IMPORTANCE Dexmedetomidine provides sedation for patients undergoing ventilation; however, its effects on mortality and ventilator-free days have not been well studied among patients with sepsis.OBJECTIVES To examine whether a sedation strategy with dexmedetomidine can improve clinical outcomes in patients with sepsis undergoing ventilation. INTERVENTIONS Patients were randomized to receive either sedation with dexmedetomidine (n = 100) or sedation without dexmedetomidine (control group; n = 101). Other agents used in both groups were fentanyl, propofol, and midazolam. MAIN OUTCOMES AND MEASURESThe co-primary outcomes were mortality and ventilator-free days (over a 28-day duration). Sequential Organ Failure Assessment score (days 1, 2, 4, 6, 8), sedation control, occurrence of delirium and coma, intensive care unit stay duration, renal function, inflammation, and nutrition state were assessed as secondary outcomes. RESULTSOf the 203 screened patients, 201 were randomized. The mean age was 69 years (SD, 14 years); 63% were male. Mortality at 28 days was not significantly different in the dexmedetomidine group vs the control group (19 patients [22.8%] vs 28 patients [30.8%]; hazard ratio, 0.69; 95% CI, 0.38-1.22; P = .20). Ventilator-free days over 28 days were not significantly different between groups (dexmedetomidine group: median, 20 [interquartile range, 5-24] days; control group: median, 18 [interquartile range, 0.5-23] days; P = .20). The dexmedetomidine group had a significantly higher rate of well-controlled sedation during mechanical ventilation (range, 17%-58% vs 20%-39%; P = .01); other outcomes were not significantly different between groups. Adverse events occurred in 8 (8%) and 3 (3%) patients in the dexmedetomidine and control groups, respectively. CONCLUSIONS AND RELEVANCE Among patients requiring mechanical ventilation, the use of dexmedetomidine compared with no dexmedetomidine did not result in statistically significant improvement in mortality or ventilator-free days. However, the study may have been underpowered for mortality, and additional research may be needed to evaluate this further.

The ubiquitin E3 protein ligase Nedd4-2 is a physiological regulator of the epithelial sodium channel ENaC, which is essential for transepithelial Na ؉ transport and is linked to Liddle's syndrome, an autosomal dominant disorder of human salt-sensitive hypertension. Nedd4-2 function is negatively regulated by phosphorylation via a serum-and glucocorticoid-inducible protein kinase (Sgk1), which serves as a mechanism to inhibit the ubiquitination-dependent degradation of ENaC. We report here that 14-3-3 proteins participate in this regulatory process through a direct interaction with a phosphorylated form of human Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2). The interaction is dependent on Sgk1-catalyzed phosphorylation of hNedd4-2 at Ser-468. We found that this interaction preserved the activity of the Sgk1-stimulated ENaC-dependent Na ؉ current while disrupting the interaction decreased ENaC density on the Xenopus laevis oocytes surface possibly by enhancing Nedd4-2-mediated ubiquitination that leads to ENaC degradation. Our findings suggest that 14-3-3 proteins modulate the cell surface density of ENaC cooperatively with Sgk1 kinase by maintaining hNedd4-2 in an inactive phosphorylated state.

Bitter taste perception is a conserved chemical sense against the ingestion of poisonous substances in mammals. A multigene family of G-protein-coupled receptors, T2R (so-called TAS2R or TRB) receptors and a G-protein alpha subunit (Galpha), gustducin, are believed to be key molecules for its perception, but little is known about the molecular basis for its interaction. Here, we use a heterologous expression system to determine a specific domain of gustducin necessary for T2R coupling. Two chimeric Galpha16 proteins harboring 37 and 44 gustducin-specific sequences at their C termini (G16/gust37 and G16/gust44) responded to different T2R receptors with known ligands, but G16/gust 23, G16/gust11, and G16/gust5 did not. The former two chimeras contained a predicted beta6 sheet, an alpha5 helix, and an extreme C terminus of gustducin, and all the domains were indispensable to the expression of T2R activity. We also expressed G16 protein chimeras with the corresponding domain from other Galpha(i) proteins, cone-transducin (Galpha(t2)), Galpha(i2), and Galpha(z) (G16/t2, G16/i2, and G16/z). As a result, G16/t2 and G16/i2 produced specific responses of T2Rs, but G16/z did not. Because Galpha(t2) and Galpha(i2) are expressed in the taste receptor cells, these G-protein alpha(i) subunits may also be involved in bitter taste perception via T2R receptors. The present Galpha16-based chimeras could be useful tools to analyze the functions of many orphan G-protein-coupled taste receptors.

Peak serum concentration of creatine kinase as well as the number of injured extremities serve to estimate the severity of crush syndrome.

Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) and mitochondria play a critical role in growth factor-induced switch from a quiescent to an angiogenic phenotype in endothelial cells (ECs). However, how highly diffusible ROS produced from different sources can coordinate to stimulate VEGF signaling and drive the angiogenic process remains unknown. Using the cytosol- and mitochondria-targeted redox-sensitive RoGFP biosensors with real-time imaging, here we show that VEGF stimulation in human ECs rapidly increases cytosolic RoGFP oxidation within 1 min, followed by mitochondrial RoGFP oxidation within 5 min, which continues at least for 60 min. Silencing of Nox4 or Nox2 or overexpression of mitochondria-targeted catalase significantly inhibits VEGF-induced tyrosine phosphorylation of VEGF receptor type 2 (VEGFR2-pY), EC migration and proliferation at the similar extent. Exogenous hydrogen peroxide (HO) or overexpression of Nox4, which produces HO, increases mitochondrial ROS (mtROS), which is prevented by Nox2 siRNA, suggesting that Nox2 senses Nox4-derived HO to promote mtROS production. Mechanistically, HO increases S36 phosphorylation of p66Shc, a key mtROS regulator, which is inhibited by siNox2, but not by siNox4. Moreover, Nox2 or Nox4 knockdown or overexpression of S36 phosphorylation-defective mutant p66Shc(S36A) inhibits VEGF-induced mtROS, VEGFR2-pY, EC migration, and proliferation. In summary, Nox4-derived HO in part activates Nox2 to increase mtROS via pSer36-p66Shc, thereby enhancing VEGFR2 signaling and angiogenesis in ECs. This may represent a novel feed-forward mechanism of ROS-induced ROS release orchestrated by the Nox4/Nox2/pSer36-p66Shc/mtROS axis, which drives sustained activation of angiogenesis signaling program.

