Efficient regulation of intracellular pH (pHi) is of paramount importance for all human cells. Large changes in pHi can elicit a wide range of changes in physiological processes, including protein synthesis, DNA replication and ion transport and, consequentially, cells must possess mechanisms by which they can resist changes in pHi. Diffusion of CO2 from the blood to epithelia places an acid load on the cells, a process which is augmented in patients with lung disease who exhibit a high partial pressure of CO2 in arterial blood (PaCO2, hypercapnia). We sought to assess the effect of hypercapnia on the pHi regulatory mechanisms of a model of human airway epithelia. Calu-3 cells were loaded with the pH-sensitive dye, BCECF-AM, and pHi was measured by fluorescent microscopy. Cells were initially perfused with solutions gassed with 5% CO2 (normocapnia). Application of 10% CO2 (hypercapnia) to both the apical and basolateral membrane caused a rapid, intracellular acidosis of 0.24 ± 0.02 units (n=6). Cells were able to recover pHi from intracellular acidosis; a response which took ~20 mins. We observed that pHi recovery in the absence of basolateral Na+ was -10.5 ± 21.4% of control (n=5; p<0.001) yet pHi recovery in the presence of the Na+/H+ exchanger inhibitor EIPA (3µM) remained at a substantial 85.0 ± 8.4% of control (n=4; p>0.05) whilst the H+ channel inhibitor ZnCl2 (100µM) also did not significantly alter pHi recovery (n=3; p>0.05). Conversely, the Na+/HCO3- cotransporter (NBC) inhibitor DIDS (100µM) caused a 56.7 ± 3.8% decrease in pHi recovery (n=3; p<0.05). Together, these data implicated Na+-dependent HCO3- influx, as opposed to Na+-dependent H+ efflux, underlied pHi recovery. Interestingly, BAPTA-AM (50µM), the phospholipase C inhibitor U73122 (10µM) and intracellular Ca2+ store depletion all reduced pHi recovery by 63.1 ± 6.8% (n=5; p<0.001), 75.8 ± 9.0% (n=3; p<0.001) and 74.8 ± 8.1% (n=4; p<0.001) respectively, implying a Ca2+-signalling component was also involved in mediating pHi recovery. End-point PCR revealed that both SLC4A4 (NBCe1) and SLC4A7 (NBCn1) were expressed in Calu-3 cells. Upon measuring NBC activity per se, we observed a 2.7 ± 0.3 fold increase (n=5; p<0.01) in activity in hypercapnic conditions. These findings suggest that CO2 stimulates an increase in NBC activity to facilitate increased HCO3- influx; a mechanism by which cells can maintain intracellular pH from CO2-induced acidosis. This work further reinforces the role of CO2 as a cell signalling molecule in human epithelia and demonstrates the adaptive mechanisms by which cells are able to maintain intracellular pH in response to acid loads.
Physiology 2015 (Cardiff, UK) (2015) Proc Physiol Soc 34, C51
Oral Communications: CO2-dependent stimulation of Na+/HCO3- cotransporter is a mechanism to facilitate recovery from CO2-induced intracellular acidosis in human airway epithelia
M. J. Turner2,1, M. Cann3, M. Gray2
1. Department of Physiology, McGill University, Montreal, Quebec, Canada. 2. Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom. 3. School of Biological and Biomedical Sciences, Durham University, Durham, United Kingdom.
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