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ABSTRACT: Muscle wasting in various catabolic conditions is at least in part regulated by glucocorticoids. Increased calcium levels have been reported in atrophying muscle. Mechanisms regulating calcium homeostasis in muscle wasting, in particular the role of glucocorticoids, are poorly understood. Here we tested the hypothesis that glucocorticoids increase intracellular calcium concentrations in skeletal muscle and stimulate store-operated calcium entry (SOCE) and that these effects of glucocorticoids may at least in part be responsible for glucocorticoid-induced protein degradation. Treatment of cultured myotubes with dexamethasone, a frequently used in vitro model of muscle wasting, resulted in increased intracellular calcium concentrations determined by fura-2 AM fluorescence measurements. When SOCE was measured by using calcium "add-back" to muscle cells after depletion of intracellular calcium stores, results showed that SOCE was increased 15-25% by dexamethasone and that this response to dexamethasone was inhibited by the store-operated calcium channel blocker BTP2. Dexamethasone treatment stimulated the activity of calcium-independent phospholipase A(2) (iPLA(2)), and dexamethasone-induced increase in SOCE was reduced by the iPLA(2) inhibitor bromoenol lactone (BEL). In additional experiments, treatment of myotubes with the store-operated calcium channel inhibitor gadolinium ion or BEL reduced dexamethasone-induced increase in protein degradation. Taken together, the results suggest that glucocorticoids increase calcium concentrations in myocytes and stimulate iPLA(2)-dependent SOCE and that glucocorticoid-induced muscle protein degradation may at least in part be regulated by increased iPLA(2) activity, SOCE, and cellular calcium levels.
AJP Cell Physiology 05/2010; 298(5):C1127-39. · 3.54 Impact Factor
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Yu Chen,
Yongli Yao,
Yuka Sumi,
Andrew Li,
Uyen Kim To,
Abdallah Elkhal,
Yoshiaki Inoue,
Tobias Woehrle,
Qin Zhang, Carl Hauser,
Wolfgang G Junger
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ABSTRACT: Efficient activation of neutrophils is a key requirement for effective immune responses. We found that neutrophils released cellular adenosine triphosphate (ATP) in response to exogenous stimuli such as formylated bacterial peptides and inflammatory mediators that activated Fcgamma, interleukin-8, C5a complement, and leukotriene B(4) receptors. Stimulation of the formyl peptide receptor (FPR) led to ATP release through pannexin-1 (panx1) hemichannels, and FPRs colocalized with P2Y2 nucleotide receptors on the cell surface to form a purinergic signaling system that facilitated neutrophil activation. Disruption of this purinergic signaling system by inhibiting or silencing panx1 hemichannels or P2Y2 receptors blocked neutrophil activation and impaired innate host responses to bacterial infection. Thus, purinergic signaling is a fundamental mechanism required for neutrophil activation and immune defense.
Science Signaling 01/2010; 3(125):ra45. · 7.50 Impact Factor
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ABSTRACT: Bacterial DNA (bDNA) can activate an innate-immune stimulatory "danger" response via toll-like receptor 9 (TLR9). Mitochondrial DNA (mtDNA) is unique among endogenous molecules in that mitochondria evolved from prokaryotic ancestors. Thus, mtDNA retains molecular motifs similar to bDNA. It is unknown, however, whether mtDNA is released by shock or is capable of eliciting immune responses like bDNA. We hypothesized shock-injured tissues might release mtDNA and that mtDNA might act as a danger-associated molecular pattern (or "alarmin") that can activate neutrophils (PMNs) and contribute to systemic inflammatory response syndrome. Standardized trauma/hemorrhagic shock caused circulation of mtDNA as well as nuclear DNA. Human PMNs were incubated in vitro with purified mtDNA or nuclear DNA, with or without pretreatment by chloroquine (an inhibitor of endosomal receptors like TLR9). Neutrophil activation was assessed as matrix metalloproteinase (MMP) 8 and MMP-9 release as well as p38 and p44/42 mitogen-activated protein kinase (MAPK) phosphorylation. Mitochondrial DNA induced PMN MMP-8/MMP-9 release and p38 phosphorylation but did not activate p44/42. Responses were inhibited by chloroquine. Nuclear DNA did not induce PMN activation. Intravenous injection of disrupted mitochondria (mitochondrial debris) into rats induced p38 MAPK activation and IL-6 and TNF-alpha accumulation in the liver. In summary, mtDNA is released into the circulation by shock. Mitochondrial DNA activates PMN p38 MAPK, probably via TLR9, inducing an inflammatory phenotype. Mitochondrial DNA may act as a danger-associated molecular pattern or alarmin after shock, contributing to the initiation of systemic inflammatory response syndrome.
