Uptake and release of Ca2+ from isolated liver nuclei were studied with fluorescent probes. We show with the help of digital imaging and confocal microscopy that the Ca(2+)-sensitive fluorescent probe Fura 2 is concentrated in or around the nuclear envelope and that the distribution of Fura 2 fluorescence is similar to that of an endoplasmic reticulum marker. The previously demonstrated ATP-dependent uptake of Ca2+ into isolated nuclei and release of the accumulated Ca2+ by inositol 1,4,5-trisphosphate (IP3) are therefore due to transport of Ca2+ into and out of the nuclear envelope and not the nucleoplasm. Dextrans labeled with fluorescent Ca2+ indicators (calcium-Green 1 and Fura 2) are distributed uniformly in the nucleoplasm and can be used to show that changes in the external Ca2+ concentration produce rapid changes in the nucleoplasmic Ca2+ concentration. Nevertheless, IP3 and cyclic ADP-ribose evoke transient intranuclear Ca2+ elevations. The release from the Ca2+ stores in or around the nuclear envelope appears to be directed into the nucleoplasm from where it can diffuse out through the permeable nuclear pore complexes.

Agonist-evoked cytosolic Ca 2⍣ spikes in mouse pancreatic acinar cells are specifically initiated in the apical secretory pole and are mostly confined to this region. The role played by mitochondria in this process has been investigated. Using the mitochondria-specific fluorescent dyes MitoTracker Green and Rhodamine 123, these organelles appeared as a bright belt concentrated mainly around the secretory granule area. We tested the effects of two different types of mitochondrial inhibitor on the cytosolic Ca 2⍣ concentration using simultaneous imaging of Ca 2⍣ -sensitive fluorescence (Fura 2) and electrophysiology. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied in the presence of the Ca 2⍣ -releasing messenger inositol 1,4,5-trisphosphate (IP 3 ), the local repetitive Ca 2⍣ responses in the granule area were transformed into a global rise in the cellular Ca 2⍣ concentration. In the absence of IP 3 , CCCP had no effect on the cytosolic Ca 2⍣ levels. Antimycin and antimycin ⍣ oligomycin had the same effect as CCCP. Active mitochondria, strategically placed around the secretory pole, block Ca 2⍣ diffusion from the primary Ca 2⍣ release sites in the granule-rich area in the apical pole to the basal part of the cell containing the nucleus. When mitochondrial function is inhibited, this barrier disappears and the Ca 2⍣ signals spread all over the cytosol.

A number of specific cellular Ca2+ uptake pathways have been described in many different cell types [1] [2] [3]. The possibility that substantial quantities of Ca2+ could be imported via endocytosis has essentially been ignored, although it has been recognized that endosomes can store Ca2+ [4] [5]. Exocrine cells can release significant amounts of Ca2+ via exocytosis [6], so we have investigated the fate of Ca2+ taken up via endocytosis into endosomes. Ca2+-sensitive and H+-sensitive fluorescent probes were placed in the extracellular solution and subsequently taken up into fibroblasts by endocytosis. Confocal microscopy was used to assess the distribution of fluorescence intensity. Ca2+ taken up by endocytosis was lost from the endosomes within a few minutes, over the same period as endosomal acidification took place. The acidification was inhibited by reducing the extracellular Ca2+ concentration, and Ca2+ loss from the endosomes was blocked by bafilomycin (100 nM), a specific inhibitor of the vacuolar proton ATPase. Quantitative evaluation indicated that endocytosis causes substantial import of Ca2+ because of rapid loss from early endosomes.