Airway hyperreactivity in diseases such as asthma involves enhanced airway smooth muscle (ASM) contraction due either to increased intracellular Ca2+ ([Ca2+]i) and/or increased Ca2+ sensitivity (force for a given [Ca2+]i). Airway inflammation is a key aspect of airways disease, and exposure to several inflammatory mediators (such as tumor necrosis factor (TNFa) and interleukin-13 (IL-13)) increase ASM contractility. Several studies showed that cytokines increase agonist-induced [Ca2+]i responses in ASM, thus linking In the current proposal, we will explore inflammation-induced changes in [Ca2+]i regulatory mechanisms that result in overall increased [Ca2+]i responses. In ASM, Ca2+ influx in response to SR depletion (store-operated Ca2+ entry;SOCE) is enhanced by cytokines. Preliminary data suggests that Na+/Ca2+ exchange (NCX)-mediated influx is enhanced by cytokines. Such enhancement of SOCE and influx-mode NCX would lead to increased [Ca2+]i levels. Reduction in [Ca2+]i is normally achieved by plasma membrane (PM) efflux mechanisms (perhaps including NCX-mediated efflux), and by SR Ca2+ reuptake via SR ATPase (SERCA). Organelles such as mitochondria can buffer Ca2+ and alter Ca2+ availability for SR refilling. Normally, these mechanisms help maintain basal [Ca2+]i at low levels, while SR Ca2+ stores are replete until agonist stimulation when [Ca2+]i rises as SR stores deplete. Preliminary studies suggest that SERCA and mitochondrial Ca2+ buffering are impaired by inflammation. Based on these contrasting preliminary findings of enhanced Ca2+ influx, but decreased sequestration or efflux, our central hypothesis is that inflammation promotes mechanisms that increase [Ca2+]i, but impairs those that decrease [Ca2+]i. This leads to an overall increase in basal [Ca2+]i as well as enhanced [Ca2+]i responses to agonist stimulation. In this regard, we propose that PM vs. intracellular mechanisms functionally interact in [Ca2+]i homeostasis under normal circumstances, and that disruption of these mechanisms and their interactions with inflammation leads to increased [Ca2+]i. Our overall approach will be to use human ASM cells or tissue strips to examine the above mechanisms with or without exposure to pro-inflammatory cytokines (TNFa, IL-13). Studies will use complementary techniques including molecular biology (siRNA;overexpression), imaging of [Ca2+]i, [Na+]i and luminal (SR) Ca2+, real-time confocal imaging of fluorescently- tagged proteins, as well as force measurements to address these aims.
Our Specific Aims are: decrease [Ca2+]i Aim 1: To determine the influence of inflammatory cytokines on STIM1, STIM2 and Orai1 interactions in human ASM regulation;
Aim 2 : To determine the influence of inflammatory cytokines on NCX and its role in [Ca2+]i regulation in human ASM;
Aim 3 : To determine the influence of inflammatory cytokines on mitochondria and its role in [Ca2+]i regulation in human ASM;
Aim 4 : To determine the influence of inflammatory cytokines on SERCA and its role in [Ca2+]i regulation in human ASM;[Ca2+]i Aim 5: To determine the influence of inflammatory cytokines on the overall contribution of [Ca2+]i regulatory mechanisms to contractility in human ASM.

Public Health Relevance

There is increasing recognition that abnormalities in airway smooth muscle contractility contribute to exaggerated airway narrowing and accompanying shortness of breath in clinically-important diseases such as asthma and chronic bronchitis. Airway contractility is highly dependent on intracellular calcium levels. The proposed studies will examine the mechanisms by which intracellular calcium is controlled in human airway smooth muscle. These studies will the foundation for better understanding of airway diseases, and potential development of new therapeutic targets.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL074309-09
Application #
8435542
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Banks-Schlegel, Susan P
Project Start
2003-07-01
Project End
2015-02-28
Budget Start
2013-03-01
Budget End
2015-02-28
Support Year
9
Fiscal Year
2013
Total Cost
$366,548
Indirect Cost
$123,961
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
Sathish, Venkatachalem; Thompson, Michael A; Sinha, Sutapa et al. (2014) Inflammation, caveolae and CD38-mediated calcium regulation in human airway smooth muscle. Biochim Biophys Acta 1843:346-51
Aravamudan, Bharathi; Kiel, Alexander; Freeman, Michelle et al. (2014) Cigarette smoke-induced mitochondrial fragmentation and dysfunction in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 306:L840-54
Wylam, Mark E; Xue, Ailing; Sieck, Gary C (2012) Mechanisms of intrinsic force in small human airways. Respir Physiol Neurobiol 181:99-108
Sathish, Venkatachalem; Delmotte, Philippe F; Thompson, Michael A et al. (2011) Sodium-calcium exchange in intracellular calcium handling of human airway smooth muscle. PLoS One 6:e23662
Ijpma, Gijs; Al-Jumaily, Ahmed M; Cairns, Simeon P et al. (2011) Myosin filament polymerization and depolymerization in a model of partial length adaptation in airway smooth muscle. J Appl Physiol 111:735-42
Palmer, Melissa L; Peitzman, Elizabeth R; Maniak, Peter J et al. (2011) K(Ca)3.1 channels facilitate K+ secretion or Na+ absorption depending on apical or basolateral P2Y receptor stimulation. J Physiol 589:3483-94
Sathish, Venkatachalem; Thompson, Michael A; Bailey, Jeffrey P et al. (2009) Effect of proinflammatory cytokines on regulation of sarcoplasmic reticulum Ca2+ reuptake in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 297:L26-34
Prakash, Y S; Sathish, Venkatachalem; Thompson, Michael A et al. (2009) Asthma and sarcoplasmic reticulum Ca2+ reuptake in airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 297:L794
Prakash, Y S; Thompson, Michael A; Pabelick, Christina M (2009) Brain-derived neurotrophic factor in TNF-alpha modulation of Ca2+ in human airway smooth muscle. Am J Respir Cell Mol Biol 41:603-11
Sathish, Venkatachalem; Leblebici, Figen; Kip, Sertac N et al. (2008) Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 294:L787-96

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