Propidium uptake and ATP release in A549 cells share similar transport mechanisms

Abstract

The lipid bilayer of eukaryotic cells' plasma membrane is almost impermeable to small ions and large polar molecules, but its miniscule basal permeability in intact cells is poorly characterized. This report describes the intrinsic membrane permeability of A549 cells toward the charged molecules propidium (Pr²⁺) and ATP^4-. Under isotonic conditions, we detected with quantitative fluorescence microscopy, a continuous low-rate uptake of Pr (∼150 × 10^-21 moles (zmol)/h/cell, [Pr]_o = 150 μM, 32°C). It was stimulated transiently but strongly by 66% hypotonic cell swelling reaching an influx amplitude of ∼1500 (zmol/h)/cell. The progressive Pr uptake with increasing [Pr]_o (30, 150, and 750 μM) suggested a permeation mechanism by simple diffusion. We quantified separately ATP release with custom wide-field-of-view chemiluminescence imaging. The strong proportionality between ATP efflux and Pr²⁺ influx during hypotonic challenge, and the absence of stimulation of transmembrane transport following 300% hypertonic shock, indicated that ATP and Pr travel the same conductive pathway. The fluorescence images revealed a homogeneously distributed intracellular uptake of Pr not consistent with high-conductance channels expressed at low density on the plasma membrane. We hypothesized that the pathway consists of transiently formed water pores evenly spread across the plasma membrane. The abolition of cell swelling-induced Pr uptake with 500 μM gadolinium, a known modulator of membrane fluidity, supported the involvement of water pores whose formation depends on the membrane fluidity. Our study suggests an alternative model of a direct permeation of ATP (and other molecules) through the phospholipid bilayer, which may have important physiological implications.

Keywords: ATP release; imaging; osmotic shock; propidium uptake.

Figure 1

Extracellular ATP correction. (A) Postmounting residual ATP-dependent chemiluminescence. Example of luminescent signal (ΣLU, dark trace) generated by ATP_o immediately after A549 cells, grown on 25-mm glass coverslip, were mounted inside the imaging chamber (isotonic). Extracellular ATP originated from cell mechanical disruption upon mounting (e.g., lysis). The decaying signal intensity can be fitted with a single exponential decay function (red trace). (B) Example of ATP accumulation curve corrected for ATP degradation. Cell-normalized ATP accumulation from 90% hypotonic challenged A549 cells without correction (dark trace) and with correction (red trace). The ATP degradation rate was derived by iterating –b parameter (from dark trace) until finding the value that brings the dATP_o/dt toward zero when reaching the end of the 90% shock-induced release. To see this figure in color, go online.

Figure 2

Intracellular propidium calibration with permeabilized A549. (A) Pr_i fluorescence changes in permeabilized A549. Average fluorescence emitted by Pr_i with increasing [Pr]_o at 1.5, 15, and 150 nM (DMEM) from n = 50–100 swollen (66% hypotonic, 15 min) and 0.05% Triton X-100 permeabilized A549 cells (32°C). Pr_i fluorescence signal stabilization took 60 min indicating slow intracellular diffusion. Note that, since we used more than 1000-fold lower [Pr]_o than our standard of 150 μM used with intact cells, the background fluorescence from unbound Pr in solution was negligible here, and therefore allowed us to continuously monitor the changes in fluorescence emission. (B) Calibration curve. Fluorescence levels at the end of the 60 min incubation period were plotted against Pr_i (n = 3, each inverted triangle is an independent experiment). The quantity of Pr_i (in zmol, 10⁻²¹ moles) was calculated with an estimated volume of 30 pL (theoretical threefold increase from V_o = 10 pL) times [Pr]_o = [Pr]_i. The calibration factor is 0.4 zmol Pr/(LU/pixel).

Figure 3

Kinetics of Pr_i accumulation in intact A549. (A) Representative bright-field (BF) and Pr_i fluorescence images. Left columns: BF images (objective 20×/0.8) of A549 (32°C) before and 30 min after switching to a hypotonic 66%, isotonic (control), or hypertonic 300% solutions. The cellular outline is slightly changed in 50% hypotonic, but unchanged with 300% hypertonic since the bulk of volume changes occur in a plane vertical to the glass coverslip. (Note that the contrast in these BF images comes from reducing the condenser NA.) Right columns: Pr_i fluorescence from corresponding cells in BF images. The Pr uptake in intact A549 is clearly visible in 66% hypotonically challenged cells. Note that, although the emission varied from cell-to-cell, all cells emitted fluorescence, and also note the prominent emission from the DNA-rich nucleus alongside a diffuse signal within the cytoplasm. The increase in Pr_i fluorescence was much smaller in isotonic and 300% hypertonic conditions, although still discernible. (B) Kinetics of Pr_i uptake (Mean ± SD) changes in (zmol) over time for isotonic, 66% hypotonic, and 300% hypertonic (n = 3 independent experiments with each an average of 50–100 cells). The incubation with Pr_o began 15 min before the change in tonicity at 0 min. To see this figure in color, go online.

