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In airway epithelia, purinergic receptor (P2[Y.sub.2]-R) stimulation of intracellular calcium ([Ca.sup.2+.sub.i])-regulated ion transport is restricted to the membrane domain ipsilateral to receptor activation, implying compartmentalization of [Ca.sup.2+.sub.i] signaling. Because mitochondria can spatially restrict cellular [Ca.sup.2+.sub.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 P2[Y.sub.2]-R-induced [Ca.sup.2+.sub.i] signals was investigated by measuring changes in mitochondrial [Ca.sup.2+] ([Ca.sup.2+.sub.m]) in human airway epithelial monolayers. P2[Y.sub.2]-R activation induced [Ca.sup.2+.sub.m] accumulation in mitochondria confined to the domain ipsilateral to P2[Y.sub.2]-R stimulation, which was blocked by mitochondrial uncoupling with 1 [micro]M CCCP and 2.5 [micro] g/ml oligomycin. The role of mitochondria in restricting the cellular cross-talk between basolateral P2[Y.sub.2]-R-dependent [Ca.sup.2+.sub.i] mobilization and apical membrane [Ca.sup.2+]-activated [Cl.sup.-] secretion was investigated in studies simultaneously measuring [Ca.sup.2+.sub.i] and [Cl.sup.-] secretion in cystic fibrosis human airway epithelial monolayers. Activation of basolateral P2[Y.sub.2]-Rs produced similar increases in [Ca.sup.2+.sub.i] in monolayers without and with pretreatment with uncouplers, whereas [Ca.sup.2+.sub.i]-activated [Cl.sup.-] secretion was only efficiently triggered in mitochondria-uncoupled conditions. We conclude that (a) mitochondria function as a [Ca.sup.2+.sub.i]-buffering system in airway epithelia, compartmentalizing [Ca.sup.2+.sub.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 [Ca.sup.2+.sub.i]-modulated functions associated with [Ca.sup.2+.sub.i] signals emanating from the apical or the basolateral membrane domains. KEY WORDS: calcium signaling * mitochondria * endoplasmic reticulum * chloride secretion * purinergic receptors

Chloride concentration ([[Cl.sup.-]]) was measured in defined organellar compartments using fluorescently labeled transferrin, [[alpha].sub.2]-macroglobulin, and cholera toxin B-subunit conjugated with [Cl.sup.-]-sensitive and -insensitive dyes. In pulse-chase experiments, [[Cl.sup.-]] in Tf-labeled early/ recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over ~10 min in parallel to a drop in pH from 6.91 to 6.05. The low [[Cl.sup.-]] just after internalization (compared with 137 mM solution [[Cl.sup.-]]) was prevented by reducing the interior-negative Donnan potential. [[Cl.sup.-]] in [[alpha].sub.2]-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20. [Cl.sup.-] accumulation was prevented by bafilomycin but restored by valinomycin. A [Cl.sup.-] channel inhibitor slowed endosomal acidification and [Cl.sup.-] accumulation by ~2.5-fold. [[Cl.sup.-]] was 49 mM and pH was 6.42 in cholera toxin B subunit-labeled Golgi complex in Vero cells; Golgi compartment [Cl.sup.-] accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that [[Cl.sup.-] is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [[Cl.sup.-]] early after internalization. We propose that reduced [[Cl.sup.-]] and volume in early endosomes permits endosomal acidification and [[Cl.sup.-]] accumulation without lysis.

Indicator molecules for caspase-3 activation have been reported that use fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein (the donor) and enhanced yellow fluorescent protein (EYFP; the acceptor). Because EYFP is highly sensitive to proton ([H.sup.+]) and chloride ion ([Cl.sup.-]) levels, which can change during apoptosis, this indicator's ability to trace the precise dynamics of caspase activation is limited, especially in vivo. Here, we generated an [H.sup.+]- and [Cl.sup.-]--insensitive indicator for caspase activation, SCAT, in which EYFP was replaced with Venus, and monitored the spatio-temporal activation of caspases in living cells. Caspase-3 activation was initiated first in the cytosol and then in the nucleus, and rapidly reached maximum activation in 10 min or less. Furthermore, the nuclear activation of caspase-3 preceded the nuclear apoptotic morphological changes. In contrast, the completion of caspase-9 activation took much longer and its activation was attenuated in the nucleus. However, the time between the initiation of caspase-9 activation and the morphological changes was quite similar to that seen for caspase-3, indicating the activation of both caspases occurred essentially simultaneously during the initiation of apoptosis.

The removal of extracellular Cl- ions blocks the release of the photoreceptor transmitter L-glutamate from the photoreceptors to the horizontal, ON bipolar and OFF bipolar cells in the mudpuppy retina. The block of L-glutamate suppresses the inward currents in the horizontal and OFF bipolar cells. However, the OFF cells still show light-induced voltage responses due to an increase in the input resistance. The absence of extracellular Cl- blocks the outward current in some cells.

Atrial myocytes isolated from guinea pig hearts were studied for the effects of various intracellular anions on the G protein-mediated (Gk) activation of the muscarinic potassium ion (KAch) channel. Results show that intracellular chloride ion concentration does not affect the turn-on reaction but slows the turn-off reaction of Gk resulting in higher sensitivity of the KAch for glutamine triphosphate.