A role for zinc in regulating hypoxia-induced contractile events in pulmonary endothelium
ABSTRACT We previously reported that zinc thiolate signaling contributes to hypoxic contraction of small, nonmuscularized arteries of the lung. The present studies were designed to investigate mechanisms by which hypoxia-released zinc induces contraction in isolated pulmonary endothelial cells and to delineate the signaling pathways involved in zinc-mediated changes in the actin cytoskeleton. We used fluorescence-based imaging to show that hypoxia induced time-dependent increases in actin stress fibers that were reversed by the zinc chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). We further showed that hypoxia-induced phosphorylation of the contractile protein myosin light chain (MLC) and assembly of actin stress fibers were each TPEN sensitive. Hypoxia and zinc-induced inhibition of MLC phosphatase (MLCP) were independent of the regulatory subunit (MYPT1) of MLCP, and therefore hypoxia-released zinc likely inhibits MLCP at its catalytic (PP1) subunit. Inhibition of PKC by Ro-31-8220 and a dominant-negative construct of PKC-ε attenuated hypoxia-induced contraction of isolated pulmonary endothelial cells. Furthermore, zinc-induced phosphorylation of MLC (secondary to inhibition of MLCP) was PKC dependent, and hypoxia-released zinc promoted the phosphorylation of the PKC substrate, CPI-17. Collectively, these data suggest a link between hypoxia, elevations in labile zinc, and activation of PKC, which in turn acts through CPI-17 to inhibit MLCP activity and promote MLC phosphorylation, ultimately inducing stress fiber formation and endothelial cell contraction.
SourceAvailable from: Kalidasan Thambiayya[Show abstract] [Hide abstract]
ABSTRACT: After iron, zinc is the most abundant essential trace metal. Intracellular zinc ([Zn](i)) is maintained across a wide range of cells and species in a tight quota (100 to 500 μM) by a dynamic process of transport, intracellular vesicular storage, and binding to a large number of proteins (estimated at 3-10% of human proteome). As such, zinc is an integral component of numerous metalloenzymes, structural proteins, and transcription factors. It is generally assumed that a vanishingly small component of [Zn](i,) referred to as free or labile zinc, and operationally defined as the pool sensitive to chelation (by agents such as N, N, N', N'-tetrakis [2-pyridylmethyl] ethylenediamine [TPEN]) and capable of detection by a variety of chemical and genetic sensors, participates in signal transduction pathways. Zinc deficiencies, per se, can arise from acquired (malnutrition, alcoholism) or genetic (mutations in molecules affecting zinc homeostasis, the informative and first example being acrodermatitis enteropathica) factors or as a component of various diseases (e.g., sickle cell disease, cystic fibrosis, sepsis). Hypozincemia has profound effects on developing humans, and all facets of physiological function (neuronal, endocrine, immunological) are affected, although considerably less is known regarding cardiovascular pathophysiology. In this review, we provide an update on current knowledge of molecular and cellular aspects of zinc homeostasis and then focus on implications of zinc signaling in pulmonary endothelium as it relates to programmed cell death, altered contractility, and septic and aseptic injury to this segment of the lung.03/2012; 2(4):443-451. DOI:10.4103/2045-8932.105032
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ABSTRACT: Studies on normal physiological and pathological processes that take place in the lung are hampered by the architecture and behaviour of a living, breathing lung. The lung has a very complex three-dimensional structure that is constantly in motion. Not only do the airways and vasculature constrict and relax, but the alveolar airspaces regularly and rapidly contract and expand, appreciably changing the size and volume of the lung at regular intervals during normal lung function. Both the development of the lung, as well as normal lung function, are dependent upon the intactness and coordinated movement of the lung, making in vitro studies on intact lungs difficult. Although the proximal regions of the intact, breathing lung are relatively accessible via the trachea, the most distal regions of the lung, the alveolar units, are relatively inaccessible, being reachable only through the tiny alveolar ducts which provide passage into the alveolar sacs. Recently, there has been an explosion of interest in imaging physiological processes in the lung in real-time, and several notable advances have been made, which have been facilitated by new methodological approaches, novel microscopic and radiological imaging technologies, as well as the development of genetically modified animal models which facilitate imaging approaches. The pressing need for further advances in the development or refinement of experimental approaches for the real-time assessment of lung structure and function, together with an explosion of interest in this area of pre-clinical and clinical pulmonary research, has prompted this Special Call for Papers on "Real-time Visualization of Lung Function: from Micro to Macro" from the American Journal of Physiology - Lung Cellular and Molecular Physiology.AJP Lung Cellular and Molecular Physiology 10/2012; 304(1). DOI:10.1152/ajplung.00279.2012 · 4.04 Impact Factor
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ABSTRACT: We previously noted an important signaling role for decreased labile intracellular zinc ([ Zn ] (i)) in LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) (Tang ZL, Wasserloos KJ, Liu X, Stitt MS, Reynolds IJ, Pitt BR, St Croix CM. Mol Cell Biochem 234-235: 211-217, 2002; Thambiayya K, Wasserloos KJ, Huang Z, Kagan VE, St Croix CM, Pitt BR. Am J Physiol Lung Cell Mol Physiol 300: L624-632, 2011). In the present study, we used small interfering RNA (siRNA) to important contributors of zinc homeostasis [ SLC39A14 or Zrt/Irt-like protein 14 (ZIP14), a zinc importer; metallothionein (MT), a zinc binding protein ] to define molecular pathways by which extracellular zinc or nitric oxide (NO) increase labile [ Zn ] (i) [ e.g., zinc-sensitive fluorophore (FluoZin-3) detectable and/or chelatable by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine ] and reduce the sensitivity of SPAEC to LPS. Addition of 10 μM zinc to serum-free medium of SPAEC increased [ Zn ] (i) and abolished LPS-induced apoptosis (e.g., increased annexin V binding). The increase in [ Zn ] (i) and the protective effect of extracellular zinc were sensitive to reduction in ZIP14 expression (by siRNA), but not affected by collectively knocking down major isoforms of sheep MT (sMT-Ia, -Ib, -Ic, and -II). Pretreatment of wild-type SPAEC with 250 μM of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased labile zinc in a relatively similar fashion to addition of extracellular zinc and reduced sensitivity of SPAEC to LPS-induced apoptosis (e.g., caspase-3/7 activation) in a N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine-sensitive fashion. The antiapoptotic effects of SNAP were insensitive to siRNA knockdown of ZIP14, but were abolished (along with SNAP-induced increase in [ Zn ] (i)) when SPAEC were pretreated with siRNA to sheep MT. Zinc was able to directly inhibit recombinant caspase-3 activity in an in vitro assay. Collectively, these data show that increases in labile [ Zn ] (i) are an important component of ZIP14- or NO-mediated resistance to LPS-induced apoptosis. Cytoprotection via ZIP14 appeared to be secondary to transcellular movement of extracellular zinc, whereas NO-mediated protection was secondary to S-nitrosation of MT and redistribution of [ Zn ] (i).AJP Lung Cellular and Molecular Physiology 04/2012; 302(12):L1287-95. DOI:10.1152/ajplung.00385.2011 · 4.04 Impact Factor