Variable stretch pattern enhances surfactant secretion in alveolar type II cells in culture

Abstract

Secretion of pulmonary surfactant that maintains low surface tension within the lung is primarily mediated by mechanical stretching of alveolar epithelial type II (AEII) cells. We have shown that guinea pigs ventilated with random variations in frequency and tidal volume had significantly larger pools of surfactant in the lung than animals ventilated in a monotonous manner. Here, we test the hypothesis that variable stretch patterns imparted on the AEII cells results in enhanced surfactant secretion. AEII cells isolated from rat lungs were exposed to equibiaxial strains of 12.5, 25, or 50% change in surface area (DeltaSA) at 3 cycles/min for 15, 30, or 60 min. (3)H-labeled phosphatidylcholine release and cell viability were measured 60 min following the onset of stretch. Whereas secretion increased following 15-min stretch at 50% DeltaSA and 30-min stretch at 12.5% DeltaSA, 60 min of cyclic stretch diminished surfactant secretion regardless of strain. When cells were stretched using a variable strain profile in which the amplitude of each stretch was randomly pulled from a uniform distribution, surfactant secretion was enhanced both at 25 and 50% mean DeltaSA with no additional cell injury. Furthermore, at 50% mean DeltaSA, there was an optimum level of variability that maximized secretion implying that mechanotransduction in these cells exhibits a phenomenon similar to stochastic resonance. These results suggest that application of variable stretch may enhance surfactant secretion, possibly reducing the risk of ventilator-induced lung injury. Variable stretch-induced mechanotransduction may also have implications for other areas of mechanobiology.

Fig. 1.

A: schematic demonstrating the stretch protocol for alveolar epithelial type II (AEII) cells. All media and cell lysates were collected at 60 min following the onset of stretch. Thus cells devoted to 15 min of stretch would undergo the appropriate period of cyclic stretch followed by 45-min incubation, whereas cells devoted to 60 min of stretch would undergo stretch without any incubation time and control cells would simply undergo 60-min incubation. B: %change in surface area (ΔSA) composed of a sequence of half sine waves used to stretch AEII cells. Solid line represents monotonous cyclic stretching [half-width (W) = 0%] with amplitude A₀ = 50%. The dashed line represents variable stretching with A = A₀ + η, where η was taken from a uniform distribution on a cycle-to-cycle basis (W = 15%). Frequency was adjusted with each cycle to maintain an average rate of stretching of 3 cycles/min. C: histograms demonstrating the probability distributions from which A was pulled on a cycle-by-cycle basis with W = 0, ±2.5, ±5, ±10, and ±15% superimposed on the mean amplitude of A₀ = 50%. For W = 0 (thin line), the straight line extends to 100%, that is each stretch will be delivered to 50% ΔSA.

Fig. 2.

A: baseline phosphatidylcholine (PC) secretion in AEII cells as a function of time in nonstimulated cells (*P < 0.05). B: PC secretion in AEII cells exposed to TPA at concentrations of 10 and 100 nM for 3 h.

Fig. 3.

³H-labeled phosphatidylcholine secretion of AEII cells as a function of stretch amplitude for 15 min (A), 30 min (B), and 60 min (C) of monotonous stretch. *P < 0.05 increase over unstretched control cells; #P < 0.05 decrease with respect to unstretched control cells.

Fig. 4.

A: fluorescent images (×20 objective) of living cells stained green with calcein alveolar macrophages (AM) (top) and injured cells stained red with ethidium homodimer (bottom) for unstretched cells (left), cells stretched at A₀ = 50% ΔSA at 3 cycles/min (middle), and those stretched at A₀ = 50% ΔSA 12 cycles/min (right). B: calculated percentage of dead cells as a function of duration of stretch for cells stretched at A₀ = 12.5, 25, and 50% ΔSA at 3 cycles/min. In each group, 5 wells were imaged in 5 independent regions for a total of 25 measurements (*P < 0.05 compared with unstretched cells).

Fig. 5.

PC secretion as a function of variability W for A₀ = 12.5% (dashed line) and A₀ = 25% (solid lines) mean stretch amplitude (*P < 0.05 increase over cells exposed to monotonous stretch; W = 0).

Fig. 6.

PC secretion as a function of variability W for A₀ = 50% stretch amplitude for 30 min (solid line) or 60 (dashed line) min (*P < 0.05 increase over cells exposed to monotonous stretch; W = 0).