Abstract
Purinergic receptor stimulation has potential therapeutic effects for cystic fibrosis (CF). Thus, we explored roles for P2Y and P2X receptors in stably increasing [Ca(2+)](i) in human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Cytosolic Ca(2+) was measured by fluorospectrometry using the fluorescent dye Fura-2/AM. Expression of P2X receptor (P2XR) subtypes was assessed by immunoblotting and biotinylation. In IB3-1 cells, ATP and other P2Y agonists caused only a transient increase in [Ca(2+)](i) derived from intracellular stores in a Na(+)-rich environment. In contrast, ATP induced an increase in [Ca(2+)](i) that had transient and sustained components in a Na(+)-free medium; the sustained plateau was potentiated by zinc or increasing extracellular pH. Benzoyl-benzoyl-ATP, a P2XR-selective agonist, increased [Ca(2+)](i) only in Na(+)-free medium, suggesting competition between Na(+) and Ca(2+) through P2XRs. Biochemical evidence showed that the P2X(4) receptor is the major subtype shared by these airway epithelial cells. A role for store-operated Ca(2+) channels, voltage-dependent Ca(2+) channels, or Na(+)/Ca(2+) exchanger in the ATP-induced sustained Ca(2+) signal was ruled out. In conclusion, these data show that epithelial P2X(4) receptors serve as ATP-gated calcium entry channels that induce a sustained increase in [Ca(2+)](i). In airway epithelia, a P2XR-mediated Ca(2+) signal may have therapeutic benefit for CF.
Highlights
In cystic fibrosis (CF),1 cyclic AMP- and protein kinase A-dependent transepithelial ClϪ transport is impaired because of mutations in the CF gene that encodes for the protein, the “cystic fibrosis transmembrane conductance regulator” or CFTR [1]
The exact mechanisms of the regulation of these proteins by CFTR are not yet fully understood, it is clear that impaired ClϪ transport is shared as a key disease phenotype by CF epithelia from all affected tissues and that this pathway is lost in CF
Recent data [57] indicate that 2MeSATP and, possibly, ADPS at a concentration of 100 M may activate P2Y11 receptors, we believe it is very unlikely that the increase in [Ca2ϩ]i observed in this study was due to the activation of P2Y11 receptors
Summary
In cystic fibrosis (CF), cyclic AMP- and protein kinase A-dependent transepithelial ClϪ transport is impaired because of mutations in the CF gene that encodes for the protein, the “cystic fibrosis transmembrane conductance regulator” or CFTR [1]. The exact mechanisms of the regulation of these proteins by CFTR are not yet fully understood, it is clear that impaired ClϪ transport is shared as a key disease phenotype by CF epithelia from all affected tissues and that this pathway is lost in CF. Stimulation of Ca2ϩ-dependent ClϪ channels can correct the impaired HCO3Ϫ secretion in CF cells [6, 7]. ATP is released into the extracellular space, it can bind to purinoceptors regulating a variety of functions in different epithelia [13,14,15]. ATP and other agonists of purinoceptors are known to increase intracellular Ca2ϩ concentration ([Ca2ϩ]i) potently in airway epithelial cells which, in turn, leads to stimulation of ClϪ secretion (14 –17) and inhibition of Naϩ absorption (18 –22).
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