Formation and biological action of inositol 1,4,5-trisphosphate

A wide variety of receptors appear to be coupled to a phospholipase C (EC 3.1.4.3) that hydrolyzes inositol lipids. This reaction is believed to provide a link between receptor activation and cellular Ca2+ mobilization. The mechanisms by which this occurs are believed to involve inositol 1,4,5-trisphosphate (1,4,5-IP3), which signals release of Ca2+ from the endoplasmic reticulum. In rat parotid acinar cells made permeable with saponin, 1,4,5-IP3 induced rapid release of sequestered Ca2+. In intact parotid cells, the concentration-response relationship for methacholine-induced IP3 formation was similar to the relationship for muscarinic receptor occupancy by methacholine. About 10-fold lower concentrations of methacholine were sufficient to increase cytosolic [Ca2+] and to activate secretion, indicating an excess IP3 forming capacity for the muscarinic receptor. The mechanisms for the coupling of receptors to IP3 formation were studied in pancreatic acinar cells made permeable electrically. In this preparation, nonhydrolyzable derivatives of GTP potentiated agonist-induced IP3 production, which suggests the involvement of a guanine nucleotide-dependent regulatory protein. The effects of agonists and guanine nucleotides were not altered by pretreating the acinar cells with cholera or pertussis toxins, which indicated that the regulatory protein linking receptors to IP3 formation is distinct from the ones involved in the regulation of adenylate cyclase.     

Homer 2 tunes G protein-coupled receptors stimulus intensity by regulating RGS proteins and PLCbeta GAP activities

Homers are scaffolding proteins that bind G protein-coupled receptors (GPCRs), inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), ryanodine receptors, and TRP channels. However, their role in Ca2+ signaling in vivo is not known. Characterization of Ca2+ signaling in pancreatic acinar cells from Homer2-/- and Homer3-/- mice showed that Homer 3 has no discernible role in Ca2+ signaling in these cells. In contrast, we found that Homer 2 tunes intensity of Ca2+ signaling by GPCRs to regulate the frequency of [Ca2+]i oscillations. Thus, deletion of Homer 2 increased stimulus intensity by increasing the potency for agonists acting on various GPCRs to activate PLCbeta and evoke Ca2+ release and oscillations. This was not due to aberrant localization of IP3Rs in cellular microdomains or IP3R channel activity. Rather, deletion of Homer 2 reduced the effectiveness of exogenous regulators of G proteins signaling proteins (RGS) to inhibit Ca2+ signaling in vivo. Moreover, Homer 2 preferentially bound to PLCbeta in pancreatic acini and brain extracts and stimulated GAP activity of RGS4 and of PLCbeta in an in vitro reconstitution system, with minimal effect on PLCbeta-mediated PIP2 hydrolysis. These findings describe a novel, unexpected function of Homer proteins, demonstrate that RGS proteins and PLCbeta GAP activities are regulated functions, and provide a molecular mechanism for tuning signal intensity generated by GPCRs and, thus, the characteristics of [Ca2+]i oscillations.     

NAADP-induced calcium release in sea urchin eggs

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores described. It acts on a mechanism distinct from inositol trisphosphate and ryanodine receptors, the two major Ca2+ release channels characterised. NAADP-gated Ca2+ release channels do not appear to be regulated by Ca2+ and may be better suited for triggering Ca2+ signals rather than propagating them. They exhibit a remarkable pharmacology for a putative intracellular Ca2+ release channel in that they are selectively blocked by potassium and L-type Ca2+ channel antagonists. Furthermore, in contrast to microsomal Ca2+ stores expressing IP3Rs and RyRs, those sensitive to NAADP are thapsigargin-insensitive, suggesting that they may be expressed on a different part of the endoplasmic reticulum. Perhaps the most unusual feature of the NAADP-gated Ca2+ release mechanisms is its inactivation properties. Unlike the mechanisms regulated by IP3 and cADPR in sea urchin eggs which after induction of Ca2+ release appear to become refractory to subsequent activation, very low concentrations of NAADP are able to inactivate NAADP-induced Ca2+ release fully at concentrations well below those required to activate Ca2+ release. The mechanism and physiological significance of this most unusual desensitisation phenomenon are unclear. More recently, NAADP has been shown to mobilise Ca2+ in ascidian oocytes, brain microsomes and pancreatic acinar cells suggesting a more widespread role in Ca2+ signalling. A possible role for this novel Ca2+ release mechanism in sea urchin egg fertilisation is discussed.     

