Cellular mechanisms for apical ATP effects on intracellular pH in human bronchial epithelium.
The effect of external ATP on intracellular pH (pH(i)) was investigated using a pH imaging system in a human bronchial epithelial cell line (16HBE14o-) loaded with BCECF-AM. The steady-state pH(i) of 16HBE14o- epithelial monolayers was 7.137 +/- 0.027 (n = 46). Apical addition of ATP (10(-4) M) to epithelial monolayers induced a rapid and sustained pH(i) decrease of 0.164 +/- 0.024 pH units (n = 17; P < 0.001). The intracellular acidification was rapidly reversed upon removal of external ATP. In contrast, the non-hydrolysable ATP analogue AMP-PNP did not produce any significant change in pH(i). Inhibition of purinoreceptors by suramin did not affect the acidification induced by apical ATP. Inhibition of Na+-H+ exchange by apical Na+ removal or addition of amiloride (0.5 mM) reduced the apical ATP-induced pH(i) decrease, suggesting the involvement of a Na+-H+ exchanger or surface pH effects on the ATP-induced pH(i) response. Inhibitors of proton channels such as ZnCl2 (10(-4) M) also partially inhibited the ATP response. The pH(i) response to ATP was dependent on the external pH (pH(o)), with increasing acidification produced at lower pH(o) values. Neither the basal pH(i) nor the ATP-induced intracellular acidification was affected by thapsigargin (a Ca2+-ATPase inhibitor), chelerythrine chloride (a protein kinase C (PKC) inhibitor), RpcAMP (a protein kinase A (PKA) inhibitor) or PMA (a PKC activator). Therefore, the intracellular acidification of human bronchial epithelial cells induced by apical ATP does not involve signalling via Ca2+, PKC or PKA nor binding to a purinoreceptor. We interpret the effect of ATP to produce an intracellular acidification as a three step process: activation of H+ channels, inhibition of Na+-H+ exchange and influx of protonated ATP.
INSERM U454, CHU A. de Villeneuve, 34295 Montpellier, France. urbach@montp.inserm.fr
V
V
Urbach
N
N
Hélix
B
B
Renaudon
B J
BJ
Harvey
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D001980:Bronchi / Q000166:cytology; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D016764:Cell Polarity / Q000502:physiology; D002712:Chlorides / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects*; D011493:Protein Kinase C / Q000378:metabolism; D011983:Receptors, Purinergic / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D011983:Receptors, Purinergic; D017923:Sodium-Hydrogen Exchangers; D017967:Zinc Compounds; D000266:Adenylyl Imidodiphosphate; C016837:zinc chloride; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium