtained after femoral artery resection. Evaluation of the ischemic tissue perfusion and histochemical analyses revealed that contrary to Cav ϩ/ϩ mice, Cav Ϫ/Ϫ mice failed to recover a functional vasculature and actually lost part of the ligated limbs, thereby recapitulating the effects of the NOS inhibitor L-NAME administered to operated Cav ϩ/ϩ mice. We also isolated endothelial cells (ECs) from Cav Ϫ/Ϫ aorta and showed that on VEGF stimulation, NO production and endothelial tube formation were dramatically abrogated when compared with Cav ϩ/ϩ ECs. The Ser1177 eNOS phosphorylation and Thr495 dephosphorylation but also the ERK phosphorylation were similarly altered in VEGF-treated Cav Ϫ/Ϫ ECs. Interestingly, caveolin transfection in Cav Ϫ/Ϫ ECs redirected the VEGFR-2 in caveolar membranes and restored the VEGF-induced ERK and eNOS activation. However, when high levels of recombinant caveolin were reached, VEGF exposure failed to activate ERK and eNOS. These results emphasize the critical role of caveolae in ensuring the coupling between VEGFR-2 stimulation and downstream mediators of angiogenesis. This study also provides new insights to understand the paradoxical roles of caveolin (eg, repressing basal enzyme activity but facilitating activation on agonist stimulation) in cardiovascular pathophysiology. Key Words: caveolin-1 Ⅲ nitric oxide Ⅲ vascular endothelial growth factor Ⅲ angiogenesis Ⅲ ischemia C aveolae are 50-to 100-nm cell surface invaginations playing key roles in vesicular transport and signal transduction. 1 The structural protein of these plasmalemmal microdomains, caveolin, acts as a scaffold for many caveolar residents. 2 The caveolin-1 isoform is particularly abundant in endothelial cells (ECs) where it regulates various functions including transcytosis, permeability, vascular tone, and angiogenesis. 3 Recently, Woodman et al 4 documented that in a model of tumor cell injection in caveolin-deficient mice (Cav Ϫ/Ϫ ), angiogenesis was markedly reduced in comparison with wild-type (WT) animals. Although the same authors showed that the reduction in vessel density could be reproduced in a model of Matrigel plugs supplemented with bFGF, 4 the mechanisms supporting the role of caveolin in the angiogenic response to exogenous stimuli remain poorly understood and, based on previous publications, 5-9 a matter of debate.For instance, the well-established inhibitory interaction between caveolin and the endothelial nitric oxide (NO) synthase (eNOS) 10,11 le...

Background-The therapeutic effects of nonspecific ␤-blockers are limited by vasoconstriction, thus justifying the interest in molecules with ancillary vasodilating properties. Nebivolol is a selective ␤ 1 -adrenoreceptor antagonist that releases nitric oxide (NO) through incompletely characterized mechanisms. We identified endothelial ␤ 3 -adrenoreceptors in human coronary microarteries that mediate endothelium-and NO-dependent relaxation and hypothesized that nebivolol activates these ␤ 3 -adrenoreceptors. Methods and Results-Nebivolol dose-dependently relaxed rodent coronary resistance microarteries studied by videomicroscopy (10 mol/L, Ϫ86Ϯ6% of prostaglandin F2␣ contraction); this was sensitive to NO synthase (NOS) inhibition, unaffected by the ␤ 1-2 -blocker nadolol, and prevented by the ␤ 1-2-3 -blocker bupranolol (PϽ0.05; nϭ3 to 8). Importantly, nebivolol failed to relax microarteries from ␤ 3 -adrenoreceptor-deficient mice. Nebivolol (10 mol/L) also relaxed human coronary microvessels (Ϫ71Ϯ5% of KCl contraction); this was dependent on a functional endothelium and NO synthase but insensitive to ␤ 1-2 -blockade (all PϽ0.05). In a mouse aortic ring assay of neoangiogenesis, nebivolol induced neocapillary tube formation in rings from wild-type but not ␤ 3 -adrenoreceptor-or endothelial NOS-deficient mice. In cultured endothelial cells, 10 mol/L nebivolol increased NO release by 200% as measured by electron paramagnetic spin trapping, which was also reversed by NOS inhibition. In parallel, endothelial NOS was dephosphorylated on threonine 495 , and fura-2 calcium fluorescence increased by 91.8Ϯ23.7%; this effect was unaffected by ␤ 1-2 -blockade but abrogated by ␤ 1-2-3 -blockade (all PϽ0.05). Conclusions-Nebivolol dilates human and rodent coronary resistance microarteries through an agonist effect on endothelial ␤ 3 -adrenoreceptors to release NO and promote neoangiogenesis. These properties may prove particularly beneficial for the treatment of ischemic and cardiac failure diseases through preservation of coronary reserve.

