Tarik Smani

A novel mechanism for the store-operated calcium influx pathway

Activation of store-operated channels (SOCs) and capacitative calcium influx are triggered by depletion of intracellular calcium stores. However, the exact molecular mechanism of such communication remains unclear. Recently, we demonstrated that native SOC channels can be activated by calcium influx factor (CIF) that is produced upon depletion of calcium stores, and showed that Ca2+-independent phospholipase A2 (iPLA2) has an important role in the store-operated calcium influx pathway. Here, we identify the key plasma-membrane-delimited events that result in activation of SOC channels. We also propose a novel molecular mechanism in which CIF displaces inhibitory calmodulin (CaM) from iPLA2, resulting in activation of iPLA2 and generation of lysophospholipids that in turn activate soc channels and capacitative calcium influx. Upon refilling of the stores and termination of CIF production, CaM rebinds to iPLA2, inhibits it, and the activity of SOC channels and capacitative calcium influx is terminated.

Ca2+-independent Phospholipase A2 Is a Novel Determinant of Store-operated Ca2+ Entry

Store-operated cation (SOC) channels and capacitative Ca2+ entry (CCE) play very important role in cellular function, but the mechanism of their activation remains one of the most intriguing and long lasting mysteries in the field of Ca2+ signaling. Here, we present the first evidence that Ca2+-independent phospholipase A2(iPLA2) is a crucial molecular determinant in activation of SOC channels and store-operated Ca2+ entry pathway. Using molecular, imaging, and electrophysiological techniques, we show that directed molecular or pharmacological impairment of the functional activity of iPLA2 leads to irreversible inhibition of CCE mediated by nonselective SOC channels and by Ca2+-release-activated Ca2+ (CRAC) channels. Transfection of vascular smooth muscle cells (SMC) with antisense, but not sense, oligonucleotides for iPLA2 impaired thapsigargin (TG)-induced activation of iPLA2 and TG-induced Ca2+ and Mn2+ influx. Identical inhibition of TG-induced Ca2+ and Mn2+ influx (but not Ca2+ release) was observed in SMC, human platelets, and Jurkat T-lymphocytes when functional activity of iPLA2 was inhibited by its mechanism-based suicidal substrate, bromoenol lactone (BEL). Moreover, irreversible inhibition of iPLA2impaired TG-induced activation of single nonselective SOC channels in SMC and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid)-induced activation of whole-cell CRAC current in rat basophilic leukemia cells. Thus, functional iPLA2 is required for activation of store-operated channels and capacitative Ca2+influx in wide variety of cell types.

K+ and Ca2+ channel activity and cytosolic [Ca2+] in oxygen-sensing tissues.

Ion channels are known to participate in the secretory or mechanical responses of chemoreceptor cells to changes in oxygen tension (P(O2)). We review here the modifications of K+ and Ca2+ channel activity and the resulting changes in cytosolic [Ca2+] induced by low P(O2) in glomus cells and arterial smooth muscle which are well known examples of O2-sensitive cells. Glomus cells of the carotid body behave as presynaptic-like elements where hypoxia produces a reduction of K+ conductance leading to enhanced membrane excitability, Ca2+ entry and release of dopamine and other neurotransmitters. In arterial myocytes, hypoxia can inhibit or potentiate Ca2+ channel activity, thus regulating cytosolic [Ca2+] and contraction. Ca2+ channel inhibition is observed in systemic myocytes and most conduit pulmonary myocytes, whereas potentiation is seen in a population of resistance pulmonary myocytes. The mechanism whereby O2 modulates ion channel activity could depend on either the direct allosteric modulation by O2-sensing molecules or redox modification by reactive chemical species.

Activation Mechanism for CRAC Current and Store-operated Ca2+ Entry CALCIUM INFLUX FACTOR AND Ca2+-INDEPENDENT PHOSPHOLIPASE A2β-MEDIATED PATHWAY

Here we tested the role of calcium influx factor (CIF) and calcium-independent phospholipase A2 (iPLA2) in activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry in rat basophilic leukemia (RBL-2H3) cells. We demonstrate that 1) endogenous CIF production may be triggered by Ca2+ release (net loss) as well as by simple buffering of free Ca2+ within the stores, 2) a specific 82-kDa variant of iPLA2β and its corresponding activity are present in membrane fraction of RBL cells, 3) exogenous CIF (extracted from other species) mimics the effects of endogenous CIF and activates iPLA2β when applied to cell homogenates but not intact cells, 4) activation of ICRAC can be triggered in resting RBL cells by dialysis with exogenous CIF, 5) molecular or functional inhibition of iPLA2β prevents activation of ICRAC, which could be rescued by cell dialysis with a human recombinant iPLA2β, 6) dependence of ICRAC on intracellular pH strictly follows pH dependence of iPLA2β activity, and 7) (S)-BEL, a chiral enantiomer of suicidal substrate specific for iPLA2β, could be effectively used for pharmacological inhibition of ICRAC and store-operated Ca2+ entry. These findings validate and significantly advance our understanding of the CIF-iPLA2-dependent mechanism of activation of ICRAC and store-operated Ca2+ entry.

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.

