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- 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.
Department of Molecular Pharmacology, Stanford University School of Medicine, CA 94305-5246.
A C
AC
Chao
J B
JB
Zifferblatt
J A
JA
Wagner
Y J
YJ
Dong
D C
DC
Gruenert
P
P
Gardner
D000227:Adenine Nucleotides / Q000494:pharmacology; D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism*; D004594:Electrophysiology; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007455:Iodine / Q000378:metabolism; D007457:Iodine Radioisotopes; D010641:Phenotype; D058915:Purinergic P1 Receptor Antagonists; D018047:Receptors, Purinergic P1 / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013805:Theobromine / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000494:pharmacology
D000227:Adenine Nucleotides; D018118:Chloride Channels; D002712:Chlorides; D007457:Iodine Radioisotopes; D058915:Purinergic P1 Receptor Antagonists; D018047:Receptors, Purinergic P1; C024311:N-cyclopropyl adenosine-5'-carboxamide; C057837:3,7-dimethyl-1-propargylxanthine; D007455:Iodine; D000241:Adenosine; D013805:Theobromine; D002118:Calcium