Increased Activation of Stromal Interaction Molecule-1/Orai-1 in Aorta From Hypertensive Rats A Novel Insight Into Vascular Dysfunction

Disturbances in the regulation of cytosolic calcium (Ca(2+)) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca(2+) entry. Although STIMs act as Ca(2+) sensors for the intracellular Ca(2+) stores, Orai is the putative pore-forming component of Ca(2+) release-activated Ca(2+) channels at the plasma membrane. We hypothesized that augmented activation of Ca(2+) release-activated Ca(2+)/Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca(2+) influx. Depletion of intracellular Ca(2+) stores, which induces Ca(2+) release-activated Ca(2+) activation, was performed by placing arteries in Ca(2+) free-EGTA buffer. The addition of the Ca(2+) regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 mu mol/L), an inhibitor of the sarcoplasmic reticulum Ca(2+) ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca(2+) release-activated Ca(2+) channel inhibitors 2-aminoethoxydiphenyl borate (100 mu mol/L) or gadolinium (100 mu mol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca(2+) levels in hypertension. (Hypertension. 2009; 53[part 2]: 409-416.)

Expression and characterization of LTx2, a neurotoxin from Lasiodora sp effecting on calcium channels

Here, we described the expression and characterization of the recombinant toxin LTx2, which was previously isolated from the venomous cDNA library of a Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae). The recombinant toxin found in the soluble and insoluble fractions was purified by reverse phase high-performance liquid chromatography (HPLC). Ca2+ imaging analysis revealed that the recombinant LTx2 acts on calcium channels of BC3H1 cells, blocking L-type calcium channels. (C) 2008 Elsevier Inc. All rights reserved.

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha 7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca(2+) influx. However, the study of recombinant alpha 7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha 3, alpha 5, alpha 7, beta 2 and beta 4 subunits was present during the course of differentiation, while mRNAs coding for alpha 2, alpha 4 and beta 3 subunits were not expressed in PC12 cells. alpha 7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha 7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha 7 agonist choline. Increased alpha 7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin gamma 1 chain

The prion protein (PrP(C)) is highly expressed in the nervous system, and its abnormal conformer is associated with prion diseases. PrP(C) is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP(C)-mediated intracellular signaling. Binding of laminin (Ln) to PrP(C) modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP(C)-Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP(C)-Ln signals. The Ln gamma 1-chain peptide, which contains the Ln binding site for PrP(C), induced neuritogenesis through activation of phospholipase C (PLC), Ca(2+) mobilization from intracellular stores, and protein kinase C and extracellular signal-regulated kinase (ERK1/2) activation in primary cultures of neurons from wild-type, but not PrP(C)-null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluR1/5) associate with PrP(C). Expression of either mGluR1 or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP(C)-Ln gamma 1 peptide interaction. Specific inhibitors of these receptors impaired PrP(C)-Ln gamma 1 peptide-induced signaling and neuritogenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP(C)-Ln, and they support the notion that PrP(C) participates in the assembly of multiprotein complexes with physiological functions on neurons.-Beraldo, F. H., Arantes, C. P., Santos, T. G., Machado, C. F., Roffe, M., Hajj, G. N., Lee, K. S., Magalhaes, A. C., Caetano, F. A., Mancini, G. L., Lopes, M. H., Americo, T. A., Magdesian, M. H., Ferguson, S. S. G., Linden, R., Prado, M. A. M., Martins, V. R. Metabotropic glutamate receptors trans-duce signals for neurite outgrowth after binding of the prion protein to laminin gamma 1 chain. FASEB J. 25, 265-279 (2011). www.fasebj.org

Tumor Necrosis Factor alpha and Interleukin-1 beta Modulate Calcium and Nitric Oxide Signaling in Mechanically Stimulated Osteocytes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Subfornical organ mediates pressor effect of angiotensin: Influence of nitric oxide synthase inhibitors, AT(1) and AT(2) angiotensin antagonist's receptors

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effects of indoramin in rat vas deferens and aorta: concomitant alpha(1)-adrenoceptor and neuronal uptake blockade

