Cell to cell communication in response to mechanical stress via bilateral release of ATP and UTP in polarized epithelia

Abstract

Airway epithelia are positioned at the interface between the body and the environment, and generate complex signaling responses to inhaled toxins and other stresses. Luminal mechanical stimulation of airway epithelial cells produces a propagating wave of elevated intracellular Ca(2+) that coordinates components of the integrated epithelial stress response. In polarized airway epithelia, this response has been attributed to IP(3) permeation through gap junctions. Using a combination of approaches, including enzymes that destroy extracellular nucleotides, purinergic receptor desensitization, and airway cells deficient in purinoceptors, we demonstrated that Ca(2+) waves induced by luminal mechanical stimulation in polarized airway epithelia were initiated by the release of the 5' nucleotides, ATP and UTP, across both apical and basolateral membranes. The nucleotides released into the extracellular compartment interacted with purinoceptors at both membranes to trigger Ca(2+) mobilization. Physiologically, apical membrane nucleotide-release coordinates airway mucociliary clearance responses (mucin and salt, water secretion, increased ciliary beat frequency), whereas basolateral release constitutes a paracrine mechanism by which mechanical stresses signal adjacent cells not only within the epithelium, but other cell types (nerves, inflammatory cells) in the submucosa. Nucleotide-release ipsilateral and contralateral to the surface stimulated constitutes a unique mechanism by which epithelia coordinate local and distant airway defense responses to mechanical stimuli.

Figure 1

Propagation of Ca²⁺ signals in polarized cultures of normal mouse nasal epithelial cells. a, Ca²⁺ waves: a single cell in a confluent, Fura-2 loaded culture grown on a permeable membrane support was mechanically stimulated with a micropipette. This stimulation resulted in a radially propagating Ca²⁺ wave. The numbers above the pseudocolor images indicate the time in seconds relative to the stimulus. The first image in which an elevation in Ca²⁺ level was observed is considered as time 0. Subsequent exposure to 1 mM carbachol (Carb) induced a second Ca²⁺ response in the cells (including the stimulated one), showing that the cells remain viable and responsive. Color bar shows the calibration for [Ca²⁺]_i. Bar, 100 μm. b, Cell viability: viability of the cells after mechanical stimulation was also tested by the addition of 10 mM ethidium-homodimer (EthD-1). Only one cell (distinct from the mechanically stimulated one) showed dye uptake after a 5-min incubation. As control for the viability assay, all the cells were lysed with 80 M digitonin in the continuous presence of the dye (EthD-1 + dig). c, Quantitation of Ca²⁺ waves: Ca²⁺ concentrations were quantitated in three circular regions of interest 70 μm apart (ROI_0, ROI_70 and ROI_140) shown in the bright-field image (left). An arrow indicates the stimulated cell. Bar, 100 μm. Time course of [Ca²⁺]_i for the three regions of interest in the same experiment is shown in the central panel. The color of each tracing corresponds to the color of circles denoting each ROI shown in the bright-field image on the left. The right panel depicts the mean change in Ca²⁺ levels (± SEM) for the ROI_0, ROI_70 and ROI_140 at 8, 12, and 16 s after the stimulus, respectively (n = 20).

Figure 2

Cell–cell contact is not required for the mechanically induced Ca²⁺ wave in mouse nasal epithelial cells. a, Propagating Ca²⁺ waves in not-contacting cells. Mechanical stimulation of a single cell in a nonpolarized culture grown on a glass coverslip resulted in a Ca²⁺ wave propagation (top). Elevation in Ca²⁺ level was observed in cells that were not in physical contact with the stimulated cell. Apyrase (10 U/ml) abolished the spread of Ca²⁺ wave (bottom). Low intensity pixels (<32) of the background were omitted on the pseudocolor images. Bar graph on the right depicts summary data for multiple experiments (n = 4 and 6). For other details, see Fig. 1 c. b, The Ca²⁺ wave propagates over a physical gap: a confluent culture of mouse epithelial cells grown on permeable membrane support was gently scraped with a micropipette 1 h before the experiment to produce a gap in the epithelial sheet. A single cell close to the scrape (marked with a red arrow on the bright-field image on the left) was mechanically stimulated in the absence (top) and presence (bottom) of apyrase (10 U/ml, grade V). The Ca²⁺ wave propagated over the gap (top) indicating that the signal transmission mechanism involves extracellular substance(s). The mediator was sensitive to apyrase (bottom). Bar, 100 μm. To avoid artificial background noise effects of low intensity fields, the gap was masked on the basis of the bright-field image. The mean changes in Ca²⁺ level (± SEM) are shown in the bar graph in the right panels (n = 4 and 4, respectively). The solid bars refer to the ROIs on the stimulated side, whereas hatched bars refer to the ROIs on the opposite side of the gap. No significant differences between the two sides were found in the spread of the Ca²⁺ wave.

Figure 3

Inhibition of intercellular Ca²⁺ waves with extracellular nucleotidase activity. Pseudocolor images on the left demonstrate the intracellular Ca²⁺ levels at designated time points in representative experiments. In the center, time courses of [Ca²⁺]_i in these experiments are shown. Summary data for multiple experiments are shown on the right (n = 6 and 13). Apical addition of apyrase (10 U/ml) attenuated, but did not block, the mechanically induced intercellular Ca²⁺ wave (top), whereas bilateral addition of apyrase completely prevented the Ca²⁺ spread (bottom). Subsequent carbachol-induced Ca²⁺ responses were not affected as compared with control experiments shown in Fig. 1 c (middle).

