Alejandro Domínguez-Rodríguez

Role of Ca2+-Independent Phospholipase A2 and Store-Operated Pathway in Urocortin-Induced Vasodilatation of Rat Coronary Artery

Urocortin has been shown to produce vasodilatation in several arteries, but the precise mechanism of its action is still poorly understood. Here we demonstrate the role of store operated Ca2+ entry (SOCE) regulated by Ca2+-independent phospholipase A2 (iPLA2) in phenylephrine hydrochloride (PE)-induced vasoconstriction, and we present the first evidence that urocortin induces relaxation by the modulation of SOCE and iPLA2 in rat coronary artery. Urocortin produces an endothelium independent relaxation, and its effect is concentration-dependent (IC50≈4.5 nmol/L). We show in coronary smooth muscle cells (SMCs) that urocortin inhibits iPLA2 activation, a crucial step for SOC channel activation, and prevents Ca2+ influx evoked by the emptying of the stores via a cAMP and protein kinase A (PKA)-dependent mechanism. Lysophophatidylcholine and lysophosphatidylinositol, products of iPLA2, exactly mimic the effect of the depletion of the stores in presence of urocortin. Furthermore, we report that long treatment with urocortin downregulates iPLA2 mRNA and proteins expression in rat coronary smooth muscle cells. In summary, we propose a new mechanism of vasodilatation by urocortin which involves the regulation of iPLA2 and SOCE via the stimulation of a cAMP/PKA-dependent signal transduction cascade in rat coronary artery.

Urocortin induces positive inotropic effect in rat heart.

AIMS The aim of this study is to evaluate the positive inotropic effect of urocortin (Ucn) and to characterize its signalling pathways. METHODS AND RESULTS Contractility was measured in ex vivo Langendorff-perfused hearts isolated from Wistar rats. Isolated ventricular cardiomyocytes were used to analyse intracellular calcium ([Ca(2+)](i)) transients evoked by electrical stimulation and L-type Ca(2+) current by confocal microscopy and whole-cell patch-clamping, respectively. The application of Ucn to perfused hearts induced progressive, sustained, and potent inotropic and lusitropic effects that were dose-dependent with an EC(50) of approximately 8 nM. Ucn effects were independent of protein kinase A (PKA) activation but were significantly reduced by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors and by brefeldin A, an antagonist of guanine nucleotide exchange factor, suggested to be an inhibitor of exchange protein activated by cAMP (Epac). These whole-organ effects were correlated with the inotropic effects observed in isolated cells: Ucn increased I(CaL) density, [Ca(2+)](i) transients, cell shortening and Ca(2+) content of sarcoplasmic reticulum. CONCLUSION Our results show that Ucn evokes potent positive inotropic and lusitropic effects mediated, at least in part, by an increase in I(CaL) and [Ca(2+)](i) transient amplitude. These effects may involve the activation of Epac, PKC, and MAPK signalling pathways.

Ultra-fast and energy-efficient sintering of ceramics by electric current concentration

Electric current activated/assisted sintering (ECAS) techniques, such as electrical discharge sintering (EDS) or resistive sintering (RS), have been intensively investigated for longer than 50 years. In this work, a novel system including an electrically insulated graphite die for Spark Plasma Sintering (SPS) is described, which allows the sintering of any refractory ceramic material in less than 1 minute starting from room temperature with heating rates higher than 2000°C/min and an energy consumption up to 100 times lower than with SPS. The system alternates or combines direct resistive sintering (DRS) and indirect resistive sintering (IRS). Electrical insulation of the die has been achieved through the insertion of a film made of alumina fibers between the graphite die and the graphite punches, which are protected from the alumina fiber film by a graphite foil. This system localized the electric current directly through the sample (conductive materials) as in DRS and EDS, or through the thin graphite foil (non-conductive materials) as in IRS, and is the first system capable of being used under EDS or RS conditions independently combining current concentration/localization phenomena.