Background-Coronary vessel tone is modulated in part by ␤-adrenergic relaxation. However, the implication of specific ␤-adrenoceptor subtypes and their downstream vasorelaxing mechanism(s) in human coronary resistance arteries is poorly defined. ␤ 3 -Adrenoceptors were recently shown to vasodilate animal vessels and are expressed in human hearts. Methods and Results-We examined the expression and functional role of ␤ 3 -adrenoceptors in human coronary microarteries and their coupling to vasodilating nitric oxide (NO) and/or hyperpolarization mechanisms. The expression of ␤ 3 -adrenoceptor mRNA and protein was demonstrated in extracts of human coronary microarteries. Immunohistochemical analysis revealed their exclusive localization in the endothelium, with no staining of vascular smooth muscle.In contractility experiments in which videomicroscopy was used, the nonspecific ␤-agonist isoproterenol and the ␤ 3 -preferential agonist BRL37344 evoked an Ϸ50% relaxation of endothelin-1-preconstricted human coronary microarteries. Relaxations were blocked by the ␤ 1 /␤ 2 /␤ 3 -adrenoceptor antagonist bupranolol but were insensitive to the ␤ 1 /␤ 2 -adrenoceptor antagonist nadolol, confirming a ␤ 3 -adrenoceptor-mediated pathway. Relaxation in response to BRL37344 was absent in human coronary microarteries devoid of functional endothelium. When human coronary microarteries were precontracted with KCl (thereby preventing vessel hyperpolarization), the relaxation to BRL37344 was reduced to 15.5% and totally abrogated by the NO synthase inhibitor L--nitroarginine, confirming the participation of a NO synthase-mediated relaxation. The NO synthase-independent relaxation was completely inhibited by the Ca 2ϩ -activated K ϩ channel inhibitors apamin and charybdotoxin, consistent with an additional endothelium-derived hyperpolarizing factor-like response. Accordingly, membrane potential recordings demonstrated vessel hyperpolarization in response to ␤ 3 -adrenoceptor stimulation. Conclusions-␤

Background-In endothelial cells, caveolin-1, the structural protein of caveolae, acts as a scaffolding protein to cluster lipids and signaling molecules within caveolae and, in some instances, regulates the activity of proteins targeted to caveolae. Specifically, different putative mediators of the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation are located in caveolae and/or regulated by the structural protein caveolin-1, such as potassium channels, calcium regulatory proteins, and connexin 43, a molecular component of gap junctions. Methods and Results-Comparing relaxation in vessels from caveolin-1 knockout mice and their wild-type littermates, we observed a complete absence of EDHF-mediated vasodilation in isolated mesenteric arteries from caveolin-1 knockout mice. The absence of caveolin-1 is associated with an impairment of calcium homeostasis in endothelial cells, notably, a decreased activity of Ca 2ϩ -permeable TRPV4 cation channels that participate in nitric oxide-and EDHF-mediated relaxation. Moreover, morphological characterization of caveolin-1 knockout and wild-type arteries showed fewer gap junctions in vessels from knockout animals associated with a lower expression of connexins 37, 40, and 43 and altered myoendothelial communication. Finally, we showed that TRPV4 channels and connexins colocalize with caveolin-1 in the caveolar compartment of the plasma membrane. Conclusions-We demonstrated that expression of caveolin-1 is required for EDHF-related relaxation by modulating membrane location and activity of TRPV4 channels and connexins, which are both implicated at different steps in the EDHF-signaling pathway.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence in the brain of senile plaques which contain an amyloid core made of b-amyloid peptide (Ab). Ab is produced by the cleavage of the amyloid precursor protein (APP). Since impairment of neuronal calcium signalling has been causally implicated in ageing and AD, we have investigated the influence of an influx of extracellular calcium on the metabolism of human APP in rat cortical neurones. We report that a high cytosolic calcium concentration, induced by neuronal depolarization, inhibits the a-secretase cleavage of APP and triggers the accumulation of intraneuronal C-terminal fragments produced by the b-cleavage of the protein (CTFb). Increase in cytosolic calcium concentration specifically induces the production of large amounts of intraneuronal Ab1-42, which is inhibited by nimodipine, a specific antagonist of L-type calcium channels. Moreover, calcium release from endoplasmic reticulum is not sufficient to induce the production of intraneuronal Ab, which requires influx of extracellular calcium mediated by the capacitative calcium entry mechanism. Therefore, a sustained high concentration of cytosolic calcium is needed to induce the production of intraneuronal Ab1-42 from human APP. Our results show that this accumulation of intraneuronal Ab1-42 induces neuronal death, which is prevented by a functional c-secretase inhibitor. Keywords: Alzheimer's disease, amyloid peptide, amyloid precursor protein, calcium, neuronal death, processing. A typical hallmark of AD is the deposition of b-amyloid or Ab peptide into senile plaques in the cerebral cortex (Hardy and Allsop 1991). The Ab peptide (39-43 amino acids) is generated by the proteolytic processing of the amyloid precursor protein (APP). In neurones, APP is a 695-amino acid type I transmembrane protein (APP695) (Kang et al. 1987) with a large N-terminal ectodomain and a short intracellular C-terminus. APP is processed by two distinct intracellular metabolic pathways, the non-amyloidogenic and the amyloidogenic pathways. In the non-amyloidogenic pathway, APP is cleaved, within the Ab sequence, by a-secretase to generate extracellular soluble APP (sAPPa) and intracellular C-terminal fragment (CTFa) corresponding to the 83 C-terminal amino acids of the protein (Oltersdorf et al. 1990). This a-secretase cleavage, which is performed by proteases of the ADAM family (Schlondorff and Blobel 1999), precludes the formation of Ab peptide. In the amyloidogenic pathway, Ab is generated by the sequential cleavages of APP by b-and c-secretases. The b-secretase cleavage, performed by BACE (Vassar et al. 1999), produces a C-terminal fragment of APP (CTFb) corresponding to the 99 C-terminal amino acids of the protein. The CTFb is further cleaved by a c-secretase activity to generate Ab. Intracellular factor responsible for c-cleavage needs to be unambiguously identified, but it has been demonstrated to be part of a multiprotein complex containing presenilin 1, nicastrin, Pen-2 and Aph1 (D...

