Of fundamental importance to the optimization of pulmonary gas exchange is the role of the alveolar epithelium in regulation of its micro-environment. In a healthy lung, alveolar efficiency is dependent upon both the degree of hydration and rate of pulmonary surfactant production. Both of these factors are critically regulated by the alveolar epithelial cells. Although much information has been gleaned from isolated cellular studies, little is known about the temporo-spatial relationship of many of the homeostatic processes occurring within an intact epithelium and the impact that intercellular communication has on such processes is currently unknown. Similarly, little information is available on the differential location of key transport proteins within the alveolar epithelium itself. In order to address these controversies we aimed to: (a) to develop a lung slice preparation suitable for physiological study; (b) develop differential live staining of alveolar type I and type II cells within the lung slice; and (c) study Ca²⁺ homeostasis in the identified cells.

Neonatal rats were humanely killed. Lungs were was stabilized by tracheal infusion of 2 % agarose followed by immersion in ice-cold, oxygenated solution and then embedded in 4 % agarose. Lung slices of 200 µm were processed for live immuno- and histochemical determination of cell localization and viability before being placed in a laser scanning confocal microscope perfusion chamber or being cultured for up to 30 h. Living lung slices were imaged following treatment with a combination of the following reagents: (a) 1:4 mVIIIB2 primary antibody, FITC-labelled anti-mouse IgG secondary (Alveolar type I cells); (b) 1 µg ml^-1 Nile Red (Alveolar type II cells); (c) 10 µM Hoechst 33342 (live cells); (d) 5 µM propidium iodide (dead cells). [Ca²⁺]_i was monitored using Fluo-3.

Using this combination of fluorescence histochemistry and immunohistochemistry we have positively identified alveolar type I (VIIIB2) and type II (Nile Red) cells within a living lung slice. Furthermore, we have shown that measurement of dynamic changes of [Ca²⁺]_i in identified cells within an intact alveolar epithelium is feasible with good temporal and spatial resolution; type II cells demonstrate oscillatory behaviour which is often synchronous and is maintained for up to 30 h in culture. This novel technique will provide the experimental framework upon which to interrogate fully the physiological regulation of the alveolar micro-environment in health and disease.

This work was funded by Wellcome Trust, British Heart Foundation and National Institutes of Health.