Urotensin-II Signaling Mechanism in Rat Coronary Artery: Role of STIM1 and Orai1-Dependent Store Operated Calcium Influx in Vasoconstriction

Objective—Human urotensin-II (UII) is considered the most potentendogenous vasoconstrictor discovered to date, although the precise mechanism activated downstream of its receptor UTS2R in blood vessels remains elusive. The aim of this study was to determine the role of the store operated Ca2+ entry (SOCE) signaling pathway in UII-induced coronary artery vasoconstriction. Methods and Results—We used a combination of isometric tension measurement, Ca2+ imaging, pharmacology, and molecular approaches to study UII-mediated rat coronary artery vasoconstriction and intracellular Ca2+ mobilization in coronary smooth muscle cells. We found that UII promoted dose-dependent vasoconstriction and elicited Ca2+ and Mn2+ influx, which were sensitive to classical SOCE inhibitors. In addition, knockdown of either STIM1 or Orai1 essentially inhibited UII-mediated SOCE and prevented UII but not high-KCL evoked contraction in transfected coronary artery. Moreover, we found that Ca2+-independent phospholipase A2&bgr; was involved in UII effects and that is colocalized with STIM1 in different submembrane compartments. Importantly, STIM1 but not Orai1 downregulation inhibits significantly independent phospholipase A2 activation. Furthermore, lysophosphatidylcholine, an independent phospholipase A2 product, activated Orai1 but not STIM1-dependent contraction and SOCE. Conclusion—Here, we demonstrated that different critical players of SOCE signaling pathway are required for UII-induced vasoconstriction of rat coronary artery.

Ca2+ Fluxes Involvement in Gene Expression During Cardiac Hypertrophy

Cardiac hypertrophy arises as a response of the heart to many different pathological stimuli that challenge its work. Regardless of the initial pathologic cause, cardiac hypertrophy shares some characteristics resulting from a genetic reprogramming of several proteins. Recent studies point to Ca2+ as a key signaling element in the initiation of this genetic reprogramming. In fact, besides its important role in excitation-contraction coupling, Ca2+ regulates cardiac growth by activation of Ca2+-dependent transcription factors. This mechanism has been termed excitation-transcription (ET) coupling. Some information about cardiac ET coupling is being gathered from the analysis of cardiac hypertrophy development, where two Ca2+ dependent enzymes are key actors: the Ca2+/calmodulin kinase II (CaMKII) and the phosphatase calcineurin, both activated by Ca2+/Calmodulin. In this review we focus on some neurohormonal signaling pathways involved in cardiac hypertrophy, which could be ascribed as activators of ET coupling, for instance, adrenergic stimulation and the renin-angiotensin-aldosterone system. β-adrenergic receptor (β-AR) produces cAMP, which directly, (through cAMP response element) or indirectly (through activating Epac) induces cardiac hypertrophy. α1 AR and angiotensin receptor type 1 are Gq protein coupled receptors, which when activated, stimulate phospholipase C producing inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG). IP3 promotes elevation of [Ca2+] in the nucleus, activating CaMKII/MEF2 (myocyte enhancer factor 2) pathway and may indirectly induce Ca2+ entry through transient receptor potential channels (TRPC). Other TRPC channels are activated by DAG. Ca2+ entry activates calcineurin/NFAT hypertrophic signaling. By promoting L-type Ca2+ channel expression, aldosterone may also have an important role in the genetic reprogramming during hypertrophy.

Urocortin-2 induces vasorelaxation of coronary arteries isolated from patients with heart failure.

1. Urocortin‐2 (Ucn2) is a vasoactive peptide belonging to the corticotrophin‐releasing factor (CRF) family that has potent cardiovascular actions. It has been suggested that Ucn2 participates in the pathophysiology of heart failure. However, little is known about the mechanisms underlying the action of Ucn2 in human coronary arteries. The aim of the present study was to assess the effects of Ucn2 on the vascular tone of human coronary arteries dissected from heart failure patients.

