Identification and function of A1 adenosine receptors in normal and cystic fibrosis human airway epithelial cells.
A role for adenosine in the regulation of ion transport in pulmonary epithelial cells has recently been proposed. Although evidence exists documenting the presence and function of adenosine A2 receptors in airway epithelia, the presence of adenosine A1 receptors remains controversial. The present study used reverse transcriptase-polymerase chain reaction (PCR) and whole cell patch-clamp analysis to investigate A1 receptor presence and function in normal and cystic fibrosis (CF) human airway epithelial cells. Oligonucleotide primers complementary to the human brain A1 receptor sequence generated a PCR product of the predicted size (311 bp) in normal tracheal (9HTEo-) and CF submucosal (2CFSMEo-) airway cell lines and in primary cultures of CF nasal polyp epithelial cells. An oligonucleotide probe internal to the PCR primers hybridized with the 311-bp cDNAs by Southern blot analysis. cDNA sequencing demonstrated that the normal and CF airway cell PCR products are 100% identical to the corresponding sequence of the human brain adenosine A1 receptor. Northern blot analysis of 9HTEo-and 2CFSMEo- poly(A)+ RNA revealed the presence of two bands of approximately 3.0 and approximately 5.5 kb corresponding to the A1 receptor. Whole cell patch-clamp analyses demonstrated that 8-cyclopentyl-1,3-dipropylxanthine, a specific A1 receptor antagonist, increases adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- conductance in 9HTEo-airway cells and allows cAMP to increase Cl- conductance in 2CFSMEo- CF airway cells and CF nasal polyp epithelial cells in primary culture. These results provide evidence for the presence and function of A1 receptors in normal and CF airway epithelial cells and provide support for a role of adenosine A1 receptors in modulating airway epithelial cell Cl- transport.
McCoy
D E
DE
Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Schwiebert
E M
EM
Karlson
K H
KH
Spielman
W S
WS
Stanton
B A
BA
D001483:Base Sequence; D015139:Blotting, Southern; D001921:Brain / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism*; D004247:DNA / Q000032:analysis / Q000096:biosynthesis; D017931:DNA Primers; D004848:Epithelium / Q000378:metabolism / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009092:Mucous Membrane / Q000378:metabolism; D009298:Nasal Polyps / Q000378:metabolism; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D018047:Receptors, Purinergic P1 / Q000096:biosynthesis* / Q000502:physiology; D012016:Reference Values; D012689:Sequence Homology, Nucleic Acid; D014132:Trachea / Q000378:metabolism*
D017931:DNA Primers; D018047:Receptors, Purinergic P1; D004247:DNA