Measurement of delta pH and membrane potential in isolated neurosecretory vesicles from bovine neurohypophyses.

The steady state concentration ratios of [3H]methylamine and [14C]SCN- were measured to determine the pH gradient and electrical potential, respectively, across the membrane of purified, intact, neurosecretory vesicles from bovine posterior pituitary. The intravesicular pH was 5.8 when the medium pH was 7. Varying the external pH had only a small effect on the intravesicular pH and resulted in a change in the pH gradient. Extrapolation to zero pH gradient yielded a value of 5.5 for the intravesicular pH. When the medium pH was 7, the pH gradient collapsed in the presence of ammonium sulfate or the combination of nigericin and K+. Measurement of the thiocyanate distribution across the vesicle membrane suggested the existence of an inside negative membrane potential. Mg2+ ATP caused a marked shift in the potential to +50 mV (inside positive) without altering the intravesicular pH. This shift was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The possible roles of the large pH gradient and membrane potential in the storage of peptide hormones and their secretion is discussed.