Electrical events and intracellular calcium concentration ([Ca2+]) imaged using fluo‐3 and laser scanning confocal microscopy were simultaneously monitored in single smooth muscle cells freshly isolated from guinea‐pig vas deferens or urinary bladder. Images obtained every 8 ms, during stepping from ‐60 to 0 or +10 mV for 50 ms under voltage clamp, showed that a rise in [Ca2+] could be detected within 20 ms of depolarization in five to twenty small (< 2 μm diameter) ‘hot spots’, over 95 % of which were located within 1.5 μm of the cell membrane. Depolarization at 30 s intervals activated hot spots at the same places. Cd2+ or verapamil abolished both hot spots and Ca2+‐activated K+ current (IK,Ca). Caffeine almost abolished hot spots and markedly reduced IK,Ca. Cyclopiazonic acid, which raised basal global [Ca2+], decreased the rise in hot spot [Ca2+] and IK,Ca amplitude during depolarization. These results suggest that Ca2+ entry caused Ca2+‐induced Ca2+ release (CICR). Under voltage clamp, hot spot [Ca2+] closely paralleled the rise in IK,Ca and reached a peak within 20 ms of the start of depolarization, but the rise in global [Ca2+] over the whole cell area was much slower. Step depolarization to potentials positive to ‐20 mV caused hot spots to grow in size and coalesce, leading to a rise in global [Ca2+] and contraction. Ca2+ hot spots also occurred during the up‐stroke of an evoked action potential under current clamp. It is concluded that the entry of Ca2+ in the early stages of an action potential evokes CICR from discrete subplasmalemma Ca2+ storage sites and generates hot spots that spread to initiate a contraction. The activation of Ca2+‐dependent K+ channels in the plasmalemma over hot spots initiates IK,Ca and action potential repolarization.

Rationale A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased resting [Ca2+]cyt and enhanced Ca2+ influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH). Objective We examined whether the extracellular Ca2+-sensing receptor (CaSR) is involved in the enhanced Ca2+ influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension. Methods and Results In normal PASMC superfused with Ca2+-free solution, addition of 2.2 mM Ca2+ to the perfusate had little effect on [Ca2+]cyt. In IPAH-PASMC, however, restoration of extracellular Ca2+ induced a significant increase in [Ca2+]cyt. Extracellular application of spermine also markedly raised [Ca2+]cyt in IPAH-PASMC, but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca2+-induced [Ca2+]cyt rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca2+-mediated [Ca2+]cyt increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia. Conclusions The extracellular Ca2+-induced increase in [Ca2+]cyt due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca2+ influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension.

The relationship between Ca2+ sparks spontaneously occurring at rest and local Ca2+ transients elicited by depolarization was analysed using two‐dimensional confocal Ca2+ images of single smooth muscle cells isolated from guinea‐pig vas deferens and urinary bladder. The current activation by these Ca2+ events was also recorded simultaneously under whole‐cell voltage clamp. Spontaneous transient outward currents (STOCs) and Ca2+ sparks were simultaneously detected at ‐40 mV in approximately 50 % of myocytes of either type. Ca2+ sparks and corresponding STOCs occurred repetitively in several discrete sites in the subplasmalemmal area. Large conductance Ca2+‐dependent K+ (BK) channel density in the plasmalemma near the Ca2+ spark sites generating STOCs was calculated to be 21 channels μm−2. When myocytes were depolarized from ‐60 to 0 mV, several local Ca2+ transients were elicited within 20 ms in exactly the same peripheral sites where sparks occurred at rest. The local Ca2+ transients often lasted over 300 ms and spread into other areas. The appearance of local Ca2+ transients occurred synchronously with the activation of Ca2+‐dependent K+ current (IK,Ca). Immunofluorescence staining of the BK channel α‐subunit (BKα) revealed a spot‐like pattern on the plasmalemma, in contrast to the uniform staining of voltage‐dependent Ca2+ channel α1C subunits along the plasmalemma. Ryanodine receptor (RyR) immunostaining also suggested punctate localization predominantly in the periphery. Double staining of BKα and RyRs revealed spot‐like co‐localization on/beneath the plasmalemma. Using pipettes of relatively low resistance, inside‐out patches that included both clustered BK channels at a density of over 20 channels μm−2 and functional Ca2+ storage sites were obtained at a low probability of ≈5 %. The averaged BK channel density was 3‐4 channels μm−2 in both types of myocyte. These results support the idea that a limited number of discrete sarcoplasmic reticulum (SR) fragments in the subplasmalemmal area play key roles in the control of BK channel activity in two ways: (i) by generating Ca2+ sparks at rest to activate STOCs and (ii) by generating Ca2+ transients presumably triggered by sparks during an action potential to activate a large IK,Ca and also induce a contraction. BK channels and RyRs may co‐localize densely at the junctional areas of plasmalemma and SR fragments, where Ca2+ sparks occur to elicit STOCs.