Seeing is believing: Recent trends in the measurement of Ca2+ in subcellular domains and intracellular organelles

The role of Ca2+ in the regulation of the cell cycle has been investigated mostly in studies assessing global cytosolic free Ca2+. Recent studies, however, have used unique techniques to assess Ca2+ in subcellular organelles, such as mitochondria, and in discrete regions of the cytoplasm. These studies have used advanced fluorescence digital imaging techniques and Ca2+-sensitive fluorescence probes, and/or targeting of Ca2+-sensitive proteins to intracellular organelles. The present review describes the results of some of these studies and the techniques used. The novel techniques used to measure Ca2+ in microdomains and intracellular organelles are likely to be of great use in future investigations assessing Ca2+ homeostasis during the cell cycle.

Removal from the Membrane Affects the Interaction of Rat Osseous Plate Ecto-Nucleosidetriphosphate Diphosphohydrolase-1 with Substrates and Ions

We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane-bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site-site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min(-1) mg(-1). The solubilized enzyme has M(r) of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always < 2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases.

Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein

Chang S, Gomes CM, Hypolite JA, Marx J, Alanzi J, Zderic SA, Malkowicz B, Wein AJ, Chacko S. Detrusor overactivity is associated with downregulation of large-conductance calcium-and voltage-activated potassium channel protein. Am J Physiol Renal Physiol 298: F1416-F1423, 2010. First published April 14, 2010; doi: 10.1152/ajprenal.00595.2009.-Large-conductance voltage-and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstructioninduced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC(20)) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel alpha- and beta-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BK beta mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK beta-subunit was greater than that of the BK alpha-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK beta-subunit was employed to study the effect of BK depletion on MLC(20) phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC(20) phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC(20) phosphorylation.

Giant protein kinases: Domain interactions and structural basis of autoregulation

The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules, We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain, The structure of the longer fragment shoes that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues, Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I.

cDNA microarray analysis of differentially expressed genes in penile tissue after treatment with tadalafil

To evaluate differential gene expression in penile tissue after treatment with the phosphodiesterase 5 (PDE5) inhibitor tadalafil, as of the three clinically available PDE5 inhibitors (sildenafil, tadalafil, and vardenafil) used for the treatment of erectile dysfunction (ED), tadalafil has a long half-life and low incidence of side-effects. In all, 32 adult rats were divided into two groups. The control group received 0.5 mL of drinking water alone, while the tadalafil group was treated with tadalafil at a dose of 0.27 mg/kg. At 4 h after treatment with water or tadalafil the rats were killed and the penile tissue was removed. The total RNA was isolated from the penile tissue from both groups and differentially expressed genes were identified by cDNA microarray analysis. To validate the expression data from the microarray analysis, quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry were used. In all, 153 genes were differentially expressed between the control group and the tadalafil group. We validated the microarray results by quantitative PCR for the insulin-like growth factor binding protein 6 (IGFBP-6) gene and the neuronal calcium sensor 1 (NCS-1) gene, both of which were up-regulated in the tadalafil group, and for the natriuretic peptide receptor 1 (NPR-1) gene that was down-regulated in this group. Immunohistochemistry showed localization of the NCS-1 protein in sinusoid trabeculae of the corpus cavernosum in control and tadalafil-treated rats. There was differential expression in 153 genes after tadalafil treatment. Some of these genes such as IGFBP-6, NPR-1 and NCS-1, might result in new targets in the treatment of ED.

Effects of chronic L-NAME treatment lung tissue mechanics, eosinophilic and extracellular matrix responses induced by chronic pulmonary inflammation

The importance of lung tissue in asthma pathophysiology has been recently recognized. Although nitric oxide mediates smooth muscle tonus control in airways, its effects on lung tissue responsiveness have not been investigated previously. We hypothesized that chronic nitric oxide synthase (NOS) inhibition by N-omega-nitro-L-arginine methyl ester (L-NAME) may modulate lung tissue mechanics and eosinophil and extracellular matrix remodeling in guinea pigs with chronic pulmonary inflammation. Animals were submitted to seven saline or ovalbumin exposures with increasing doses (1 similar to 5 mg/ml for 4 wk) and treated or not with L-NAME in drinking water. After the seventh inhalation (72 h), animals were anesthetized and exsanguinated, and oscillatory mechanics of lung tissue strips were performed in baseline condition and after ovalbumin challenge (0.1%). Using morphometry, we assessed the density of eosinophils, neuronal NOS (nNOS)- and inducible NOS (iNOS)-positive distal lung cells, smooth muscle cells, as well as collagen and elastic fibers in lung tissue. Ovalbumin-exposed animals had an increase in baseline and maximal tissue resistance and elastance, eosinophil density, nNOS- and iNOS-positive cells, the amount of collagen and elastic fibers, and isoprostane-8-PGF(2 alpha) expression in the alveolar septa compared with controls (P < 0.05). L-NAME treatment in ovalbumin-exposed animals attenuated lung tissue mechanical responses (P < 0.01), nNOS- and iNOS-positive cells, elastic fiber content (P < 0.001), and isoprostane-8-PGF(2 alpha) in the alveolar septa (P < 0.001). However, this treatment did not affect the total number of eosinophils and collagen deposition. These data suggest that NO contributes to distal lung parenchyma constriction and to elastic fiber deposition in this model. One possibility may be related to the effects of NO activating the oxidative stress pathway.

