Stimulation of chloride secretion by P1 purinoceptor agonists in cystic fibrosis phenotype airway epithelial cell line CFPEo

1 P1 purinoceptor agonists like adenosine have been shown to stimulate Cl−1 transport in secretory epithelia. In the present study, we investigated whether P1 agonist‐induced Cl−1 secretion is preserved in cystic fibrosis airway epithelium and which signalling mechanism is involved. The effects of purinoceptor agonists on Cl−1 secretion were examined in a transformed cystic fibrosis airway phenotype epithelial cell line, CFPEo‐. 2 Addition of adenosine (ADO; 0.1 – 1 mm) markedly increased 125I efflux rate. The rank order of potency of purinoceptor agonists in stimulating 125I efflux was ADO > AMP > ADP ≃ ATP. A similar order of potency was seen in transformed cystic fibrosis nasal polyp cells, CFNPEo‐ (ADO > ATP > AMP > ADP). These results are consistent with the activation of Cl−1 secretion via a P1 purinoceptor. 3 The P1 agonists tested (at 0.01 and 0.1 mm) revealed a rank order of potency of 5′‐N‐ethylcarboxamine adenosine (NECA) > 2‐chloro‐adenosine (2‐Cl‐ADO) > R‐phenylisopropyl adenosine (R‐PIA). 4 The known potent A2 adenosine receptor (A2AR) agonist, 5′‐(N‐cyclopropyl) carboxamidoadenosine (CPCA, 2 μm) but not the A1 adenosine receptor agonist, N6‐phenyl adenosine (N6‐phenyl ADO, 10 μm) markedly increased 125I efflux rate (baseline, 5.9 ± 2.0% min−1, + CPCA, 10.9 ± 0.6% min−1; P < 0.01). The stimulant effect of CPCA (10 μm) was abolished by addition of the A2AR antagonist 3,7‐dimethyl‐1‐propargylxanthine (DMPX) (100 μm; reported K1 = 11 ± 3 μm). These results favour the involvement of A2AR. 5 ADO (0.1 – 1 mm) and CPCA (2 μm) both induced a marked increase in intracellular [Ca2+] ([Ca2+]i); the effect of the latter was again abolished by pretreatment of the cells with DMPX. By contrast, N6‐phenyl ADO did not affect [Ca2+]i. 6 In patch‐clamp experiments, ADO (1 mm) induced an outwardly‐rectified whole‐cell Cl−1 current (baseline, 2.5 ± 0.8 pA pF−1, + ADO, 78.4 ± 23.8 pA pF−1; P < 0.02), which was largely inhibited in cells internally perfused with a selective inhibitory peptide of the multifunctional Ca2+/calmodulin‐dependent protein kinase, CaMK [273–302] (20 μm), as compared to a control peptide, CaMK [284–302]. Addition of BAPTA (10 mm), a Ca2+ chelator, to the perfusion pipette also abolished the ADO‐elicited Cl−1 current. 7 In conclusion, our results suggest that A2AR participates in regulation of airway Cl−1 secretion via a Ca2+‐dependent signalling pathway, which involves CaMK and appears to be at least partially conserved in cystic fibrosis a