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.

In pancreatic acinar cells low (physiological) agonist concentrations evoke cytosolic Ca2+ spikes specifically in the apical secretory pole that contains a high density of secretory (zymogen) granules (ZGs). Inositol 1,4,5-trisphosphate (IP3) is believed to release Ca2+ from the endoplasmic reticulum, but we have now tested whether the Ca(2+)-releasing messengers IP3 and cyclic ADP-ribose (cADPr) can liberate Ca2+ from AGs. In experiments on single isolated ZGs, we show using confocal microscopy that IP3 and cADPr evoke a marked decrease in the free intragranular Ca2+ concentration. Using a novel high resolution method, we have measured changes in the Ca2+ concentration in the vicinity of an isolated AG and show that IP3 and cADPr cause rapid Ca2+ release from the granule, explaining the agonist-evoked cytosolic Ca2+ rise in the secretory pole.

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca 2+ concentration inside pancreatic acinar cells ([Ca 2+ ] i ), due to excessive release of Ca 2+ stored inside the cells followed by Ca 2+ entry from the interstitial fluid. The sustained [Ca 2+ ] i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca 2+ release-activated Ca 2+ channels (CRAC) would prevent sustained elevation of [Ca 2+ ] i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca 2+ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca 2+ . Ca 2+ entry then occurred upon admission of Ca 2+ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca 2+ entry in a concentrationdependent manner within the range of 1 to 50 μM (IC 50 = 3.4 μM), but had little or no effect on the physiological Ca 2+ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca 2+ ] i , which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca 2+ ] i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.capacitative Ca 2+ entry | alcohol metabolite | pancreas | hepatocyte Ca 2+ entry | AR42JA cute pancreatitis is a human disease mostly caused by alcohol abuse or complications from biliary disease. In this disease, against which there is currently no effective therapy, digestive proenzymes are prematurely activated inside the acinar cells leading to autodigestion and necrosis (1-3). Intracellular Ca 2+ plays a critical role in the initiation of this disease process (2-4), but intracellular Ca 2+ also plays a critical role in the physiological regulation of the normal exocytotic secretion of the digestive proenzymes (5).The pancreatic acinar cells are capable of generating multiple patterns of cytosolic Ca 2+ signals depending on the type and concentration of the stimulating agent (5). The physiological Ca 2+ signals regulating secretion-evoked by the neurotransmitter acetylcholine (ACh) or the hormone cholecystokinin (CCK)-consist of repetitive short-lasting rises in the cytosolic Ca 2+ concentration ([Ca 2+ ] i ). These are mostly confined to the apical area, in which the secretory (zymogen) granules (ZGs) are concentrated, by a belt of perigranular mitochondria operating as a firewall against...

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.

Hormones and neurotransmitters mobilize Ca 2+ from the endoplasmic reticulum via inositol trisphosphate (IP 3 ) receptors, but how a single target cell encodes different extracellular signals to generate speci®c cyto-solic Ca 2+ responses is unknown. In pancreatic acinar cells, acetylcholine evokes local Ca 2+ spiking in the apical granular pole, whereas cholecystokinin elicits a mixture of local and global cytosolic Ca 2+ signals. We show that IP 3 , cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) evoke cytosolic Ca 2+ spiking by activating common oscillator units composed of IP 3 and ryanodine receptors. Acetylcholine activation of these common oscillator units is triggered via IP 3 receptors, whereas cholecystokinin responses are triggered via a different but converging pathway with NAADP and cyclic ADP-ribose receptors. Cholecystokinin potentiates the response to acetylcholine, making it global rather than local, an effect mediated speci®cally by cyclic ADPribose receptors. In the apical pole there is a common early activation site for Ca 2+ release, indicating that the three types of Ca 2+ release channels are clustered together and that the appropriate receptors are selected at the earliest step of signal generation.

SummaryNAADP, cADPR and IP 3 all release Ca 2+ from the endoplasmic reticulum and an acidic store in the secretory granule area