The mitochondrial barriers segregate agonist-induced calcium-dependent functions in human airway epithelia

Abstract

In airway epithelia, purinergic receptor (P2Y2-R) stimulation of intracellular calcium (Ca2+i)-regulated ion transport is restricted to the membrane domain ipsilateral to receptor activation, implying compartmentalization of Ca2+i signaling. Because mitochondria can spatially restrict cellular Ca2+i signals, immunocytochemical, electron microscopic, and fluorescent studies of mitochondria localization were performed in human airway epithelia. Although concentrated at the apical domain, mitochondria were found distributed at both the apical and the basolateral poles and in close association with the endoplasmic reticulum. The role of mitochondria in locally restricting P2Y2-R-induced Ca2+i signals was investigated by measuring changes in mitochondrial Ca2+ (Ca2+m) in human airway epithelial monolayers. P2Y2-R activation induced Ca2+m accumulation in mitochondria confined to the domain ipsilateral to P2Y2-R stimulation, which was blocked by mitochondrial uncoupling with 1 microM CCCP and 2.5 microg/ml oligomycin. The role of mitochondria in restricting the cellular cross-talk between basolateral P2Y2-R-dependent Ca2+i mobilization and apical membrane Ca2+-activated Cl- secretion was investigated in studies simultaneously measuring Ca2+i and Cl- secretion in cystic fibrosis human airway epithelial monolayers. Activation of basolateral P2Y2-Rs produced similar increases in Ca2+i in monolayers without and with pretreatment with uncouplers, whereas Ca2+i-activated Cl- secretion was only efficiently triggered in mitochondria-uncoupled conditions. We conclude that (a) mitochondria function as a Ca2+i-buffering system in airway epithelia, compartmentalizing Ca2+i-dependent functions to the membrane ipsilateral to receptor stimulation; and (b) the mitochondria provide structural barriers that protect the airway epithelia against nonspecific activation of Ca2+i-modulated functions associated with Ca2+i signals emanating from the apical or the basolateral membrane domains.

Figure 1.

Mitochondrial distribution in airway epithelia. (A) Immunostaining of the mitochondrial resident protein, mitochondrial heat shock protein 70, in freshly excised human bronchial airway epithelia. Bar, 10 μm. (B) Transmission electron micrograph displaying mitochondrial distribution in freshly excised human bronchial airway epithelia. Arrows indicate mitochondria. Magnification, 4,400×. (C) The mitochondrial distribution in human bronchial airway epithelia in primary culture (monolayer) labeled with MitoTracker Red CMX Ros. Bar, 10 μm. Immunostain is representative of five tissue codes; micrograph is representative of three tissue codes and mitotracker staining is representative of four tissue codes.

Figure 2.

Mitochondrial calcium (Ca²⁺ _m) uptake elicited by apical P2Y2-R activation is inhibited by mitochondrial uncouplers. Representative time series of XY confocal scans from the apical epithelial domain. (A, top) Time course for Ca²⁺ _m uptake (visualized as increases in rhod-2 fluorescence) after addition of 100 μM mucosal UTP to a polarized primary culture monolayer of CF human bronchial airway epithelia. (A, bottom) Effect of subsequent mitochondrial uncoupling with 1 μM CCCP and 2.5 μg/ml oligomycin on Ca²⁺ _m accumulation induced by apical P2Y₂-R activation. Bar, 10 μm. (B) Summary of the time series studies depicted in A. Data are expressed as a percentage of baseline fluorescence and represent the mean of four experiments ± SEM. *, P < 0.05, 100 μM mucosal UTP-induced fluorescence vs. baseline (t = 0) fluorescence.

Figure 3.

Mitochondria and ER are closely associated in human airway epithelia. Representative electron micrograph from a CF primary culture monolayer illustrating, as denoted by the arrows, that mitochondria and ER (depicted as rough ER membranes decorated with ribosomes) are intimately associated. Magnification, 20,000×. Micrograph is representative of four tissue codes.