1 the actions of the alpha(1)-adrenoceptor antagonist indoramin have been examined against the contractions induced by noradrenaline in the rat vas deferens and aorta taking into account a putative neuronal uptake blocking activity of this antagonist which could. result in self-cancelling actions.2 Indoramin behaved as a simple competitive antagonist of the contractions induced by noradrenaline in the vas deferens and aorta yielding pA(2) values of 7.38 +/- 0.05 (slope = 0.98 +/- 0.03) and 6.78 +/- 0.14 (slope = 1.08 +/- 0.06), respectively.3 When the experiments were repeated in the presence of cocaine (6 mu M) the potency (pA(2)) of indoramin in antagonizing the contractions of the vas deferens to noradrenaline was increased to 8.72 +/- 0.07 (slope = 1.10 +/- 0.05) while its potency remained unchanged in the aorta (pA(2) = 6.69 +/- 0.12; slope = 1.04 +/- 0.05).4 In denervated vas deferens, indoramin antagonized the contractions to noradrenaline with a potency similar to that found in the presence of cocaine (8.79 +/- 0.07; slope = 1.09 +/- 0.06).5 It is suggested that indoramin blocks alpha(1)-adrenoceptors and neuronal uptake in rat vas deferens resulting in Schild plots with slopes not different from unity even in the absence of selective inhibition of neuronal uptake. As a major consequence of this double mechanism of action, the pA(2) values for this antagonist are underestimated when calculated in situations where the neuronal uptake is active, yielding spurious pK(B) values.

Linear readout of dynamic phase modulation index in an optical voltage sensor using the new J(1) ... J(3) spectral method

The J(1)...J(3) is a recent optical method for linear readout of dynamic phase modulation index in homodyne interferometers. In this work, the J(1)... J(3) method is applied to measure voltage in an optical voltage sensor. Based on the classical J(1)...J(4) method, the J(1)... J(3) technique shows to be more stable to phase drift and simpler for implementation than the original one. The sensor dynamic range is enhanced. The agreement between theoretical and experimental results, based on 1/f noise, is demonstrated.

Nitrergic pathways and L-type calcium channel of MnPO influencing cardiovascular homeostasis

The median preoptic nucleus (MnPO) is one of most important site of the lamina terminalis implicated in the regulation of hydro electrolytic and cardiovascular balance. The purpose of this study was to determine the effect of L-Type calcium channel antagonist, nifedipine, on the increase of median arterial blood pressure (MAP) induce by angiotensin II (ANG II) injected into the MnPO. The influence of nitric oxide (NO) on nifedipine antipressor action has also been studied by utilizing N W-nitro-L-arginine methyl ester (L-NAME) (40 μg 0.2 μL -1) a NO synthase inhibitor (NOSI), 7-nitroindazole (7-NIT) (40 μg 0.2 μL -1), a specific neuronal NO synthase inhibitor (nNOSI) and sodium nitroprusside (SNP) (20 μg 0.2 μL -1) a NO donor agent. We have also investigated the central role of losartan and PD123349 (20 nmol 0.2 μL -1), AT 1 and AT 2, respectively (selective non peptide ANG II receptor antagonists), in the pressor effect of ANG II (25 pmol 0.2 μL -1) injected into the MnPO. Male Wistar rats weighting 200-250 g, with cannulae implanted into the MnPO were utilized. Losartan injected into the MnPO, prior to ANG II, blocked the pressor effect of ANGII. PD 123319 only decreased the pressor effect of ANG II. Rats pre-treated with either 50 μg 0.2 μL -1 or 100 μg 0.2 μL -1 of nifedipine, followed by 25 pmol 0.2 μL -1 of ANG II, decreased ANG II-pressor effect. L-NAME potentiated the pressor effect of ANG II. 7-NIT injected prior to ANG II into the MnPO also potentiated the pressor effect of ANGII but with less intensity than that of L-NAME. SNP injected prior to ANG II blocked the pressor effect of ANG II. The potentiation action of L-NAME and 7-NIT on ANG II-pressor effect was blocked by prior injection of nifedipine. The results described in this study provide evidence that calcium channels play important roles in central ANG II-induced pressor effect. The structures containing NO in the brain, such as MnPO, include both endothelial and neuronal cells, which might be responsible for the influence of nifedipine on the pressor effect of ANG II. These data have shown the functional relationship between L-Type calcium channel and a free radical gas NO in the MnPO, on the control of ANG II-induced pressor effect acting in AT 1 and AT 2 receptors.

