Cellular mechanisms for apical ATP effects on intracellular pH in human bronchial epithelium

Abstract

The effect of external ATP on intracellular pH (pHi) was investigated using a pH imaging system in a human bronchial epithelial cell line (16HBE14o‐) loaded with BCECF‐AM. The steady‐state pHi of 16HBE14o‐ epithelial monolayers was 7.137 ± 0.027 (n= 46). Apical addition of ATP (10−4m) to epithelial monolayers induced a rapid and sustained pHi 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 pHi. 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 pHi decrease, suggesting the involvement of a Na+‐H+ exchanger or surface pH effects on the ATP‐induced pHi response. Inhibitors of proton channels such as ZnCl2 (10−4m) also partially inhibited the ATP response. The pHi response to ATP was dependent on the external pH (pHo), with increasing acidification produced at lower pHo values. Neither the basal pHi 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.

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“…This result is consistent with previous observations on the role of Na ϩ /H ϩ exchanger activity in maintaining the steady-state pH i in a wide variety of epithelia such as renal cells (23)(24)(25), retinal pigment epithelial cells (26 -28), corneal epithelium (29), human esophageal epithelial cells (30), nasal epithelial cells (18), and lung epithelial cells (31)(32)(33)(34)(35).…”

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“…In addition, the range of concentrations of the inhibitors used in the present study were the same as those reported in previous studies that have used human bronchial epithelial cells as targets (20,43). These results are also consistent with those of other studies which used these inhibitors and showed a role for PKC in viral infection by several enveloped viruses, such as the vesicular stomatitis virus, herpes simplex I virus, turkey herpes virus, vaccinia virus, Sindbis virus, human herpesvirus 8, and NHBE cells per ml in suspension were placed in the absence or presence of different concentrations of the myr-PKC peptide for 30 min at 37°C.…”

Amyloid precursor protein and amyloid β-peptide bind to ATP synthase and regulate its activity at the surface of neural cells

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