The Mitochondrial Barriers Segregate Agonist-induced Calcium-dependent Functions in Human Airway Epithelia

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 μM CCCP and 2.5 μg/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.