Uncoupled IP3 receptor can function as a Ca2+-leak channel: cell biological and pathological consequences

Ca(2+) release via intracellular release channels, IP(3)Rs (inositol 1,4,5-trisphosphate receptors) and RyRs (ryanodine receptors), is perhaps the most ubiquitous and versatile cellular signalling mechanism, and is involved in a vast number of cellular processes. In addition to this classical release pathway there is limited, but yet persistent, information about less well-defined Ca(2+)-leak pathways that may play an important role in the control of the Ca(2+) load of the endo(sarco)plasmic reticulum. The mechanisms responsible for this 'basal' leak are not known, but recent data suggest that both IP(3)Rs and RyRs may also operate as Ca(2+)-leak channels, particularly in pathological conditions. Proteolytic cleavage or biochemical modification (such as hyperphosphorylation or nitrosylation), for example, occurring during conditions of cell stress or apoptosis, can functionally uncouple the cytoplasmic control domains from the channel domain of the receptor. Highly significant information has been obtained from studies of malfunctioning channels in various disorders; for example, RyRs in cardiac malfunction or genetic muscle diseases and IP(3)Rs in neurodegenerative diseases. In this review we aim to summarize the existing information about functionally uncoupled IP(3)R and RyR channels, and to discuss the concept that those channels can participate in Ca(2+)-leak pathways.     

CCK-JMV-180, an analog of cholecystokinin, releases intracellular calcium from an inositol trisphosphate-independent pool in rat pancreatic acini.

In pancreatic acinar cells cholecystokinin and its analogs, caerulein and CCK-JMV-180, stimulate an increase in intracellular free [Ca2+] by releasing Ca2+ from non-mitochondrial intracellular pools. It is generally believed that the caerulein-induced release of Ca2+ is mediated by phospholipase C-catalyzed production of 1,4,5-inositol triphosphate (IP3). In this study we have investigated the source and mechanism of Ca2+ release induced by CCK-JMV-180 using streptolysin O-permeabilized pancreatic acinar cells. Caerulein-stimulated release of Ca2+ was completely blocked by either neomycin, an inhibitor of phospholipase C, or by heparin, an IP3 receptor antagonist. These observations are compatible with the conclusion that caerulein releases Ca2+ from an IP3-sensitive pool. In contrast to caerulein, however, CCK-JMV-180-stimulated release of Ca2+ was not blocked by either neomycin or by heparin, indicating that CCK-JMV-180 releases Ca2+ by mechanisms which do not involve the generation or action of IP3. CCK-JMV-180 stimulated the release of Ca2+ even after the IP3-sensitive pool had been completely emptied by prior exposure to a supramaximally stimulating concentration of IP3 (40 microM). Prestimulation of permeabilized acini with 20 mM caffeine did not abolish the CCK-JMV-180-induced Ca2+ release. These results indicate that CCK-JMV-180 stimulates release of Ca2+ from a hitherto uncharacterized intracellular storage pool which is insensitive to either IP3 or caffeine.     

Spatial microenvironment defines Ca2+ entry and Ca2+ release in salivary gland cells

The difference of Ca(2+) mobilization induced by muscarinic receptor activation between parotid acinar and duct cells was examined. Oxotremorine, a muscarinic-cholinergic agonist, induced intracellular Ca(2+) release and extracellular Ca(2+) entry through store-operated Ca(2+) entry (SOC) and non-SOC channels in acinar cells, but it activated only Ca(2+) entry from non-SOC channels in duct cells. RT-PCR experiments showed that both types of cells expressed the same muscarinic receptor, M3. Given that ATP activated the intracellular Ca(2+) stores, the machinery for intracellular Ca(2+) release was intact in the duct cells. By immunocytochemical experiments, IP(3)R2 colocalized with M3 receptors in the plasma membrane area of acinar cells; in duct cells, IP(3)R2 resided in the region on the opposite side of the M3 receptors. On the other hand, purinergic P2Y2 receptors were found in the apical area of duct cells where they colocalized with IP(3)R2. These results suggest that the expression of the IP(3)Rs near G-protein-coupled receptors is necessary for the activation of intracellular Ca(2+) stores. Therefore, the microenvironment probably affects intracellular Ca(2+) release and Ca(2+) entry.     

