One of the tragic aspects of CF is the fact that a defect in the activity of a cyclic AMP regulated chloride channel can lead to such a wide range of pathological characteristics, including thick, sticky mucus, altered cell surface biochemistry and increased bacterial binding and growth. It has been difficult to rationalize all these pathologies solely in terms of a reduction in chloride transport. Therefore, the goal of our CFRI-sponsored research has been to test the possibility that changes in acidity (normally expressed in units of pH on a logarithmic scale) in and around cells may also be involved. Recall that acidity levels increase as pH levels decline. And note as an example, a change of pH from 7.4 to 7.1 would double the level of acidity.
Acidity in cells and tissues and subcellular organelles (structures specialized for protein processing) is precisely regulated because enzymes are exquisitely pH-sensitive, and several pH-sensitive enzymes are located in cell organelles that regulate proper processing and trafficking of mucus and surface membrane structures. It has been proposed that changes in organelle pH could contribute to the altered mucus and surface membrane biochemistry that lead to increased bacterial binding and resulting pathology of CF. It also seems likely that the surface pH of tracheal cells is important in determining bacterial binding and growth. Welsh and his colleagues at the University of Iowa have shown that airway fluid in CF cells is more acidic than normal cells.
How might Cystic Fibrosis Transmembrane-conductance Regulator (CFTR), a chloride channel, affect pH of cells and organelles? One possibility is by conducting bicarbonate (HCO3) which is present in all cells. When the concentration of bicarbonate increases or decreases, the acidity decreases or increases.
My colleagues and I have used patch clamp and other electrophysiological methods to show that CFTR in the membrane of normal airway cells has a small, but finite bicarbonate conductance and that this small conductance to bicarbonate is absent in CF tracheal cells. We also showed, using digital imaging microscopy of pH-sensitive fluorescent dyes, that permeation of bicarbonate through CFTR can affect cytosolic pH in airway cells. Thus, changes in CFTR activity could play a role in the regulation of the pH of cells and airway fluid: when bicarbonate is transported from cells through CFTR to the tracheal lumen, the pH of the cells should decrease while that of the airway fluid should increase. Our findings may explain why CF airway fluid is more acidic than normal, and may also be relevant in explaining the pathology of the liver, intestine and pancreas, tissues that normally secrete a great deal of bicarbonate.
CFTR might also control indirectly the pH of organelles responsible for biochemical processing of mucins or membrane proteins that are exposed in the airway lumen. Most cell organelles are acidic, and an H-pump (acid pump) that requires chloride to maintain its activity is responsible for this acidity. CFTR might therefore control organelle pH indirectly through its effects on chloride transport. Our approach has been to utilize pH-sensitive fluorescent dyes and a digital imaging microscope that allows resolution at the cellular and subcellular level to make these measurements.
In collaboration with my Berkeley colleague Professor Hsiao-Ping Moore and several students from her lab, we have developed a method that allows direct measurement of pH in organelles in living cells. We generate DNA that code for proteins with two critical portions, one that directs the protein to a specific organelle and a second that specifically binds a fluorescent dye. We transfect cells with this DNA, and the cells that express the fusion proteins are exposed to pH-sensitive fluorescent dyes which accumulate in the organelle where the fusion protein is expressed. We then use our digital imaging microscope to measure fluorescence from the organelles that bind the dye. We can also measure rates at which the organelles move from their cytosolic location to the plasma membrane. We are particularly interested in organelles that play key roles in the biochemical maturation and processing of CFTR and other proteins that are thought to be involved in CF pathology. We have found in fibroblasts (cells giving rise to connective tissue) that two key organelles are acidic (pH 6.2-6.5), that this acidity is regulated by an H-pump (acid pump) and a leak for acid, and that inhibiting the pump leads to reductions in the rates of traffic from the organelles to the plasma membrane. This demonstrates the importance of organelle luminal acidity for membrane traffic. We are now applying this method to CF and CFTR-corrected CF epithelial cells. These studies may lead to alternate therapies (that is, in addition to gene therapy) in the treatment of CF.
Our studies have been supported by CFRI, and beginning October 1996, by the National Institutes of Health (NIH). My colleagues and I are grateful for CFRI's crucial support in establishing and maintaining a thriving CF research community in the Bay Area.
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