Shock (Augusta, Ga.) 12/2009; 34(1):55-9. · 2.87 Impact Factor
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ABSTRACT: Inflammation and microvascular thrombosis are interrelated causes of acute lung injury in the systemic inflammatory response syndrome. Neutrophils (polymorphonuclear neutrophil [PMN]) and endothelial cells (EC) activated by systemic inflammatory response syndrome interact to increase pulmonary vascular permeability, but the interactions between PMN and EC are difficult to study. Recently, we reported that sphingosine 1-phosphate is a second messenger eliciting store-operated calcium entry (SOCE) in response to inflammatory agonists in both PMN and EC. Store-operated calcium entry is therefore a target mechanism for the therapeutic modulation of inflammatory PMN-EC interactions. Here, we isolated, modeled, and studied the effects of pharmacologic SOCE inhibition using real-time systems to monitor EC permeability after exposure to activated PMN. We created systems to continuously assess permeability of human pulmonary artery endothelial cells and human microvascular endothelial cells from lung. Endothelial cells show increased permeability after challenge by activated PMN. Such permeability increases can be attenuated by exposure of the cocultures to sphingosine kinase (SK) inhibitors (SKI-2, N,N-dimethylsphingosine [DMS]) or Ca2+ entry inhibitors (Gd3+, MRS-1845). Human microvascular endothelial cells from lung pretreated with SKI-2 or DMS showed decreased permeability when later exposed to activated PMN. Likewise, when PMNs were activated with thapsigargin (TG) in the presence of SKI-2, DMS, Gd, or MRS-1845, their ability to cause EC permeability subsequently was reduced. SKI-2 also inhibited the activation of human pulmonary artery ECs by thrombin. These studies will provide a firm mechanistic foundation for understanding how systemic SOCE inhibition may be used to prevent acute lung injury in vivo.
Shock (Augusta, Ga.) 10/2009; 33(4):381-6. · 2.87 Impact Factor
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ABSTRACT: Trauma and hemorrhagic shock cause systemic inflammatory response syndrome. Neutrophils (polymorphonuclear leukocytes [PMN]) and other cells involved in acute lung injury are activated by Ca entry. Thus, inhibiting Ca entry might attenuate post-traumatic lung injury. Inhibiting voltage-operated (L-type) Ca channels during shock would cause cardiovascular collapse, but PMN are "nonexcitable" cells, lack L-type channels, and mobilize Ca via nonspecific channels. We previously showed that PMN Ca entry requires sphingosine 1-phosphate synthesis by sphingosine kinase and that both sphingosine kinase inhibition and blockade of nonspecific channels attenuate acute lung injury when begun before shock. Pretreatment for clinical injuries, however, is impractical. Therefore, we now studied whether Ca entry inhibition that begun during resuscitation from trauma and hemorrhagic shock could attenuate systemic inflammatory response syndrome and lung injury without causing hemodynamic compromise. Male Sprague-Dawley rats underwent laparotomy and fixed-pressure shock (mean arterial pressure, 35 +/- 5 mmHg; 90 min). Sphingosine kinase inhibition or nonspecific Ca channel inhibition was begun after resuscitation with 10% of shed blood. We then studied in vivo PMN activation and associated lung injury in the presence or absence of Ca entry inhibition. Neither treatment worsened shock. Each treatment decreased CD11b expression, respiratory burst, PMN P38 MAP-kinase phosphorylation, PMN sequestration, and lung capillary leak in vivo. The similar results seen with two different forms of inhibition strengthen the conclusion that the biological effects seen were specific for calcium entry inhibition. Ca entry inhibition is a candidate therapy for management of lung injury after shock.
Shock 09/2007; Publish Ahead of Print. · 2.85 Impact Factor
