Secretion of Lung Fluid by the Developing Fetal Rat Alveolar Epithelium in Organ Culture

We studied lung expiants in submersion organ culture to examine the role of the developing fetal alveolar epithelium in the production of lung fluid. Fourteen-day-gestation fetal rat lungs were grown in a collagen gel matrix supplemented with F-12 media and 10% fetal calf serum. In this model, the lung continues to grow, secrete fluid, and become progressively cystic in morphology. There is gradual thinning of the distal epithelial layer, which is lined by alveolar type II cells and their precursors. After 6 to 8 days in culture, we impaled the cyst walls with a microelectrode and continuously recorded the transepithelial potential (ψt). Stable, baseline transepithelial potentials of −1.1 to −6.2 mV (mean ± SEM = −3.3 ± 0.11 mV, lumen negative, n = 34) were measured in bicarbonate-buffered Ringer's solution, suggesting active electrolyte transport. When bumetanide, an inhibitor of chloride secretion in other systems, was added to the bathing solution, ψt decreased from a baseline of −3.5 ± 0.07 mV (mean ± SEM) to a value of −2.2 ± 0.07 mV, suggesting chloride transport contributes to the voltage (n = 18, P < 0.0005). Isoproterenol hyperpolarized ψt from a baseline of −4.3 ± 1.0 mV to −6.5 ± 1.0 mV (n = 7, P < 0.005). 8-(4-Chlorophenylthio) adenosine 3′:5′cyclic monophosphate (CPT-cAMP) plus isobutylmethylxanthine (IBMX) similarly hyperpolarized ψt from a baseline of −4.6 ± 0.4 mV to −7.3 ± 0.7 mV (n = 11, P < 0.005). Addition of bumetanide after stimulation with isoproterenol or CPT-cAMP/IBMX depolarized ψt. Bath chloride was replaced with gluconate to further test the hypothesis that the change in ψt in response to isoproterenol was due to chloride transport. In Cl− free solution, baseline ψt decreased and there was no significant change in ψt in response to isoproterenol. These findings suggest: (1) the developing fetal alveolar epithelium actively produces lung fluid by a process that is chloride dependent, (2) chloride secretion can be stimulated by a β-adrenergic agonist, and (3) chloride secretion is mediated in part through cAMP-dependent pathways.