Abstract
Lung cell injury and repair is a hallmark of the acute respiratory distress syndrome (ARDS). Lung protective mechanical ventilation strategies in these patients may lead to hypercapnia (HC). Although HC has been explored in the clinical context of ARDS, its effect upon alveolar epithelial cell (AEC) wounding and repair remains poorly understood. We have previously reported that HC alters the likelihood of AEC repair by a pH-sensitive but otherwise unknown mechanism. Adenylate cyclase (AC) is an attractive candidate as a putative AEC CO2 sensor and effector as it is bicarbonate sensitive and controls key mediators of AEC repair. The effect of HC on AC activity and plasma membrane (PM) wound repair was measured in AEC type 1 exposed to normocapnia (NC, 40 Torr) or HC (80 Torr), ± tromethamine (THAM) or sodium bicarbonate (HCO3) ± AC probes in a micropuncture model of AEC injury relevant to ARDS. Intracellular pH and AC activity were measured and correlated with repair. HC decreased intracellular pH 0.56, cAMP by 37%, and absolute PM repair rate by 26%. Buffering or pharmacologic manipulation of AC reduced or reversed the effects of HC on AC activity (THAM 103%, HCO3 113% of NC cAMP, ns; Forskolin 168%, p < 0.05) and PM repair (THAM 87%, HCO3 108% of NC likelihood to repair, ns; Forskolin 160%, p < 0.01). These findings suggest AC to be a putative AEC CO2 sensor and modulator of AEC repair, and may have implications for future pharmacologic targeting of downstream messengers of the AC-cAMP axis in experimental models of ARDS.
Highlights
Previous investigations from our laboratory[14,15] have suggested a deleterious, pH-mediated effect of HC on the repair of wounded alveolar epithelial cells (AEC)
In the current study we explore the effect of pH on specific candidate signaling mediators of the HC regulation of AEC plasma membrane (PM) repair, and in so doing hope to identify potential therapeutic targets for future testing in experimental models of acute respiratory distress syndrome (ARDS)/VILI
The use of rodents for cell isolation was previously approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC), as well as the Mayo Clinic Institutional Review Board (IRB)
Summary
Previous investigations from our laboratory[14,15] have suggested a deleterious, pH-mediated effect of HC on the repair of wounded alveolar epithelial cells (AEC). In the cytosolic compartment cAMP may activate protein kinase A (PKA), exchange proteins activated by cAMP 1 and 2 (EPAC)[19], and the cellular transcription factor cAMP response element-binding (CREB) which, through actions on GTP binding proteins (Rabs)[20] and SNAP protein and SNARE complexing[21], modulate endomembrane lipid vesicle production, trafficking, and PM docking[22] integral to successful PM repair. Such processes are Ca2+ dependent, and PKA is well established to alter intracellular calcium through direct actions on Ca2+ channels and phosphoinositide-mediated release from intracellular stores. We propose AC to be both a putative CO2 sensor and master regulator of repair in AEC, and hypothesize that HC alters AEC PM repair through CO2-derived HCO3-dependent changes in AC-cAMP axis and downstream second messenger signaling (Fig. 1)
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