Hydrogen sulphide (H 2 S), which is produced endogenously from L-cysteine in mammalian tissues, has been suggested to function as a neuromodulator in the brain. However, the role of H 2 S in microglial cells is unclear. In this study, the effect of exogenous and endogenous H 2 S on intracellular calcium homeostasis was investigated in primary cultured microglial cells. Sodium hydrosulphide (NaHS), a H 2 S donor, caused a concentration-dependent (0.1-0.5 mM) increase in intracellular calcium concentration ([Ca 21 ] i ). This effect was significantly attenuated in the presence of a calcium-free extracellular solution, Gd 31 (100 lM), a nonselective Ca 21 channel blocker, or thapsigargin (2 lM), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca 21 -ATPase. These observations suggest that the increase in [Ca 21 ] i in response to H 2 S involves both calcium influx across the plasma membrane and calcium release from intracellular stores. The H 2 S-induced calcium elevation is partly attenuated by H-89, a selective cAMP-dependent protein kinase (PKA) inhibitor, but not by U73122, a phospholipase C (PLC) inhibitor, and chelerythrine, a selective protein kinase C (PKC) inhibitor, suggesting the involvement of cAMP/PKA, but not PLC/PKC/phosphoinositol-3,4,5-inositol (IP 3 ) pathway. Using RT-PCR, only cystathionine g-lyase (CSE), a H 2 S producing enzyme, was detected in primary cultures of microglia. Lowering endogenous H 2 S level with, D,L-propargylglycine and b-cyano-L-alanine, two CSE inhibitors, significantly decreased [Ca 21 ] i , suggesting that endogenous H 2 S may have a positive tonic influence on [Ca 21 ] i homeostasis. These findings support the possibility that H 2 S may serve as a neuromodulator to facilitate signaling between neurons and microglial cells.

Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS), a refractory lung disease with an unacceptable high mortality rate. Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The specific mechanisms involved in VILI-induced pulmonary capillary leakage, a key pathologic feature of VILI are still far from resolved. The mechanoreceptor, transient receptor potential cation channel subfamily V member 4, TRPV4 plays a key role in the development of VILI through unresolved mechanism. Endothelial nitric oxide synthase (eNOS) uncoupling plays an important role in sepsis-mediated ARDS so in this study we investigated whether there is a role for eNOS uncoupling in the barrier disruption associated with TRPV4 activation during VILI. Our data indicate that the TRPV4 agonist, 4α-Phorbol 12,13-didecanoate (4αPDD) induces pulmonary arterial endothelial cell (EC) barrier disruption through the disruption of mitochondrial bioenergetics. Mechanistically, this occurs via the mitochondrial redistribution of uncoupled eNOS secondary to a PKC-dependent phosphorylation of eNOS at Threonine 495 (T495). A specific decoy peptide to prevent T495 phosphorylation reduced eNOS uncoupling and mitochondrial redistribution and preserved PAEC barrier function under 4αPDD challenge. Further, our eNOS decoy peptide was able to preserve lung vascular integrity in a mouse model of VILI. Thus, we have revealed a functional link between TRPV4 activation, PKC-dependent eNOS phosphorylation at T495, and EC barrier permeability. Reducing pT495-eNOS could be a new therapeutic approach for the prevention of VILI.

Acute lung injury (ALI) is a devastating clinical syndrome with no effective therapies. Inflammasome activation has been reported to play a critical role in the initiation and progression of ALI. The molecular mechanisms involved in regulating the activation of inflammasome in ALI remains unresolved, although increases in mitochondrial derived reactive oxygen species (mito-ROS) are involved. Our previous work has shown that the mitochondrial redistribution of uncoupled eNOS impairs mitochondrial bioenergetics and increases mito-ROS generation. Thus, the focus of our study was to determine if lipopolysaccharide (LPS)-mediated inflammasome activation involves the mitochondrial redistribution of uncoupled eNOS. Our data show that the increase in mito-ROS involved in LPS-mediated inflammasome activation is associated with the disruption of mitochondrial bioenergetics in human lung microvascular endothelial cells (HLMVEC) and the mitochondrial redistribution of eNOS. These effects are dependent on RhoA-ROCK signaling and are mediated via increased phosphorylation of eNOS at Threonine (T)-495. A derivative of the mitochondrial targeted Szeto‐Schiller peptide (SSP) attached to the antioxidant Tiron (T-SSP), significantly attenuated LPS-mediated mito-ROS generation and inflammasome activation in HLMVEC. Further, T-SSP attenuated mitochondrial superoxide production in a mouse model of sepsis induced ALI. This in turn significantly reduced the inflammatory response and attenuated lung injury. Thus, our findings show that the mitochondrial redistribution of uncoupled eNOS is intimately involved in the activation of the inflammatory response in ALI and implicate attenuating mito-ROS as a therapeutic strategy in humans.

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