Mechanical stimulation of airway epithelial cells with a microprobe leads to an increase in cytoplasmic [Ca2+] that appears to be due, in part, to release of Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive stores (Boitano et al., Science 258:292). To investigate whether intracellular IP3 concentration ([IP3]i) increases in response to mechanical stimulation, we grew confluent monolayers from rabbit tracheal mucosal explants on flexible substrates and measured [IP3]i after stretching the substrate. The effect of stretch on [IP3]i was measured in the presence of Li+, an inhibitor of IP3 degradation. In unstretched cells, IP3 measured approximately 5.1 pmol/10(6) cells, from which we estimated [IP3]i to be 1.8 microM. Addition of Li+ had no effect on resting [IP3]i. When the flexible cell support was stretched to increase its surface area by 13%, mean [IP3]i increased about 3-fold with a half-time of approximately 1 s. The increased [IP3]i was maintained in a plateau phase for approximately 8 s and then decayed to near the unstretched level over the next 10 s, despite the sustained application of stretch. A transient stretch (0.5 s) induced a similar rate of increase and peak [IP3]i; however, [IP3]i subsided without a plateau phase. The magnitude of the [IP3]i increase was proportional to stimulus intensity between 0 and 13% increase in substrate surface area. In addition, dissociated airway epithelial cells were exposed to hypotonic solution to induce cell swelling. [IP3]i increased about 4-fold above control levels after 10 s of exposure to hypotonic solution. Basal [IP3]i of dissociated cells in isotonic solution was estimated to be 0.7 microM. These results are consistent with mechanical stimulation leading to phospholipase C synthesis of IP3, which mediates intracellular and intercellular Ca2+ signaling.