Sensor for diuron quantitation based on the P450 biomimetic catalyst nickel(II) 1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine

A biomimetic sensor based on a carbon paste electrode modified with the nickel(II) 1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine complex was developed as a reliable alternative technique for the sensitive and selective analysis of the herbicide diuron in environmental media. The sensor was evaluated using cyclic voltammetry and amperometric techniques. The best amperometric responses were obtained at 750 mV vs. Ag/AgCl (KClsat), using 0.1 mol L-1 phosphate buffer solution at pH 8.0. Under these conditions, the sensor showed a linear response for diuron concentrations between 9.9 × 10-6 and 1.5 × 10-4 mol L -1, a sensitivity of 22817 (±261) μA L mol-1, and detection and quantification limits of 6.14 × 10-6 and 2 × 10-5 mol L-1, respectively. The presence of the nickel complex in the carbon paste improved selectivity, stability, and sensitivity (which increased 700%), compared to unmodified paste. The applicability of the sensor was demonstrated using enriched environmental samples (river water and soil). © 2012 Elsevier B.V. All rights reserved.

Effect of calcium on the structural properties of Ba(1-x)Ca (x) TiO3 particles synthesized by complex polymerization method

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A CURRENT EXAMINATION OF DIETARY INTAKES OF FIBER, CALCIUM, IRON, AND ZINC AND THEIR RELATIONSHIP TO BLOOD LEAD LEVELS IN U.S. CHILDREN AGED 1-5 YEARS

A CURRENT EXAMINATION OF DIETARY INTAKES OF FIBER, CALCIUM, IRON, AND ZINC AND THEIR RELATIONSHIP TO BLOOD LEAD LEVELS IN U.S. CHILDREN AGED 1-5 YEARS Stephanie Ann Melchert, M.S. University of Nebraska, 2010 Adviser: Kaye Stanek Krogstrand The effect of lead on the health and well-being of those exposed has been well documented and many efforts have been made to reduce exposure of lead to the United States population. Despite these efforts, many studies have documented cognitive impairments and behavioral problems in children with even low levels of lead in their blood. Previous studies have suggested that a proper diet may have a role in the prevention of elevated blood lead levels in children. The objective of this study was to determine if there was an inverse correlation of blood lead levels (BLL) in children to their dietary intakes of fiber, calcium, iron, and zinc considering low levels of lead exposure. This study examined 1019 children in the National Health and Nutrition Examination Survey (NHANES) conducted from 2005-2006. Data were analyzed using Spearman’s rank correlations to correlate continuous variables to BLL in children and independent samples t-tests were used to compare mean blood lead levels of categorical variables. Results indicate that BLL in children is significantly correlated with and weight, recumbent length/standing height, dietary fiber intake and continine, a marker of cigarette smoke exposure. BLL was not significantly correlated with calcium, iron, zinc, or vitamin C. A significant difference was found in the mean BLL of children who took supplements, lived in smoking homes, as well as those who lived in homes built before 1978. Overall, this study shows that children living in homes built before 1978 remain at greater risk for lead exposure, and adequate dietary fiber intake may provide benefits to children who are exposed to lead.

STIM 1/Orai 1 contributes to sex differences in vascular responses to calcium in spontaneously hypertensive rats

