Ca2+ release from the envelope of isolated pancreatic acinar nuclei could be activated by nicotinic acid adenine dinucleotide phosphate (NAADP) as well as by inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose (cADPR). Each of these agents reduced the Ca2+ concentration inside the nuclear envelope, and this was associated with a transient rise in the nucleoplasmic Ca2+ concentration. NAADP released Ca2+ from the same thapsigargin-sensitive pool as IP3. The NAADP action was specific because, for example, nicotineamide adenine dinucleotide phosphate was ineffective. The Ca2+ release was unaffected by procedures interfering with acidic organelles (bafilomycin, brefeldin, and nigericin). Ryanodine blocked the Ca2+-releasing effects of NAADP, cADPR, and caffeine, but not IP3. Ruthenium red also blocked the NAADP-elicited Ca2+ release. IP3 receptor blockade did not inhibit the Ca2+ release elicited by NAADP or cADPR. The nuclear envelope contains ryanodine and IP3 receptors that can be activated separately and independently; the ryanodine receptors by either NAADP or cADPR, and the IP3 receptors by IP3.

We have determined the localization of the Golgi with respect to other organelles in living pancreatic acinar cells and the importance of this localization to the establishment of Ca 2؉ gradients over the Golgi. Using confocal microscopy and the Golgi-specific fluorescent probe 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine, we found Golgi structures localizing to the outer edge of the secretory granular region of individual acinar cells. We also assessed Golgi positioning in acinar cells located within intact pancreatic tissue using two-photon microscopy and found a similar localization. The mitochondria segregate the Golgi from lateral regions of the plasma membrane, the nucleus, and the basal part of the cytoplasm. The Golgi is therefore placed between the principal Ca 2؉ release sites in the apical region of the cell and the important Ca 2؉ sink formed by the peri-granular mitochondria. During acetylcholine-induced cytosolic Ca 2؉ signals in the apical region, large Ca 2؉ gradients form over the Golgi (decreasing from trans-to cis-Golgi). We further describe a novel, close interaction of the peri-granular mitochondria and the Golgi apparatus. The mitochondria and the Golgi structures form very close contacts, and these contacts remain stable over time. When the cell is forced to swell, the Golgi and mitochondria remain juxtaposed up to the point of cell lysis. The strategic position of the Golgi (closer to release sites than the bulk of the mitochondrial belt) makes this organelle receptive to local apical Ca 2؉ transients. In addition the Golgi is ideally placed to be preferentially supplied by ATP from adjacent mitochondria.The pancreatic acinar cell is the classical model for studies of the secretory pathway. In the pancreatic acinar cell, Palade (1) first described the segregation, transport, and discharge of secretory proteins. Ca 2ϩ regulates processing of secretory proteins within (2) and transport along the secretory pathway (3-5). In turn many of the membrane-bound compartments of the secretory pathway serve as a source and a sink for Ca 2ϩ (6 -9). Ca 2ϩ signaling and the secretory pathway are inextricably tied in pancreatic acinar cells (10). Structural and functional polarity is an important aspect of this reciprocal relationship (11-13). Initiation of the secretory pathway occurs at the basolateral part of the cell with protein synthesis and terminates with Ca 2ϩ -dependent secretion at the apical membrane (14 -16).The endoplasmic reticulum (ER) 1 and nucleus are located in the basolateral part of the cell, whereas the secretory granules (SGs) are located in the apical pole (1,11,17). The most striking polarization is that of the mitochondria with three distinct groupings: peri-granular, sub-plasmalemmal, and peri-nuclear (18 -22).In this study we examined the localization of the Golgi with respect to other cellular organelles and the functional consequences of this localization. We found close Golgi-mitochondria contacts that remain remarkably stable over time and during dif...

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