A quantitative chemiluminescent method for studying replicative and stress-induced premature senescence in cell cultures

beta-Galactosidase (beta-Gal) activity is a widely accepted biomarker to detect senescence both in situ and in vitro. A cytochemical assay based on production of a blue-dyed precipitate that results from the cleavage of the chromogenic substrate X-Gal is commonly used. Blue and nonblue cells are counted under the microscope and a semiquantitative percentage of senescent cells can be obtained. Here, we present a quantitative, fast, and easy to use chemiluminescent assay to detect senescence. The Galacton chemiluminescent method used to detect the prokaryotic beta-Gal reporter enzyme in transfection studies was adapted to assay mammalian beta-Gal. The assay showed linear production of luminescence in a time- and cell-number-dependent manner. The chemiluminescent assay showed significant correlation with the cytochemical assay in detecting replicative senescence (Pearson r = 0.8486, p < 0.005). Moreover, the chemiluminescent method (Galacton) also detected stress-induced senescence in cells treated with H2O2 similar to the cytochemical assay (X-Gal) (Galacton: control 25.207.3 +/- 6548.6. H2O, 52,487.4 +/- 16,284.9, p < 0.05; X-Gal: control 41.31 +/- 7.0%, H2O2 92.97 +/- 2.8%, p < 0.01). Thus, our method is well suited to the detection of replicative and stress-induced senescence in cell culture. (C) 2007 Elsevier Inc. All rights reserved.

Challenges in Using Stem Cells for Cardiac Repair

Of the many diseases discussed in the context of stem cell therapy, those concerning the heart account for almost one-third of the publications in the field. However, the long-term clinical outcomes have been disappointing, in part because of preclinical studies failing to optimize the timing, number, type, and method of cell delivery and to account for shape changes that the heart undergoes during failure. In situations in which cardiomyocytes have been used in cell therapy, their alignment and integration with host tissue have not been realized. Here we review the present status of direct delivery of stem cells or their derivative cardiomyocytes to the heart and the particular challenges each cell type brings, and consider where we should go from here.

Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma

Purpose We evaluated the involvement of angiotensin II (AngII)-dependent pathways in melanoma growth, through the pharmacological blockage of AT1 receptor by the antihypertensive drug losartan (LOS). Results We showed immunolabeling for both AngII and the AT1 receptor within the human melanoma microenvironment. Like human melanomas, we showed that murine melanomas also express the AT1 receptor. Growth of murine melanoma, both locally and at distant sites, was limited in mice treated with LOS. The reduction in tumor growth was accompanied by a twofold decrease in tumorassociated microvessel density and by a decrease in CD31 mRNA levels. While no differences were found in the VEGF expression levels in tumors from treated animals, reduction in the expression of the VEGFR1 (Flt-1) at the mRNA and protein levels was observed. We also showed downregulation of mRNA levels of both Flt-4 and its ligand, VEGF-C. Conclusions Together, these results show that blockage of AT1 receptor signaling may be a promising anti-tumor strategy, interfering with angiogenesis by decreasing the expression of angiogenic factor receptors.

The cell biology of atherosclerosis - new developments

In the development of atherosclerotic lesions, three basic processes occur: 1) invasion of the artery wall by leucocytes, particularly monocytes and T-lymphocytes; 2) smooth muscle phenotypic modulation, proliferation, and synthesis of extracellular matrix; and 3) intracellular (macrophage and smooth muscle) lipoprotein uptake and lipid accumulation. Invasion of the vessel wall by leucocytes is mediated through the expression of adhesion molecules on both leucocytes and the endothelium making them 'sticky'. The adhesion molecules are induced by high serum cholesterol levels or complement fragments. Leucocytes which have adhered to the endothelium are chemo-attracted into the vessel wall by cytokines produced by early arriving leucocytes or by low density lipoprotein which has passively passed into the wall, in the process being trapped and oxidised. The oxidised low density lipoprotein is taken up by scavenger receptors (which are not subject to down-regulation) on both macrophages and smooth muscle cells. The overaccumulation of lipid is toxic to the cells and they die contributing to the central necrotic core. The macrophages and T-lymphocytes produce substances which induce smooth muscle cells of the artery wall to change from a 'contractile' (high volume fraction of myofilaments [V(v)myo]) to a 'synthetic' (low V(v)myo) phenotype. In this altered state they respond to growth factors released from macrophages, platelets, regenerating endothelial cells and smooth muscle cells; produce large amounts of matrix; express lipoprotein scavenger receptors; express adhesion molecules for leucocytes; and express HLA-DR following exposure to the T-lymphocyte product, IFN-delta, suggesting that they can become involved in a generalised immune reaction.

