1.
P1 purinoceptor agonists like
adenosine have been shown to stimulate Cl- transport in secretory epithelia. In the present study, we investigated whether P1 agonist-induced Cl- secretion is preserved in
cystic fibrosis airway epithelium and which signalling mechanism is involved. The effects of
purinoceptor agonists on Cl- 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 approximately equal to
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- 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 microM) but not the A1
adenosine receptor agonist, N6-phenyl
adenosine (N6-phenyl
ADO, 10 microM) 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 microM) was abolished by addition of the A2AR antagonist
3,7-dimethyl-1-propargylxanthine (
DMPX) (100 microM; reported K(i) = 11 +/- 3 microM). These results favour the involvement of A2AR. 5.
ADO (0.1-mM) and
CPCA (2 microM) 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- 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 microM), 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- current. 7. In conclusion, our results suggest that A2AR participates in regulation of airway C1 secretion via aCa2+-dependent signalling pathway, which involves CaMK and appears to be at least partially conserved in
cystic fibrosis airway epithelial cells.