Fetal Calcium Regulates Branching Morphogenesis in the Developing Human and Mouse Lung: Involvement of Voltage-Gated Calcium Channels

Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9-17 of human gestation, embryonic days (E)11.5-16.5 in mouse) in a hypercalcaemic environment (similar to 1.7 in the fetus vs. similar to 1.1-1.3 mM for an adult). Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca2+ channels (VGCC), inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (Ca(V)1.2 and Ca(V)1.3), P/Q type (Ca(V)2.1), N-type (Ca(V)2.2), R-type (Ca(V)2.3), and T-type (Ca(V)3.2 and Ca(V)3.3) VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Ca(v)1.2 and Ca(v)1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Ca(v)2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Ca(v)2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC(2)(3), demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to match growth and distension within the developing lung.