Figure 4

Participation of P₂-receptors in intercellular Ca²⁺ waves. a, Functional expression of P2Y-receptors in polarized mouse epithelial cells. Nucleotide-induced Ca²⁺ responses to apical versus basolateral additions of ATP (squares), UTP (circles), ADP (upright triangles), and UDP (inverted triangles) were measured. Basal to peak changes in [Ca²⁺]_i levels (mean ± SEM) are plotted (n = 3–6/point). b, Inhibition of intercellular Ca²⁺ wave by desensitization of P₂-receptors. Bilateral pretreatment with ATP (300 μM) completely prevented the propagation of Ca²⁺ signals without affecting the carbachol-induced Ca²⁺ response. c, Quantitative analysis of the inhibition of intercellular Ca²⁺ waves: Time course of [Ca²⁺]_i for the three regions of interest in the same experiment shown (left) and mean change in Ca²⁺ levels (± SEM) for several similar experiments (n = 15) (right). For details see legend of Fig. 1 c.

Figure 5

Propagating Ca²⁺ waves in P2Y₂-R (−/−) airway epithelial cells. a, Characterization of polarized cultures of nasal cells isolated from P2Y₂-receptor gene targeted (−/−) mice for residual expression of P2Y-receptors. Nucleotide-induced Ca²⁺ responses to ATP (squares), UTP (circles), ADP (upright triangles), and UDP (inverted triangles) were measured (n = 3–6/point). The effects of ATP (open squares) and ADP (open up triangles) were also assessed in the presence of 100 μM PAPS, a P2Y₁-receptor antagonist (Boyer et al. 1996). The pharmacological profiles indicate that the residual P₂-receptor (most likely P2Y₁-R) is expressed solely in the basolateral membrane. b, Mechanically induced intercellular Ca²⁺ wave in P2Y₂-R (−/−) cells in a representative experiment. The residual Ca²⁺ propagation in P2Y₂-R (−/−) cells suggests basolateral release of nucleotides interacting with P2Y₁-receptors in the basolateral membrane. For details see Fig. 1 a. c, Inhibition of Ca²⁺ propagation in P2Y₂-R (−/−) cells. The magnitude of the Ca²⁺ waves was smaller in P2Y₂-R (−/−) cells as compared with wild-type cells (Fig. 1 c, right). Basolateral pretreatment with ATP (hatched bars) or PAPS (open bars) completely abolished the intercellular Ca²⁺ wave as compared with P2Y₂-R (−/−) cells without treatment (solid bars; n = 11, 6, and 9, respectively). For details for quantitative analysis see legend of Fig. 1 c.

Figure 6

Participation of released UTP in the mechanically induced Ca²⁺ wave propagation. a, Characterization of a mouse P2Y₂-R (−/−) nasal cell line expressing the human P2Y₄-receptor. Ca²⁺ responses to nucleotides (100 μM) and carbachol (1 mM) were measured in P2Y₂-R (−/−) cells transduced with the Hygro^r-only vector (Control) and with the human P2Y₄-receptor (P2Y₄) (n = 3–4/point). Control [P2Y₂(−/−)] cells grown on glass coverslips showed no substantial Ca²⁺ responses to nucleotides but to carbachol. In contrast, UTP stimulated significant Ca²⁺ responses in P2Y₄-R transduced cells. No other nucleotide stimulated substantial Ca²⁺ response in the P2Y₄-receptor expressing cells. b, Mechanically induced intercellular Ca²⁺ wave in polarized culture of P2Y₄-R transduced cells in a representative experiment. Pseudocolor images demonstrate the intracellular Ca²⁺ levels in the empty vector-transduced P2Y₂(−/−) cells (left) and the P2Y₄-R transduced P2Y₂(−/−) cells (right) 16 s after local mechanical stimulation in the continuous presence of 100 μM PAPS in the basolateral bath. c, Quantitative analysis of Ca²⁺ wave propagation in control and P2Y₄-R transduced cells. Summary data for experiments similar to that shown in Fig. 6 b. Mechanical stimulation of a single cell did not elicit Ca²⁺ wave in the P2Y₂(−/−) cells transduced with the Hygro^r-only vector, when 100 μM PAPS was present in the basolateral bath, whereas an extensive Ca²⁺ wave was observed in P2Y₄-R transduced cells (n = 3 and 3, respectively). For details for quantitative analysis see legend of Fig. 1 c.

Figure 7

Intercellular Ca²⁺ waves in polarized human airway epithelial cells. a, A representative experiment. Local mechanical stimulation elicited cell to cell propagation of Ca²⁺ signals in well-differentiated human bronchial epithelial cell cultures. b, Block of Ca²⁺ wave propagation with extracellular nucleotidase activity. The intercellular Ca²⁺ waves were partially inhibited by apical addition of apyrase (hatched bars; n = 5) as compared with control (solid bars; n = 3). Bilateral treatment with apyrase completely abolished the spread of Ca²⁺ signals (open bars; n = 10).

Figure 8

A proposed model for intercellular Ca²⁺ waves in airway epithelia. Mechanical stimulation on the apical surface of a polarized airway epithelial cell induces ipsilateral and contralateral release of nucleotides that activates the adjacent cells via P2Y₂-receptors on the apical membrane and via both P2Y₁- and P2Y₂-receptors on the basolateral membrane.