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

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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‐.

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‐.

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.

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).

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.

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.

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.

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 airway epithelial cells.

DOI: 10.1111/j.1476-5381.1994.tb13047.x

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