BACKGROUND AND PURPOSEAvailable medications for chronic pain provide only partial relief and often cause unacceptable side effects. There is therefore a need for novel molecular targets to develop new therapeutics with improved efficacy and tolerability. Despite encouraging efficacy data in rodents with inhibitors of the neuronal glycine transporter-2 (GlyT2), there are also some reports of toxicity and their development was discontinued. EXPERIMENTAL APPROACHIn order to clarify the possibility of targeting GlyT2 for the treatment of pain, we have used an integrated approach comprising in vitro pharmacology, selectivity, bioavailability, in vivo efficacy and safety assessment to analyse the properties and efficacy of ALX-1393 and Org-25543, the two published GlyT2 inhibitors from which in vivo data are available. KEY RESULTSWe report that these compounds have a different set of undesirable properties that limit their usefulness as pharmacological tools. Importantly, we discover that inhibitors of GlyT2 can exert an apparent reversible or irreversible inhibition of the transporter and describe a new class of reversible GlyT2 inhibitors that preserves efficacy while avoiding acute toxicity. CONCLUSIONS AND IMPLICATIONSOur pharmacological comparison of two closely related GlyT2 inhibitors with different modes of inhibition provides important insights into their safety and efficacy profiles, uncovering that in the presence of a GlyT2 mechanism-based toxicity, reversible inhibitors might allow a tolerable balance between efficacy and toxicity. These findings shed light into the drawbacks associated with the early GlyT2 inhibitors and describe a new mechanism that might serve as the starting point for new drug development.

The present study was aimed at investigating whether, besides its pivotal role in Ca 2+ -independent contraction of smooth muscle, Rho-kinase is involved in the mechanisms underlying the Ca 2+ signal activated by noradrenaline in arteries. In rat aorta and mesenteric artery, the Rho-kinase inhibitor Y-27632 (10 mM) completely relaxed the contraction evoked by noradrenaline (1 mM) and simultaneously inhibited the Ca 2+ signal by 54 ± 1 % (mesenteric artery) and 71 ± 15 % (aorta), and the cell membrane depolarisation by 56 ± 11 % (mesenteric artery). A similar effect was observed in arteries contracted by AlF 4 _ , while in KCl-contracted arteries, Y-27632 decreased tension without changing cytosolic Ca 2+ . The same effects were observed with another inhibitor of Rho-kinase (HA1077) but not with an inhibitor of protein kinase C (Ro-31-8220). Effects of Y-27632 were not prevented by incubating the artery in 25 mM KCl, with K + channel blockers or with the Ca 2+ channel blocker nimodipine. Y-27632 did not affect either the increase in the production of inositol phosphates activated by noradrenaline, or the release of Ca 2+ from non-mitochondrial stores evoked by InsP 3 in permeabilised aortic cells, or the Ca 2+ signals evoked by thapsigargin or caffeine. The capacitative Ca 2+ entry activated by thapsigargin was not impaired by Y-27632, but the entry of Ba 2+ activated by noradrenaline in the presence of nimodipine was blocked by 10 mM Y-27632. These results indicate that Rho-kinase is involved in noradrenaline activation of a Ca 2+ entry distinct from voltage-or store-operated channels in rat arteries.

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