Microphysiometry: an assay for pH changes in cystic fibrosis
Research Projects
In research initiated at Stanford University, here at Michigan State we have focused on the pH abnormalities of cystic fibrosis. Our laboratory’s specific aim is to test the hypothesis that CFTR, a known Cl- channel, directly regulates extracellular pH through other processes.
We believe abnormal extracellular pH might be an important player in CF-related abnormalities (via HCO3- ion conductance and/or regulated activity of the exchangers). With a new biosensor, the silicon microphysiometer, that can detect small changes (0.001 pH unit/min.) in the acid efflux of cultured cells, our laboratory characterizes the acidification response of cell over-expressing wild-type and mutant CFTR to elucidate whether the malfunction in cystic fibrosis alters normal pHo (and pHi), bicarbonate flux, Na/H exchange, and metabolic rate of the cell.
In addition to defective chloride ion transport, cystic fibrosis (CF)-associated mutations in the CFTR channel have also been reported to lead to changes in other channels, secretions, and pH. The application of microphysiometry to cystic fibrosis research has revealed previously undetectable extracellular pH changes associated with CF. Our laboratory is using microphysiometry to study the mechanism by which CFTR influences pH regulation. Investigating pH characteristics might elucidate novel effects of CFTR malfunction, and may have even greater value in developing a simple and reliable assay for CF cells. Screening assays are needed in the development of drug and gene therapies for CF. A very sensitive assay that can differentiate responses of CF cells from cells that have been corrected by gene or drug therapy would be invaluable. The microphysiometer technology, with its capacity to assay large numbers of cells with great sensitivity, makes these kinds of experiments feasible for the first time.
CFTR alters extracellular pH in C127 and NIH-3T3 cell lines. We tested the cAMP elevating agent forskolin (1 uM), which stimulates metabolic rate and activates CFTR. Pulse application of forskolin increased the acid efflux rates of cell without CFTR by 20% over basal rate likely due to increased metabolism. In contrast, forskolin decreased the acid efflux rate of CFTR cells likely due to bicarbonate efflux. We also found that the membrane permeable cAMP analog, cpt-cAMP (100 uM), elicited the same response. Thus, in cells expressing CFTR, elevated cAMP activates a sustained inhibitory component which blunts the metabolic component of acid efflux. In control experiments, dideoxyforskolin (10 uM), an inactive forskolin analog, had no effect on the acidification rates of either cell line, while a range of hypotonic solutions stimulated acidification rates equally in both cell lines. Thus not all currents have been altered by CFTR expression in these cells. In addition to those findings in C127 cell lines, we have tested NIH/3T3 mouse fibroblast cell lines that express CFTR. Basal and forskolin stimulated NIH3T3 cells responded much like the C127 lines did. Forskolin (10 uM) increased the acid efflux rates of control cells and produced an even larger decrease response in the CFTR expressing cell line. The findings in mouse fibroblasts supports the assertion that this effect is solely CFTR-dependent. We are currently testing the mechanism of this pH effect (read more here).
