The long term goal of this project is to design combinations of metabolic and proton extrusion inhibitory drugs to selectively and specifically lower the intracellular pH (pHi) in cells in an environment acidic relative to normal tissues. Cells adapted to growth at low pHe develop an increased capacity for extrusion of metabolic protons so that they maintain a higher pHi than acutely acidified unadapted cells. Proton extrusion mechanisms in adapted cells behave differently from those in cells growing at pHe 7.3. The threshold to active proton extrusion requires greater intracellular acidification in adapted than in non- adapted cells, although inhibition of the lactate:H+ symport by CNCn eliminates that difference. Adapted cells do not require the Cl-/HCO3- exchanger as do acutely acidified non-adapted cells. Single inhibitors of proton extrusion are more effective inducing intracellular acidification in acutely acidified non-adapted cells so that both populations will exhibit the same degree of intracellular acidification. Furthermore, cells adapted to growth at low pH when subjected to the same acute extracellular acidification as cells growing at pH 7.3 will exhibit a greater response to combinations of inhibitors, in particular DIDS, cinnamic acid and mIBG. The hypotheses will be tested in human melanoma cells, adapted or non- adapted to growth at low pHe, by determining: 1) The effect of acute extracellular acidification on intracellular acidification down to pHe=6.1; 2) The effect of 42 degrees-hyperthermia on the kinetics of intracellular realkalinization after acute acidification to pHe 6.5 or 6.3; 3) The effect of inhibitors of proton """"""""demand"""""""" (extrusion) and proton """"""""supply"""""""" (metabolism) on pHi during acute extracellular acidification; 4) The effect of the inhibitors on the specific rate of proton extrusion using the ammonium chloride technique; and 5) The intracellular acidification and the rate of realkalinization in melanoma xenograft microfragments after extracellular acidification. This laboratory's unique fluorescence system allows pHi to be monitored during the actual heating of cells in the presence or absence of inhibitors while adherent to a growth substrate, in normal medium with HCO3-/CO2 buffering and under euoxic versus hypoxic conditions. The findings will provide critical insight into the nature of proton extrusion mechanisms in human melanoma as a function of physiologic state. Furthermore, the results will allow the other three projects to draw conclusions related to mechanisms of intracellular acidification and sensitization to hyperthermia.
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