responses of airway smooth muscle (ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in Ca 2ϩ homeostasis. Caveolin-1 can modulate localization and activity of signaling proteins, including trimeric G proteins, via a scaffolding domain. We investigated the role of caveolae in contraction and intracellular Ca 2ϩ ([Ca 2ϩ ]i) mobilization of ASM induced by the physiological muscarinic receptor agonist, acetylcholine (ACh). Human and canine ASM tissues and cells predominantly express caveolin-1. Muscarinic M3 receptors (M3R) and G␣q/11 cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with ␤-cyclodextrin in canine tracheal strips reduced sensitivity but not maximum isometric force induced by ACh. In fura-2-loaded canine and human ASM cells, exposure to methyl-␤-cyclodextrin (m␤CD) reduced sensitivity but not maximum [Ca 2ϩ ]i induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that m␤CD disrupted the colocalization of caveolae-1 and M 3R, but [N-methyl-3 H]scopolamine receptor-binding assay revealed no effect on muscarinic receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [Ca 2ϩ ]i flux, we disrupted its binding to signaling proteins using either a cell-permeable caveolin-1 scaffolding domain peptide mimetic or by small interfering RNA knockdown. Similar to the effects of m␤CD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca 2ϩ ]i mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca 2ϩ ]i mobilization leading to ASM contraction induced by submaximal concentrations of ACh.

Rationale: Point-of-care ultrasound (POCUS) is used to evaluate pulmonary edema in adults with acute respiratory distress syndrome (ARDS). Its use has not been validated in neonatal models. Objectives: We compared an in vivo lung ultrasound score against clinical and histological markers of acute lung injury, in a neonatal animal model, hypothesizing that POCUS would sensitively diagnose early acute lung injury in neonates and discern its severity. Methods: Fifteen anesthetized, ventilated 3-day-old neonatal piglets were divided into controls, moderate lung injury, or severe lung injury by graded treatment with oleic acid. Degree of lung injury was quantified at baseline, immediately after oleic acid administration, and 1 hour after the evolution of acute lung injury, by blood gases, ventilation parameters and calculated oxygenation deficit; hemodynamic indices by echocardiography, and lung ultrasound obtained in an 8-region grid of anterior and posterior zones, semi-quantitatively analyzed by a blinded observer. Lungs were inflation-fixed postmortem at last mean airway pressure, for histological assessment. Results: Acute lung injury manifested in oleic acid-treated groups as dosedependent capillary leak causing intravascular depletion and cardiac failure, hypoxemia with increasing intrapulmonary shunt fraction, decreased lung compliance, and resistance. Ultrasound scores of anterior regions distinguished moderate from severe injury; scores in posterior regions reached maximum values immediately after lung injury. POCUS score correlated with calculated intrapulmonary shunt fraction (R 2 = .65) and with histological injury score (R 2 = .61), P < .01. Conclusion: We conclude that POCUS may be valuable in neonates for early quantification of acute lung injury or ARDS; and that nondependent ultrasound regions clearly distinguish severity of pulmonary edema.

PPHN, caused by perinatal hypoxia or inflammation, is characterized by an increased thromboxane-prostacyclin ratio and pulmonary vasoconstriction. We examined effects of hypoxia on myocyte thromboxane responsiveness. Myocytes from 3rd-6th generation pulmonary arteries of newborn piglets were grown to confluence and synchronized in contractile phenotype by serum deprivation. On the final 3 days of culture, myocytes were exposed to 10% O2 for 3 days; control myocytes from normoxic piglets were cultured in 21% O2. PPHN was induced in newborn piglets by 3-day hypoxic exposure (Fi(O2) 0.10); pulmonary arterial myocytes from these animals were maintained in normoxia. Ca2+ mobilization to thromboxane mimetic U-46619 and ATP was quantified using fura-2 AM. Three-day hypoxic exposure in vitro results in increased basal [Ca2+]i, faster and heightened peak Ca2+ response, and decreased U-46619 EC50. These functional changes persist in myocytes exposed to hypoxia in vivo but cultured in 21% O2. Blockade of Ca2+ entry and store refilling do not alter peak U-46619 Ca2+ responses in hypoxic or normoxic myocytes. Blockade of ryanodine-sensitive or IP3-gated intracellular Ca2+ channels inhibits hypoxic augmentation of peak U-46619 response. Ca2+ response to ryanodine alone is undetectable; ATP-induced Ca2+ mobilization is unaltered by hypoxia, suggesting no independent increase in ryanodine-sensitive or IP3-linked intracellular Ca2+ pool mobilization. We conclude hypoxia has a priming effect on neonatal pulmonary arterial myocytes, resulting in increased resting Ca2+, thromboxane hypersensitivity, and hyperreactivity. We postulate that hypoxia increases agonist-induced TP-R-linked IP3 pathway activation. Myocyte thromboxane hyperresponsiveness persists in culture after removal from the initiating hypoxic stimulus, suggesting altered gene expression.

Background: Hypoxia and reactive oxygen species (ROS) including H 2 O 2 play major roles in triggering and progression of pulmonary vascular remodeling in persistent pulmonary hypertension. Catalase (CAT), the major endogenous enzyme scavenging H 2 O 2 , is regulated in a tissue-and context-specific manner. Objective: To investigate mechanisms by which hypoxia and H 2 O 2 regulate catalase expression, and the role of AMPK-FoxO pathway, in neonatal porcine pulmonary artery smooth muscle (PASMC). Design/Methods: PASMC were grown in hypoxia (10% O 2 ) or normoxia (21% O 2 ) for 72 hr. We measured catalase activity and lipid peroxidation; CAT, FoxO1, and FoxO3a expression by qPCR; protein contents of CAT, FoxOs, p-AMPK, p-AKT, p-JNK, p-ERK1/2 in whole lysates, and FoxOs in nuclear extracts, by immunoblot; and FoxO-1 nuclear localization by immunocytochemistry, quantified by laser scanning cytometry. Results: Hypoxia upregulated CAT transcription, content and activity, by increasing CAT transcription factors FoxO1 and FoxO3a mRNA, and promoting nuclear translocation of FoxO1. However, lipid peroxidation increased in hypoxic PASMC. Among candidate FoxO regulatory kinases, hypoxia activated AMPK, and decreased p-Akt and ERK1/2. AMPK activation increased FoxO1 (total and nuclear) and CAT, while AMPK inhibition inhibited FoxO1 and CAT, but not FoxO3a. Exogenous H 2 O 2 decreased p-AMPK and increased p-AKT in hypoxic PASMC. This decreased active FoxO1, and reduced mRNA and protein content of CAT. Hypoxic induction of CAT, AKT inhibition (LY294002), or addition of PEG-catalase partly ameliorated the H 2 O 2 -mediated loss of nuclear FoxO1. Conclusions: Hypoxia induces catalase expression, though this adaptation is insufficient to protect PASMC from hypoxia-induced lipid peroxidation. This occurs via hypoxic activation of AMPK, which promotes nuclear FoxO1 and thus catalase expression. Exogenous ROS may downregulate cellular antioxidant defenses; H 2 O 2 activates survival factor Akt, decreasing nuclear FoxO1 and thus catalase.