Cytochemical localization of adenylate cyclase in the sodium-transporting epithelium isolated from frog skin

Richards, P.D.G.; J., W.

doi:10.1007/bf00157895

Cited by 5 publications

(2 citation statements)

References 31 publications

(29 reference statements)

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“…AC expression in the apical membrane of epithelia is controversial (23)(24)(25)(26)(27)). Therefore, we tested whether conditions that modulate AC affect Ado regulation of CFTR in excised apical membrane patches.…”

Section: Resultsmentioning

confidence: 99%

“…Testing for such compartmentalization by biochemical methods that disrupt polarity has proved problematic (23,26,27). Similarly, immunohistochemical methods often lack the sensitivity to detect a small pool of functionally important proteins localized in a subcellular domain, such as the apical membrane (23)(24)(25). Here, we have used patch-clamp technique to physically isolate apical cell membranes, and we have used CFTR single-channel activity as a functional reporter to identify cAMP signaling elements that are present.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Compartmentalized autocrine signaling to cystic fibrosis transmembrane conductance regulator at the apical membrane of airway epithelial cells

Huang

Lazarowski

Tarran

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

191

209

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Physical stimulation of airway surfaces evokes liquid secretion, but the events that mediate this vital protective function are not understood. When cystic fibrosis transmembrane conductance regulator (CFTR) channel activity was used as a functional readout, we found signaling elements compartmentalized at both extracellular and intracellular surfaces of the apical cell membrane that activate apical Cl ؊ conductance in Calu-3 cells. At the outer surface, ATP was released by physical stimuli, locally converted to adenosine, and sensed by A2B adenosine receptors. These receptors couple to G proteins, adenylyl cyclase, and protein kinase A, at the intracellular face of the apical membrane to activate colocalized CFTR. Thus, airways have evolved highly efficient mechanisms to ''flush'' noxious stimuli from airway surfaces by selective activation of apical membrane signal transduction and effector systems.A irways continuously remove noxious materials through a mucociliary clearance process that requires liquid secretion (1, 2). cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl Ϫ channels (3, 4) are expected to participate in liquid secretion in airways, but the two key events in the activation of CFTR by local physical stimuli remain puzzling. First, how do physical stimuli initiate the classic cAMP signaling cascade, a process that is tightly regulated by Gprotein-coupled receptors (5)? Second, how does cAMP reach CFTR in the apical membrane? The dogma of G-proteincoupled receptors and adenylyl cyclase (AC) restricted to the basolateral membrane of epithelia does not adequately explain how these events occur (6).Two ongoing areas of research suggest a potentially more relevant, but as yet not fully tested model for activation of apical CFTR by a local physical stimulus. Airway surface epithelia are poorly innervated, suggesting that mucociliary clearance is subject to autocrine͞paracrine control. A leading candidate for mediating mucociliary clearance is the release of cellular nucleotides, because release occurs in response to physical stimuli and luminal nucleotide receptors stimulate apical Cl Ϫ conductance, mucus secretion, and ciliary beating (7,8). The action of ectonucleotidases extends the signaling potential of released ATP on luminal surface by producing adenosine (Ado), a ligand for A2 receptors that couple to AC (9, 10). Recent reports indicate that receptors, intracellular signaling pathways, and scaffolding molecules can form complexes that locally regulate functions in subcellular compartments (11,12). Thus, a model linking a luminal physical stimulus to activation of CFTR requires specific elements, including ATP release, ectonucleotidases, Ado receptors, G proteins, and AC, to be intimately associated with the apical cell membrane. The goal of the present study was to test this hypothesis in polarized airway epithelial cells. MethodsCells. Human Calu-3 cells were grown as previously described (13) on Costar clear transwells (for HPLC and cAMP assay) or homemade permeable ...

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