Reduction of Ca2+ channel activity by hypoxia in human and porcine coronary myocytes

OBJECTIVE Oxygen (O(2)) tension is a major regulator of blood flow in the coronary circulation. Hypoxia can produce vasodilation through activation of ATP regulated K(+) (K(ATP)) channels in the myocyte membrane, which leads to hyperpolarization and closure of voltage-gated Ca(2+) channels. However, there are other O(2)-sensitive mechanisms intrinsic to the vascular smooth muscle since hypoxia can relax vessels precontracted with high extracellular K(+), a condition that prevents hyperpolarization following opening of K(+) channels. The objective of the present study was to determine whether inhibition of Ca(2+) influx through voltage-dependent channels participates in the response of coronary myocytes to hypoxia. METHODS Experiments were performed on porcine anterior descendent coronary arterial rings and on enzymatically dispersed human and porcine myocytes of the same artery. Cytosolic [Ca(2+)] was measured by microfluorimetry and whole-cell currents were recorded with the patch clamp technique. RESULTS Hypoxia (O(2) tension approximately 20 mmHg) dilated endothelium-denuded porcine coronary arterial rings precontracted with high K(+) in the presence of glibenclamide (5 microM), a blocker of K(ATP) channels. In dispersed human and porcine myocytes, low O(2) tension decreased basal cytosolic [Ca(2+)] and transmembrane Ca(2+) influx independently of K(+) channel activation. In patch clamped cells, hypoxia reversibly inhibited L-type Ca(2+) channels. RT-PCR indicated that rHT is the predominant mRNA variant of the alpha(1C) Ca(2+) channel subunit in human coronary myocytes. CONCLUSION Our study demonstrates, for the first time in a human preparation, that voltage-gated Ca(2+)channels in coronary myocytes are under control of O(2) tension.

Urotensin-II promotes vascular smooth muscle cell proliferation through store-operated calcium entry and EGFR transactivation.

AIMS Urotensin-II (UII) is a vasoactive peptide that promotes vascular smooth muscle cells (VSMCs) proliferation and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling. This study aimed to determine the role of calcium (Ca(2+))-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation focusing on store-operated Ca(2+) entry (SOCE) and epithelium growth factor receptor (EGFR) transactivation. METHODS AND RESULTS We used primary cultures of VSMCs isolated from Wistar rat aorta to investigate the effects of UII on intracellular Ca(2+) mobilization, and proliferation determined by the 5-bromo-2-deoxyuridine (BrdU) assay. We found that UII enhanced intracellular Ca(2+) concentration ([Ca(2+)]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as stromal interaction molecule 1 (STIM1), Orai1, or TRPC1. Moreover, UII activated a Gd(3+)-sensitive current with similar features of the Ca(2+) release-activated Ca(2+) current (ICRAC). Additionally, UII stimulated VSMCs proliferation and Ca(2+)/cAMP response element-binding protein (CREB) activation through the SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Moreover, we determined that EGFR transactivation, extracellular signal-regulated kinase (ERK) and Ca(2+)/calmodulin-dependent kinase (CaMK) signalling pathways were involved in both UII-mediated Ca(2+) influx, CREB activation and VSMCs proliferation. CONCLUSION Our data show for the first time that UII-induced VSMCs proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1, and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.

The cytoskeleton plays a modulatory role in the association between STIM1 and the Ca2+ channel subunits Orai1 and TRPC1.

Store-operated Ca(2+) entry (SOCE) is a major pathway for Ca(2+) influx in non-excitable cells. Recent studies favour a conformational coupling mechanism between the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and Ca(2+) permeable channels in the plasma membrane to explain SOCE. Previous studies have reported a role for the cytoskeleton modulating the activation of SOCE; therefore, here we have investigated whether the interaction between STIM1 and the Ca(2+) permeable channels is modulated by the actin or microtubular network. In HEK-293 cells, treatment with the microtubular disrupter colchicine enhanced both the activation of SOCE and the association between STIM1 and Orai1 or TRPC1 induced by thapsigargin (TG). Conversely, stabilization of the microtubules by paclitaxel attenuated TG-evoked activation of SOCE and the interaction between STIM1 and the Ca(2+) channels Orai1 and TRPC1, altogether suggesting that the microtubules act as a negative regulator of SOCE. Stabilization of the cortical actin filament layer results in inhibition of TG-evoked both association between STIM1, Orai1 and TRPC1 and SOCE. Interestingly, disruption of the actin filament network by cytochalasin D did not significantly modify TG-evoked association between STIM1 and Orai1 or TRPC1 but enhanced TG-stimulated SOCE. Finally, inhibition of calmodulin by calmidazolium enhances TG-evoked SOCE and disruption of the actin cytoskeleton results in inhibition of TG-evoked association of calmodulin with Orai1 and TRPC1. Thus, we demonstrate that the cytoskeleton plays an essential role in the regulation of SOCE through the modulation of the interaction between their main molecular components.

Functional and physiopathological implications of TRP channels.

Transient Receptor Potential (TRP) channel proteins are a diverse family of proteins that are expressed in many organisms, tissues and cell types. TRP channels respond to a variety of stimuli, including light, mechanical or chemical stimuli, temperature, pH or osmolarity. In addition, several TRP family members have been identified as downstream molecules in the G protein-coupled receptor signaling pathway. TRP proteins are involved in a variety of cell functions both in non-excitable and excitable cells due to their diverse permeability to cations and their ability to modulate intracellular Ca2+ signaling. Emerging evidence suggests that TRP channel dysfunction significantly contributes to the physiopathology of a number of diseases, including cardiovascular, neurological, metabolic or neoplastic disorders. This review focuses on the implication of TRP proteins in the pathogenesis of some of the most prevalent disorders in human. We summarize the current findings regarding the role of TRP proteins in the development of cardiovascular disease, diabetes mellitus as well as diabetic complications, and tumorigenesis and present TRP proteins as targets of potential diagnostic and therapeutic strategies.

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.