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.
Chao
A C
AC
Department of Molecular Pharmacology, Stanford University School of Medicine, CA 94305-5246.
Zifferblatt
J B
JB
Wagner
J A
JA
Dong
Y J
YJ
Gruenert
D C
DC
Gardner
P
P
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