The effect of reduced red blood cell (RBC) deformability on microvessel recruitment attendant to a reduction in tissue PO2 was studied in rat cremaster muscle using indicator-dilution techniques. Transit times (TT) of fluorescently labeled RBCs (TTRBC) and plasma (TTPl) between functionally paired arterioles and venules were obtained from their dispersion throughout the microvascular network. Changes in PO2 were effected by superfusing the tissue with Ringer solution deoxygenated to different levels. Arteriolar blood flow (Q) was measured with the two-slit technique, and the vascular volume (V) occupied by RBCs and plasma was computed from the product of Q x TT during bolus infusions of rat and less deformable human RBCs to obtain VRBC and fluorescently labeled albumin to obtain VPl. Measurements of TTRBC and TTPl permitted computation of an average flow-weighted tissue (microvascular) hematocrit (HM) relative to systemic values (HS). During infusions of autologous rat RBCs, Q and total V increased threefold in response to hypoxia, whereas normalized RBC TT (TTRBC/TTPl) and normalized tissue hematocrit (HM/HS) did not show a significant trend, indicating an increase in the number of pathways through which the RBCs can traverse the network because of spatial recruitment of capillaries. In contrast, during infusions of human RBCs, TTRBC/TTPl and HM/HS decreased significantly in response to hypoxia. Although Q exhibited an increase similar to that during rat RBC infusions, VRBC exhibited a smaller increase compared with VPl, suggesting that reduced RBC deformability leads to a redistribution of RBCs through larger-diameter pathways within the network and exclusion of these RBCs from pathways normally recruited during hypoxia. Hence, reduced RBC deformability may adversely affect capillary recruitment and physiological mechanisms that ensure adequate delivery of oxygen to tissue.

Acute lung injury (ALI), which is associated with a mortality of 30-40%, is attributable to inflammation that develops rapidly across the lung's vast vascular surface, involving an entire lung or even both lungs. No specific mechanism explains this extensive inflammatory spread, probably because of the lack of approaches for detecting signal conduction in lung capillaries. Here, we addressed this question by applying the photolytic uncaging approach to induce focal increases in Ca 2+ levels in targeted endothelial cells of alveolar capillaries. Uncaging caused Ca 2+ levels to increase not only in the targeted cell, but also in vascular locations up to 150 mm from the target site, indicating that Ca 2+ was conducted from the capillary to adjacent vessels. No such conduction was evident in mouse lungs lacking endothelial connexin 43 (Cx43), or in rat lungs in which we pretreated vessels with peptide inhibitors of Cx43. These findings provide the first direct evidence to our knowledge that interendothelial Ca 2+ conduction occurs in the lung capillary bed and that Cx43-containing gap junctions mediate the conduction. A proinflammatory effect was evident in that induction of increases in Ca 2+ levels in the capillary activated expression of the leukocyte adherence receptor P-selectin in venules. Further, peptide inhibitors of Cx43 completely blocked thrombin-induced microvascular permeability increases. Together, our findings reveal a novel role for Cx43-mediated gap junctions, namely as conduits for the spread of proinflammatory signals in the lung capillary bed. Gap junctional mechanisms require further consideration in the understanding of ALI.

The liquid layer lining the pulmonary alveolar wall critically determines the lung's immune defense against inhaled pathogens, because it provides a liquid milieu in the air-filled alveolus for dispersal of immune cells and defensive surfactant proteins. However, mechanisms underlying formation of the liquid are unknown. We achieved visualization of the alveolar wall liquid (AWL) in situ in mouse lungs by means of optical-sectioning microscopy. Continuous liquid secretion was present in alveoli of wild-type (WT) mice under baseline conditions. This secretion was blocked by inhibitors of the cystic fibrosis transmembrane regulator (CFTR). The secretion was absent in Cftr ؊/؊ mice, and it was blocked when chloride was depleted from the perfusate of WT mice, providing the first evidence that CFTR-dependent chloride secretion causes AWL formation. Injected microparticles demonstrated flow of the AWL. The flow was blocked by CFTR inhibition and was absent in Cftr ؊/؊ mice. We conclude that CFTR-dependent liquid secretion is present in alveoli of the adult mouse. Defective alveolar secretion might impair alveolar immune defense and promote alveolar disease.Keywords: lung; mouse; CFTR; alveolar secretion; optical-sectioning microscopyThe thin liquid layer lining the alveolar wall of the lung constitutes an important component of the systemic defense against inhaled pathogens. The epithelial liquid establishes a liquid phase adjacent to the alveolar epithelium, enabling secreted phospholipids and proteins to maintain alveolar patency (1) and to establish alveolar immune defense (2). However, mechanisms underlying formation of the alveolar wall liquid (AWL) remain unidentified. In fact, the bulk of the evidence favors the view that in the adult lung, active mechanisms in the alveolar wall remove, rather than secrete, alveolar liquid (3).The difficulty is that studies of AWL have employed conventional morphologic approaches that are likely to alter physiologic liquid conditions in the alveolus (4, 5). To some extent, the liquid morphology was preserved by the application of lowtemperature electron microscopy that revealed continuity of the AWL (6). However, understanding of liquid formation and flow in the alveolar wall continues to be hampered by the lack of direct, real-time methods.Here, we address these issues through the first direct visualization of AWL by means of optical-sectioning microscopy of the mouse lung. Liquid secreted in proximal airways might drain (Received in original form September 13, 2006 and in final form January 25, 2007 ) The studies reported here were supported by NIH grants HL36024, HL64896, and HL78645 (J.B.); HL-75503 (K.P.); and HL080878 (C.E.P).Correspondence and requests for reprints should be addressed to Jahar

Elevation of lung capillary pressure causes exocytosis of the leukocyte adhesion receptor P-selectin in endothelial cells (ECs), indicating that lung ECs generate a proinflammatory response to pressure-induced stress. To define underlying mechanisms, we followed the EC signaling sequence leading to P-selectin exocytosis through application of real-time, in situ fluorescence microscopy in lung capillaries. Pressure elevation increased the amplitude of cytosolic Ca2+ oscillations that triggered increases in the amplitude of mitochondrial Ca2+ oscillations and in reactive oxygen species (ROS) production. Responses to blockers of the Ca2+ oscillations and of mitochondrial electron transport indicated that the ROS production was Ca2+ dependent and of mitochondrial origin. A new proinflammatory mechanism was revealed in that pressure-induced exocytosis of P-selectin was inhibited by both antioxidants and mitochondrial inhibitors, indicating that the exocytosis was driven by mitochondrial ROS. In this signaling pathway mitochondria coupled pressure-induced Ca2+ oscillations to the production of ROS that in turn acted as diffusible messengers to activate P-selectin exocytosis. These findings implicate mitochondrial mechanisms in the lung’s proinflammatory response to pressure elevation and identify mitochondrial ROS as critical to P-selectin exocytosis in lung capillary ECs