Functional Vascular Smooth Muscle-like Cells Derived from Adult Mouse Uterine Mesothelial Cells

In mammalian visceral organs, vascular smooth muscle cells (VSMCs) originate from an epithelial-to-mesenchymal transition (EMT) of embryonic mesothelial cells (MCs). The ability of adult MCs to recapitulate EMT and to acquire smooth muscle (SM) markers upon provasculogenic culture suggested they might retain embryonic vasculogenic differentiation potential. However, it remains unknown whether adult MCs-derived SM-like cells may acquire specific vascular SM lineage markers and the functionality of differentiated contractile VSMCs. Here, we describe how a gentle trypsinization of adult mouse uterine cords could selectively detach their outermost uterine mesothelial layer cells. As other MCs; uterine MCs (UtMCs) uniformly expressed the epithelial markers β-catenin, ZO-1, E-cadherin, CD54, CD29, and CK18. When cultured in a modified SM differentiation media (SMDM) UtMCs initiated a loss of epithelial characteristics and gained markers expression of EMT (Twist, Snail, and Slug), stem and progenitor (Nanog, Sox2, C-kit, Gata-4, Isl-1, and nestin), SM (α-SMA, calponin, caldesmon, SM22α, desmin, SM-MHC, and smoothelin-B) and cardiac (BMP2, BMP4, ACTC1, sACTN, cTnI, cTnT, ANF, Cx43, and MLC2a). UtMCs repeatedly subcultured in SMDM acquired differentiated VSM-like characteristics and expressed smoothelin-B in the typical stress-fiber pattern expression of contractile VSMCs. Relevantly, UtMCs-derived VSM-like cells could generate “mechanical force” to compact collagen lattices and displayed in diverse degree voltage (K+) and receptor (endothelin-1, oxytocin, norepinephrine, carbachol and vasopressin)-induced [Ca2+]i rises and contraction. Thus, we show for the first time that UtMCs could recapitulate in vitro differentiative events of early cardiovascular differentiation and transdifferentiate in cells exhibiting molecular and functional characteristics of VSMCs.

Resveratrol ameliorates the maturation process of β-cell-like cells obtained from an optimized differentiation protocol of human embryonic stem cells

Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process.

Phospholipase A2 as a Molecular Determinant of Store-Operated Calcium Entry

Activation of phospholipases A2 (PLA2) leads to the generation of biologically active lipid products that can affect numerous cellular events. Ca(2+)-independent PLA2 (iPLA2), also called group VI phospholipase A2, is one of the main types forming the superfamily of PLA2. Beside of its role in phospholipid remodeling, iPLA2 has been involved in intracellular Ca(2+) homeostasis regulation. Several studies proposed iPLA2 as an essential molecular player of store operated Ca(2+) entry (SOCE) in a large number of excitable and non-excitable cells. iPLA2 activation releases lysophosphatidyl products, which were suggested as agonists of store operated calcium channels (SOCC) and other TRP channels. Herein, we will review the important role of iPLA2 on the intracellular Ca(2+) handling focusing on its role in SOCE regulation and its implication in physiological and/or pathological processes.

The Complex Role of Store Operated Calcium Entry Pathways and Related Proteins in the Function of Cardiac, Skeletal and Vascular Smooth Muscle Cells

Cardiac, skeletal, and smooth muscle cells shared the common feature of contraction in response to different stimuli. Agonist-induced muscles contraction is triggered by a cytosolic free Ca2+ concentration increase due to a rapid Ca2+ release from intracellular stores and a transmembrane Ca2+ influx, mainly through L-type Ca2+ channels. Compelling evidences have demonstrated that Ca2+ might also enter through other cationic channels such as Store-Operated Ca2+ Channels (SOCCs), involved in several physiological functions and pathological conditions. The opening of SOCCs is regulated by the filling state of the intracellular Ca2+ store, the sarcoplasmic reticulum, which communicates to the plasma membrane channels through the Stromal Interaction Molecule 1/2 (STIM1/2) protein. In muscle cells, SOCCs can be mainly non-selective cation channels formed by Orai1 and other members of the Transient Receptor Potential-Canonical (TRPC) channels family, as well as highly selective Ca2+ Release-Activated Ca2+ (CRAC) channels, formed exclusively by subunits of Orai proteins likely organized in macromolecular complexes. This review summarizes the current knowledge of the complex role of Store Operated Calcium Entry (SOCE) pathways and related proteins in the function of cardiac, skeletal, and vascular smooth muscle cells.