Development of PGP 9.5‐ and calcitonin gene‐related peptide‐like immunoreactivity in organ cultured fetal rat lungs
Authors: Sorokin Sergei P. , Ebina Masahito , Hoyt Richard F. 
Published: February 2005

Knowing that small‐granule endocrine cells develop in organ cultured fetal lungs, we investigated whether the cells produce regulatory peptides in vitro, and if sufficient amounts appear to permit using the cultures as an experimental system for physiological study of secretory mechanisms. The paired lungs from 14‐day and 15‐day fetal rats were organ cultured for 1–8 days and examined daily for development of immunoreactivity against marker proteins and regulatory peptides associated with small‐granule endocrine cells and nerves. They proved reactive against protein gene product 9.5 (PGP) and calcitonin‐gene related peptide (CGRP) but not against calcitonin or neurofilament protein 200 K, although positive controls were obtained for these substances in lungs from postnatal animals. Initially PGP‐like immunoreactivity is associated with cell bodies and processes of neuroblasts which run medial to the bronchial axis on day 14 and are increasingly prevalent on day 15. In 15‐day explants PGP becomes detectable after a day in vitro in rare “clear cell” precursors of small‐granule cells located in the epithelium lining proximate parts of the lungs, although in 14‐day explants comparable reactivity is not seen until the third day (14 + 3 days). In culture PGP‐positive neuroblasts increase in number, and nerve processes gradually extend down the airway to encircle the sleeve of smooth muscle that develops as the bronchial tree expands. Concurrently, the initially small clusters of small‐granule cells increase in size, and new ones appear in the airway lining. By 15 + 5 days they extend to the boundary between a taller, more proximal epithelium and a glycogen‐rich cuboidal layer that lines one or two most‐distal generations of branches. Thereafter, the trachea and central, cartilage‐bound segments of the primary bronchi mainly contain solitary endocrine cells and the more peripheral lung a mixture of single cells and clusters, much as in near‐term lungs in vivo. At this stage PGP‐positive nerves extend as far as the entrances of the terminal sacs, and most are distributed to the airway muscle plexus. Exceptionally, they may innervate a small‐granule cell cluster, converting it into a neuroepithelial body. CGRP‐like immunoreactivity initially appears in small‐granule cells of 15 + 2‐day cultures but does not develop in ganglion cells or nerves. It localizes to endocrine cells at all conducting airway levels, increasing in staining intensity and accounting for most if not all of the PGP‐positive population between 15 + 4 − 15 + 8 days. Similar localizations with anti‐PGP and anti‐CGRP were obtained in lungs of 16‐day explants grown for 7 days. CGRP is present in sensory nerves of intact rat lungs. Its absence from nerves in vitro confirms that the cultures are deafferented and the PGP‐positive nerves are mainly parasympathetic efferents. Small‐granule cells and ganglion cells become concentrated in lung organ cultures because elongation of airway branches is severely curtailed, and this affords an advantage for functional studies on these populations. In addition, any experimentally induced fluctuation in release of CGRP to the medium can be ascribed to effects on the endocrine cells and not the sensory nerves. © 1993 Wiley‐Liss, Inc.