Figure 4.

Mitochondrial calcium (Ca²⁺ _m) uptake resulting from TG-dependent inhibition of ER Ca²⁺-ATPase activity in airway epithelia. Representative time series of XY confocal scans from the apical epithelial domain. (A, top) Time course for Ca²⁺ _m uptake (visualized as increases in rhod-2 fluorescence) after addition of 1 μM mucosal TG to a polarized primary culture monolayer of CF human bronchial airway epithelia. (A, bottom) Effect of subsequent mitochondrial uncoupling with 1 μM CCCP and 2.5 μg/ml oligomycin on Ca²⁺ _m accumulation induced by TG. Bar, 10 μm. (B) Summary of the time series studies illustrated in A. Data are expressed as a percentage of baseline fluorescence and represent the mean of 3 experiments ± SEM. *, P < 0.05, 1 μM mucosal TG-induced fluorescence vs. baseline (t = 0) fluorescence.

Figure 5.

Mitochondrial calcium (Ca²⁺ _m) uptake, as a read-out of localized Ca²⁺ _i signals resulting from apical P2Y₂-R activation, is restricted to the apical domain in airway epithelia. (A) Representative time course for apical P2Y₂-R activation-promoted Ca²⁺ _m uptake, as measured by increases in rhod-2 fluorescence, in a polarized primary culture monolayer of CF human airway epithelia. Bar, 10 μm. (B) Summary of the time series studies illustrated in A. Data are expressed as a percentage of baseline rhod-2 fluorescence and represent the mean of four experiments ± SEM.

Figure 6.

Mitochondrial calcium (Ca²⁺ _m) uptake, as a read-out of localized Ca²⁺ _i signals resulting from basolateral P2Y₂-R activation, is restricted to the basolateral domain in airway epithelia. (A) Representative time course for basolateral P2Y₂-R activation-promoted Ca²⁺ _m uptake, as measured by increases in rhod-2 fluorescence, in a polarized primary culture monolayer of CF human airway epithelia. Bar, 10 μm. (B) Summary of the time series studies depicted in A. Data are expressed as a percentage of baseline rhod-2 fluorescence and represent the mean of three experiments ± SEM.

Figure 7.

Basolateral P2Y₂ receptor activation–dependent intracellular calcium (Ca²⁺ _i) mobilization and apical Cl⁻ secretion in CF human airway epithelia. (A) Simultaneous measurements of Ca²⁺ _i and transepithelial electrical potential difference (V_t) in a CF monolayer exposed to luminal Na⁺ free/low Cl⁻ KBR and basolateral KBR. 10 μM forskolin and 100 μM basolateral UTP were added at times denoted by arrows. Representative V_t and Ca²⁺ _i tracings from 13 experiments. (B) Similar protocol as in A, but with addition of the mitochondrial uncouplers 1 μM CCCP + 2.5 μg/ml oligomycin at time depicted by arrow. Representative V_t and Ca²⁺ _i tracings from 24 experiments. (C) Average changes in Ca²⁺ _i (peak-baseline value) after basolateral UTP or basolateral UTP + uncouplers. n = 11 and n = 24 for UTP and UTP + CCCP + oligomycin, respectively. (D) Average changes in I_eq (calculated from the V_t values) from the experiments compiled in C. n = 13 and n = 24 for UTP and UTP + CCCP + oligomycin, respectively. *, P < 0.0005, UTP vs. UTP + CCCP + oligomycin.

Figure 8.

The apical and the basolateral mitochondrial barriers restrict Ca²⁺ _i-dependent functions to the membrane domain ipsilateral to receptor activation in human airway epithelia. The mitochondria distributed toward the apical or the basolateral domains provide structural barriers between the apical and basolateral poles to global Ca²⁺ waves, thereby protecting the airway epithelia against nonspecific regulation of Ca²⁺ _i-modulated functions associated with the domain contralateral to the membrane of receptor activation.