Abstract
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 (∼1.7 in the fetus vs. ∼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 (CaV1.2 and CaV1.3), P/Q type (CaV2.1), N-type (CaV2.2), R-type (CaV2.3), and T-type (CaV3.2 and CaV3.3) 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 Cav1.2 and Cav1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Cav2.3 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 Cav2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC2(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.
Highlights
Efficient gas exchange in the postnatal lung requires optimal formation of the bronchial tree in the fetus [1,2]
We have demonstrated previously that this relative fetal hypercalcemia is an important signal in the pseudoglandular lung, balancing branching morphogenesis with fluid secretion via developmentally regulated expression of the calcium-sensing receptor (CaSR) [12,14]
The existence of L-type Ca2+ channels in the fetal mouse lung has been previously hypothesised by Roman who showed that treatment of pseudoglandular lung rudiments with nifedipine led to the formation of hypoplastic lungs [19]
Summary
Efficient gas exchange in the postnatal lung requires optimal formation of the bronchial tree in the fetus [1,2]. Lung development begins around embryonic day (E)9.5 in mice and week 3–4 post-conception in humans and comprises of five stages [1]. Secretion of lung fluid into the lumen throughout gestation generates the distending pressure for normal growth [4]. Rhythmic peristaltic contractions causing transient and cyclic airway occlusions develop, persisting throughout gestation [1,7,8] and create the mechanical stimulus that propels the fluid secreted into the airway lumen towards the tips of the developing lung [8,9]. While it is well established that these airway smooth muscle waves require the presence of both intracellular calcium ions (Ca2+i) and extracellular calcium ions (Ca2+o), a firm link between generation of airway smooth muscle waves, airway peristalsis and lung development has never been established
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