A novel alveolar Krebs cycle-triggered CO2 sensing mechanism regulates regional pulmonary ventilation.

Pulmonary perfusion disorders provoke atelectasis in order to minimize ventilation/perfusion mismatch. However, the underlying mechanisms remain unknown. Because intraalveolar CO2 concentration ([CO2]iA) declines as a consequence of poor pulmonary perfusion we postulated the existence a novel alveolar CO2-sensing mechanism which adapts the ventilation to perfusion. Real-time fluorescence imaging of rat lungs revealed that low [CO2]iA decreased cytosolic and increased mitochondrial Ca2+ in alveolar epithelial cells (AEC), leading to reduction of surfactant secretion and alveolar ventilation. Mitochondrial inhibition by ruthenium red or rotenone blocked the hypocapnia-induced responses. In cultured Type 2 AEC hypocapnia decreased cytosolic Ca2+ independently from pH and increased the NADH production, the mitochondrial transmembrane potential, and subsequently the mitochondrial Ca2+ uptake. All responses were completely blocked by the gene knockdown of the NADH producing Krebs cycle enzyme isocitrate dehydrogenase. Furthermore, ligature of the pulmonary artery of rabbits decreased alveolar ventilation, surfactant secretion, and lung compliance. Addition of 5% CO2 to the inspiratory gas inhibited all responses. Accordingly, we provide evidence for a novel CO2-sensing mechanism of AEC regulated by the Krebs cycle activity in terms of a negative feedback loop adapting surfactant secretion and thus regional ventilation to pulmonary perfusion

Leukocyte sequestration in pulmonary microvessels and lung injury following systemic complement activation in rabbits

Inflammatory reactions are associated with sequestration of leukocytes in the lung. Complement activation leads to accumulation of leukocytes in alveolar septa and alveoli, to lung edema and hemorrhage. Although in organs other than the lung leukocytes interact with the vascular endothelium only in postcapillary venules, alveolar capillaries are considered to be the site of leukocyte sequestration in the lung. However, pulmonary venules and arterioles have not been investigated systematically after complement activation so far, A closed thoracic window was implanted in anesthetized rabbits; leukocytes and red blood cells were stained, and the movement of these cells was measured in superficial pulmonary arterioles, venules and alveolar capillaries using fluorescence video microscopy before and 30 and 60 min after infusion of cobra venom factor (CVF). Erythrocyte velocity and macrohemodynamic conditions did not change after CVF infusion and were not different from the sham-treated controls. The number of sticking leukocytes increased significantly compared to baseline and control: by 150% in arterioles and in venules and by 740% in alveolar capillaries within 60 min after CVF infusion. The width of alveolar septa in vivo was significantly enlarged after CVF infusion, indicating interstitial pulmonary edema. At the end of the experiments, myeloperoxidase activity was higher in the CVF group, showing leukocyte sequestration in the whole organ. It is concluded that complement activation by CVF induces leukocyte sequestration in lung arterioles, venules and alveolar capillaries and leads to mild lung injury

IDH3 mediates apoptosis of alveolar epithelial cells type 2 due to mitochondrial Ca2+ uptake during hypocapnia

In adult respiratory distress syndrome (ARDS) pulmonary perfusion failure increases physiologic dead-space (VD/VT) correlating with mortality. High VD/VT results in alveolar hypocapnia, which has been demonstrated to cause edema formation, atelectasis, and surfactant depletion, evoked, at least in part, by apoptosis of alveolar epithelial cells (AEC). However, the mechanism underlying the hypocapnia-induced AEC apoptosis is unknown. Here, using fluorescent live-cell imaging of cultured AEC type 2 we could show that in terms of CO2 sensing the tricarboxylic acid cycle enzyme isocitrate dehydrogenase (IDH) 3 seems to be an important player because hypocapnia resulted independently from pH in an elevation of IDH3 activity and subsequently in an increase of NADH, the substrate of the respiratory chain. As a consequence, the mitochondrial transmembrane potential (ΔΨ) rose causing a Ca2+ shift from cytosol into mitochondria, whereas the IDH3 knockdown inhibited these responses. Furthermore, the hypocapnia-induced mitochondrial Ca2+ uptake resulted in reactive oxygen species (ROS) production, and both the mitochondrial Ca2+ uptake and ROS production induced apoptosis. Accordingly, we provide evidence that in AEC type 2 hypocapnia induces elevation of IDH3 activity leading to apoptosis. This finding might give new insight into the pathogenesis of ARDS and may help to develop novel strategies to reduce tissue injury in ARDS

Role of P-selectin in platelet sequestration in pulmonary capillaries during endotoxemia

Background: There is growing evidence that platelets accumulate in the lung and contribute to the pathogenesis of acute lung injury during endotoxemia. The aims of the present study were to localize platelet sequestration in the pulmonary microcirculation and to investigate the role of P-selectin as a molecular mechanism of platelet endothelial cell interaction. Methods: We used in vivo fluorescence microscopy to quantify the kinetics of fluorescently labeled erythrocytes and platelets in alveolar capillary networks in rabbit lungs. Results: Six hours after onset of endotoxin infusion we observed a massive rolling along and firm adherence of platelets to lung capillary endothelial cells whereas under control conditions no platelet sequestration was detected. P-selectin was expressed on the surface of separated platelets which were incubated with endotoxin and in lung tissue. Pretreatment of platelets with fucoidin, a P-selectin antagonist, significantly attenuated the endotoxin-induced platelet rolling and adherence. In contrast, intravenous infusion of fucoidin in endotoxin-treated rabbits did not inhibit platelet sequestration in pulmonary capillaries. Conclusion: We conclude that platelets accumulate in alveolar capillaries following endotoxemia. P-selectin expressed on the surface of platelets seems to play an important role in mediating this platelet-endothelial cell interaction. Copyright (c) 2006 S. Karger AG, Basel

Paracrine purinergic signaling determines lung endothelial nitric oxide production

Although the vascular bed is a major source of nitric oxide (NO) production, factors regulating the production remain unclear. We considered the role played by paracrine signaling. Determinations by fluorescence microscopy in isolated, blood-perfused rat and mouse lungs revealed that a brief lung expansion enhanced cytosolic Ca2+ (Ca2+cyt) oscillations in alveolar epithelial (AEC) and endothelial (EC) cells, and NO production in EC. Furthermore, as assessed by a novel microlavage assay, alveolar ATP production increased. Intra-alveolar microinfusion of the purinergic receptor antagonist, PPADS, and the nucleotide hydrolyzing enzyme, apyrase, each completely blocked the Ca2+cyt and NO responses in EC. Lung expansion induced Ca2+cyt oscillations in mice lacking the P2Y1, but not the P2Y2, purinergic receptors, which were located in the perivascular interstitium basolateral to AEC. Prolonged lung expansion instituted by mechanical ventilation at high tidal volume increased EC expression of nitrotyrosine, indicating development of nitrosative stress in lung microvessels. These findings reveal a novel mechanism in which mechanically induced purinergic signaling couples cross-compartmental Ca2+cyt oscillations to microvascular NO production