Sex differences in Ca2+-dependent signalling and homoeostasis in the vasculature of hypertensive rats are well characterized. However, sex-related differences in SOCE (store-operated Ca2+ entry) have been minimally investigated. We hypothesized that vascular protection in females, compared with males, reflects decreased Ca2+ mobilization due to diminished activation of Orai 1/STIM 1 (stromal interaction molecule I). In addition, we investigated whether ovariectomy in females affects the activation of the Orai 1/STIM 1 pathway. Endothelium-denuded aortic rings from male and female SHRSP (stroke-prone spontaneously hypertensive rats) and WKY (Wistar Kyoto) rats and from OVX (ovariectomized) or sham female SHRSP and WKY rats were used to functionally evaluate Ca2+ influx-induced contractions. Compared with females, aorta from male SHRSP displayed: (i) increased contraction during the Ca2+-loading period; (ii) similar transient contraction during Ca2+ release from the intracellular stores; (iii) increased activation of STIM 1 and Orai1, as shown by the blockade of STIM 1 and Orai1 with neutralizing antibodies, which reversed the sex differences in contraction during the Ca2+-loading period; and (iv) increased expression of STIM I and Orai I. Additionally, we found that aortas from OVX-SHRSP showed increased contraction during the Ca2+-loading period and increased Orai1 expression, but no changes in the SR (sarcoplasmic reticulum)-buffering capacity or STIM I expression. These findings suggest that augmented activation of STIM 1/Orai 1 in aortas from male SHRSP represents a mechanism that contributes to sex-related impaired control of intracellular Ca2+ levels. Furthermore, female sex hormones may negatively modulate the STIM/Orai 1 pathway, contributing to vascular protection observed in female rats.

Implications of purinergic receptor-mediated intracellular calcium transients in neural differentiation

Purinergic receptors participate, in almost every cell type, in controlling metabolic activities and many physiological functions including signal transmission, proliferation and differentiation. While most of P2Y receptors induce transient elevations of intracellular calcium concentration by activation of intracellular calcium pools and forward these signals as waves which can also be transmitted into neighboring cells, P2X receptors produce calcium spikes which also include activation of voltage-operating calcium channels. P2Y and P2X receptors induce calcium transients that activate transcription factors responsible for the progress of differentiation through mediators including calmodulin and calcineurin. Expression of P2X2 as well as of P2X7 receptors increases in differentiating neurons and glial cells, respectively. Gene expression silencing assays indicate that these receptors are important for the progress of differentiation and neuronal or glial fate determination. Metabotropic receptors, mostly P2Y1 and P2Y2 subtypes, act on embryonic cells or cells at the neural progenitor stage by inducing proliferation as well as by regulation of neural differentiation through NFAT translocation. The scope of this review is to discuss the roles of purinergic receptor-induced calcium spike and wave activity and its codification in neurodevelopmental and neurodifferentiation processes.

Charakterisierung der biologischen Funktion der Plasmodium falciparum Calcium-abhängigen Proteinkinase 1

Im Rahmen dieser Arbeit wurden biologische Funktionen einer Proteinkinase des Erregers der Malaria tropica, genauer der „Plasmodium falciparum calcium dependent protein kinase 1“ (PfCDPK1), in parasitären Blutstadien untersucht.rnUm Einblicke in die Funktion der Kinase, die sie in den extrazellulären Kompartimenten des Parasiten übernimmt, zu gewinnen, wurden sechs Proteine untersucht, die dasselbe Translokationsignal wie PfCDPK1 besitzen. Es konnte gezeigt werden, dass fünf der untersuchten Proteine mit der PfCDPK1 im Bereich der parasitophoren Vakuole sowie des tubovesikulären Systems co-lokalisiert sind. Deletionsmutanten, denen das Translokationssignal fehlte, sowie ein Peptid, das lediglich aus diesem bestand, bestätigten, dass die Translokation in die extrazellulären Kompartimente von keinen weiteren Faktoren, außer dem Signalmotiv abhängt. Mit PfCAP und PfRKIP konnten zwei Regulatoren der PfCDPK1 identifiziert werden. PfARM, Pfrab_5b sowie PfGAP45 sind Substrate der PfCDPK1. Mit Hilfe von massenspektrometrischen Messungen wurde der Phosphorylierungsstatus der untersuchten Proteine durch die PfCDPK1 sowie der Autophosphorylierungsstatus der Kinase bestimmt, um Rückschlüsse auf regulatorische Prozesse ziehen zu können.rnDie Phosphorylierung von PfGAP45 durch die PfCDPK1 steht vermutlich mit dem Invasionsprozess des Parasiten in direktem Zusammenhang, da gezeigt wurde, dass eine Hemmung der Kinase mit PP1 einen 90%igen Rückgang an neu infizierten Erythrozyten zur Folge hatte.rnrn