Differential involvement of vacuolar H+-ATPase in the refilling of thapsigargin- and agonist-mobilized Ca2+ stores

Our objective was to evaluate the role of vacuolar H+-ATPase and proton gra dients in the refilling of Ca2+ stores in fura-2-loaded pancreatic acinar c ells. Once depleted with a high level of ACh, the Ca2+ stores were replenis hed with a Ca2+-containing solution. The degree of refilling was estimated with a second release in response to either ACh (ACh-releasable store) or t hapsigargin (thapsigargin-releasable store), a specific inhibitor of the en doplasmic reticulum Ca2+ pumps. Both the protonophore nigericin and folimyc in, a specific inhibitor of the vacuolar H+-ATPase, reduced reuptake into t he ACh-mobilized stores but not into the thapsigargin-releasable pools. The se treatments effectively dissipated the subcellular pH gradients (revealed by confocal observation of the distribution of a marker for acidic compart ments), and did not impair the [Ca2+](i) response to ACh in control cells. Our results indicate that thapsigargin and ACh release heterogeneous Ca2+ s tores which are differently operated by vacuolar proton ATPase. (C) 2000 Ac ademic Press.