Figure 4

Intact versus dead cells’ (lysed) Pr_i fluorescence intensity. (A) Pr_i intensity profiles. Representative FOV of Pr_i fluorescence from a dead cell (arrow) among intact cells following 30 min of 66% hypotonic shock (32°C) in the presence of 150 μM [Pr]_o. The segment a-b shows the cross section fluorescence of Pr_i in three intact cells. Note that the faint signal between pixels 50 and 90 is coming from the edge of one cell. The whole distance a-c includes, in addition to these three cells, the single dead cell in this FOV. Because of its much stronger signal, only the peak intensity of the dead cell is visible in the a-c plot (linear scale). (B) Pr_i fluorescence level (Box: Median, 25^th,75^th percentile; Whisker: min, max excluding outliers). Average Pr_i fluorescence from cells, 30 min in isotonic conditions or 66% hypotonic conditions, were divided according to their level of fluorescence: low fluorescence (<2000 LU/pixel) presumed from live and intact cells versus high fluorescence from dead/lysed cells. Note that the high-fluorescence cell level group comprised less than 1% of the cell population. To see this figure in color, go online.

Figure 5

Propidium diffusion into A549 cells. Intracellular Pr concentration within A549 cells exposed to 66% hypotonic medium (32°C) during 15 min in the presence of [Pr]_o at 30, 150, or 750 μM (n = 3). Note the quasilinear relationship between Pr_i and [Pr]_o, suggestive of a passive diffusion process for Pr translocation.

Figure 6

ATP release from osmotically challenged A549 cells. (A) ATP density images. Representative images of color-mapped ATP level in extracellular medium right before the experiment (<0 min, left column) and 2 min after the changes in solution osmolarity (right column) for 300% hypertonic and 66, 85, and 90% hypotonic (32°C). (B) Representative cell-normalized ATP accumulation (corrected for extracellular ATP degradation) in the chamber following 50, 66, 85, and 90% hypotonic as well as 300% hypertonic. (C) Rate of cellular ATP release derived from traces in (B). To see figure color go online.

Figure 7

Accumulation of intracellular Pr versus release of cellular ATP. (A) Representative BF and fluorescence A549 cell images at 50, 66, 85, and 90% hypotonic shock (32°C) after 15 min of changing the osmolarity of the solution. (B) Pr_i versus ATP secretion (Mean ± SD) after exposing A549 cells to hypotonic shock of 50, 66, 85, and 90% during 15 min (32°C). To see figure color go online.

Figure 8

Gadolinium and Pr uptake. (A) Representative BF and fluorescence images after 15 min of 90% hypotonic shock with 0 or 500 μM Gd³⁺ in extracellular medium. The Pr_i signal was nearly abolished in the presence of 500 μM Gd³⁺. The arrows point to a dead/lysed cell. Note the fusion of the cell membrane in the presence of Gd³⁺. (B) Scatter box plot of representative Pr uptake in absence or presence of 500 μM Gd³⁺(Box: Median, 25^th,75^th percentile; Whisker: min, max excluding outliers). Each point is the Pr content of one cell. Gadolinium very significantly (p < 0.001: Mann-Whitney U-test) blocks 90% hypo-stimulated Pr uptake in A549 (32°C) in these two groups of intact and dead cells from two representative experiments.

Figure 9

Water flow through cell membrane. Schematic visualization of water movement across the cell plasma membrane in isotonic, hypotonic, and hypertonic conditions where J_w(in) is water influx and J_w(out) is water efflux. To see this figure in color, go online.

Figure 10

Basal and hypo-stimulated ATP release. Peak 50% hypo-stimulated (37°C) ATP release (n = 3) as a function of basal release with NIH3T3, A549, and 16HBE14o– cells (n = 3) mounted in a perfusion chamber (Q = 1.3 mL/min). ATP release was titrated offline with luciferase-based reagent in a luminometer (Mean ± SD). Adapted from (3).

Figure 11

Water pore model. (A) Step formation of water pore model (1) formation of hydrophobic single line defect and (2) fully developed hydrophilic pore. (B) Proposed water pore mechanism formation from osmotic forces with water influx. (C) Absence of water pore formation in the case of water efflux. To see this figure in color, go online.