Experiments were performed to determine the internal pH and membrane potential of platelet alpha-granules. Fluorescence microscopy showed accumulation of weak bases, indicative of an acidic interior, inside secretory vesicles in intact platelets and in isolated alpha-granules. Weak base uptake was pH-dependent and NH+4-sensitive. In isolated alpha-granules suspended in medium buffered at pH 7.2, a delta pH, the difference between internal and external pH, of 1.2 (inside acidic) was measured by [14C]methylamine distribution. Uptake of isotopic or fluorescent amines was reduced by H+/cation exchange via ionophores and by addition of NH+4, but also by increasing the ionic strength suggesting that delta pH is partly due to a Donnan potential. Transmembrane potential measurements by fluorescent or radioactive ion distribution indicated that in the absence of ATP, granules are internally negative. When measured with 86Rb+, this potential could be entirely collapsed by increasing the ionic strength. Addition of ATP X Mg in the absence of permeating anions made the intragranular space more positive, as expected from inward electrogenic H+ pumping. The results are compatible with the coexistence of sealed and leaky subpopulations of alpha-granules. Internal acidity was generated in sealed granules in vivo by a H+-pumping ATPase, whereas in leaky granules acidity is a consequence of an internally negative Donnan potential.
Ammonia Animals