The expression of bombesin-like peptides (BLPs) by pulmonary neuroendocrine cells is transiently up-regulated during lung development. A functional role for BLPs is supported by their ability to stimulate lung growth and maturation both in vitro and in vivo during the late stages of lung development. In addition, the cell membrane-associated enzyme CD10/neutral endopeptidase 24.11 (CD10/NEP), which inactivates BLPs and other regulatory peptides, is also expressed by developing lungs and modulates the stimulatory effects of BLPs on lung growth and maturation. We hypothesized that, in addition to expressing BLPs and CD10/NEP, embryonic lungs must express BLP receptors, and that BLPs may also regulate processes that occur during early lung development such as branching morphogenesis. Using reverse transcriptase-polymerase chain reaction and oligonucleotide primers designed for amplifying a BLP receptor originally isolated from Swiss 3T3 mouse fibroblasts, we found that embryonic mouse lungs express a similar BLP receptor mRNA during the pseudoglandular stage of lung development when branching morphogenesis takes place. Subsequently, we evaluated the effects of ligands for this BLP receptor using embryonic mouse lungs in an in vitro model of lung branching morphogenesis. We found that, in comparison with control lungs, treatment with bombesin (1 to 100 nM) resulted in a modest increase in clefts or branching points. In contrast, embryonic mouse lungs treated with the BLP analog [Leu13-psi(CH2NH)-Leu14] bombesin (1 muM), which also binds to this BLP receptor but has predominantly antagonistic effects, demonstrated fewer branching points. When compared with the embryonic lungs treated with [Leu13-psi(CH2NH)Leu14]bombesin, however, embryonic mouse lungs treated with bombesin demonstrated a significant increase in branching points (P < 0.05), and when the treatment with exogenous bombesin (1 nM) was combined with phosphoramidon (1 muM), an inhibitor of CD10/NEP, there was further augmentation of the bombesin-induced branching (P < 0.05). Furthermore, phosphoramidon treatment without exogenous bombesin also increased branching (P < 0.05), and this effect was inhibited by [Leu13-psi(CH2NH)Leu14]bombesin. Of note, the effects of BLPs on branching did not correlate with cell proliferation as determined by changes in total lung DNA content (r = 0.03). Thus, we conclude that embryonic mouse lungs express at least one type of BLP receptor during branching morphogenesis, and that branching morphogenesis is partially regulated by both endogenous and exogenous BLP receptor ligands, as well as by phosphoramidon-sensitive peptidases, possibly mouse CD10/NEP. In addition, our data suggest that such regulation of early branching morphogenesis may involve mechanisms responsible for cleft formation but perhaps unrelated to cell proliferation.