Fibroblastic rheumatism

Fibroblastic rheumatism (FR) was first described in 1980 by Chaouat et al., and there have been few cases reported to date. The cause remains unknown. We report the first Latin-American patient with FR, to our knowledge, who is also the patient with the most striking dermatological features described in the literature. The diagnosis was based on the presence of a number of typical features. Clinically, the patient presented skin nodules and polyarthropathy with flexion contractures of the fingers. The histological findings compressed fibroblastic proliferation, thickened collagen fibres, dermal fibrosis and a decreased number of elastic fibres. Immunoreactivity for beta-catenin, alpha-smooth muscle actin and the monoclonal antibody HHF-35 showed myofibroblastic differentiation. Treatment with prednisone slightly reduced the number of nodules but did not improve the rheumatological symptoms. This condition has shown a poor response to many treatments proposed by previous authors. Further study will be necessary to identify effective treatment.

Aerobic training reverses airway inflammation and remodelling in an asthma murine model

Aerobic training (AT) decreases dyspnoea and exercise-induced bronchospasm, and improves aerobic capacity and quality of life; however, the mechanisms for such benefits remain poorly understood. The aim of the present study was to evaluate the AT effects in a chronic model of allergic lung inflammation in mice after the establishment of airway inflammation and remodelling. Mice were divided into the control group, AT group, ovalbumin (OVA) group or OVA+AT group and exposed to saline or OVA. AT was started on day 28 for 60 min five times per week for 4 weeks. Respiratory mechanics, specific immunoglobulin (Ig)E and IgG(1), collagen and elastic fibres deposition, smooth muscle thickness, epithelial mucus, and peribronchial density of eosinophils, CD3+ and CD4+, IL-4, IL-5, IL-13, interferon-gamma, IL-2, IL-1ra, IL-10, nuclear factor (NF)-kappa B and Foxp3 were evaluated. The OVA group showed an increase in IgE and IgG1, eosinophils, CD3+, CD4+, IL-4, IL-5, IL-13, NF-kappa B, collagen and elastic, mucus synthesis, smooth muscle thickness and lung tissue resistance and elastance. The OVA+AT group demonstrated an increase of IgE and IgG(1), and reduction of eosinophils, CD3+, CD4+, IL-4, IL-5, IL-13, NF-kappa B, airway remodelling, mucus synthesis, smooth muscle thickness and tissue resistance and elastance compared with the OVA roup (p < 0.05). The OVA+AT group also showed an increase in IL-10 and IL-1ra (p < 0.05), independently of Foxp3. AT reversed airway inflammation and remodelling and T-helper cell 2 response, and improved respiratory mechanics. These results seem to occur due to an increase in the expression of IL-10 and IL-1ra and a decrease of NF-kappa B.

Exercise Reduces Effects of Creatine on Lung

We recently demonstrated that creatine supplementation increased some features of lung allergic sensitization in mice. On the other hand, other studies have shown that aerobic exercise inhibited allergic airway inflammation and remodeling. We hypothesized that aerobic exercise may decrease the exacerbatory effects of the creatine supplementation in a murine model of asthma. Balb/c mice were divided into six groups: Control, Creatine (Cr), Low Intensity Exercise + Creatine (Low + Cr), Ovalbumin (OVA), Ovalbumin + Creatine (OVA + Cr) and Ovalbumin + Creatine + Low Intensity Exercise (OVA + Cr + Low). OVA-sensitized groups were sensitized with OVA intraperitoneal injections (days 0, 14, 28, and 42). Aerosol challenge (OVA 1 %) and Cr treatment (0.5 g/kg/day) were initiated on Day 21 until Day 53. Low intensity exercise began on day 22 and was sustained until day 50. Low intensity exercise in the presence of creatine supplementation in sensitized mice resulted in a decreased number of eosinophils in BALF (p < 0.001) and in the airways (P < 0.001), and a decreased density of inflammatory cells positive to IL-4 (p < 0.001) and IL-5 (p < 0.001), airway collagen (p < 0.001) and elastic fibers (p < 0.001) content, airway smooth muscle thickness (p < 0.001) and bronchoconstriction index (p < 0.05) when compared with OVA + Cr group. These results suggest that aerobic exercise reduces the exacerbatory effects of creatine supplementation in chronically sensitized mice.