The predominant role of IP₃ type 1 receptors in activation of store-operated Ca²+ entry in liver cells

Physiologically, hormone induced release of Ca2+ from intracellular stores occurs in response to inositol 1,4,5-trisphosphate (IP?) binding to its receptors expressed on the membranes of intracellular organelles, mainly endoplasmic reticulum. These IP? receptors act as channels, releasing Ca2+ into the cytoplasmic space where it is responsible for regulating a host of distinct cellular processes. The depletion of intracellular Ca2+ stores leads to activation of store-operated Ca2+ channels on the plasma membrane which replenishes lost Ca2+ and sustain Ca2+ signalling. There are three isoforms of IP? receptor, each exhibiting distinctive properties, however, little is known about the role of each isoform in the activation of store-operated Ca2+ entry. Recent evidence suggest that at least in some cell types the endoplasmic reticulum is not a homogeneous Ca2+ store, and there might be a sub-compartment specifically linked to the activation of store-operated Ca2+ channels, and Ca2+ release activated Ca2+ (CRAC) channel in particular. Furthermore, this sub-compartment might express only certain types of IP? receptor but not the others. Here we show that H4IIE liver cells express all three types of IP? receptor, but only type 1 and to a lesser extent type 3, but not type 2, participate in the activation of CRAC current (I(CRAC)), while type 1 and type 2, but not type 3, participate in observed Ca2+ release in response to receptor stimulation. Presented results suggest that in H4IIE rat liver cells the sub-compartment of intracellular Ca2+ store linked to the activation of I(CRAC) predominantly expresses type 1 IP? receptors.     

Two neuropeptides recruit different messenger pathways to evoke Ca2+ signals in the same cell

Bombesin and cholecystokinin (CCK) peptides act as signalling molecules in both the central nervous system and gastrointestinal tract [1-4]. It was reported recently that nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca2+ from mammalian brain microsomes [5] and triggers Ca2+ signals in pancreatic acinar cells, where it is proposed to mediate CCK-evoked Ca2+ signals [6]. Here, for the first time, we have finely resolved bombesin-induced cytosolic Ca2+ oscillations in single pancreatic acinar cells by whole-cell patch-clamp monitoring of Ca2+-dependent ionic currents [6-8]. Picomolar concentrations of bombesin and CCK evoked similar patterns of cytosolic Ca2+ oscillations, but high, desensitising, NAADP concentrations selectively inhibited CCK, but not bombesin-evoked signals. Inhibiting inositol trisphosphate (IP3) receptors with a high concentration of caffeine blocked both types of oscillations. We further tested whether NAADP is involved in Ca2+ signals triggered by activation of the low-affinity CCK receptor sites. Nanomolar concentrations of CCK evoked non-oscillatory Ca2+ signals, which were not affected by desensitising NAADP receptors. Our results suggest that Ca2+-release channels gated by the novel Ca2+-mobilising molecule NAADP are only essential in specific Ca2+-mobilising pathways, whereas the IP3 receptors are generally required for Ca2+ signals. Thus, the same cell may use different combinations of intracellular Ca2+-releasing messengers to encode different external messages.     

Effect of GTP and Ca2+ on inositol 1,4,5-trisphosphate induced Ca2+ release from permeabilized rat exocrine pancreatic acinar cells

The effects of Ca2+ and GTP on the release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3) sensitive Ca2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca2+ released due to IP3 directly correlated with the amount of stored Ca2+ and was found to be inversely proportional to the medium free Ca2+ concentration. Ca2+ release induced by 0.18 microM IP3 was half maximally inhibited at 0.5 microM free Ca2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca2+ release on its own, but a single addition of GTP (20 microM) abolished the apparent desensitization of the Ca2+ release which was observed during repeated IP3 applications. This effect of GTP was reversible. GTP gamma S could not replace GTP. Desensitization still occurred when GTP gamma S was added prior to GTP. The reported data indicate that GTP, stored Ca2+ and cytosolic free Ca2+ modulate the IP3 induced Ca2+ release.     

Bile acids induce Ca2+ release from both the endoplasmic reticulum and acidic intracellular calcium stores through activation of inositol trisphosphate receptors and ryanodine receptors

Gallstones can cause acute pancreatitis, an often fatal disease in which the pancreas digests itself. This is probably because of biliary reflux into the pancreatic duct and subsequent bile acid action on the acinar cells. Because Ca(2+) toxicity is important for the cellular damage in pancreatitis, we have studied the mechanisms by which the bile acid taurolithocholic acid 3-sulfate (TLC-S) liberates Ca(2+). Using two-photon plasma membrane permeabilization and measurement of [Ca(2+)] inside intracellular stores at the cell base (dominated by ER) and near the apex (dominated by secretory granules), we have characterized the Ca(2+) release pathways. Inhibition of inositol trisphosphate receptors (IP(3)Rs), by caffeine and 2-APB, reduced Ca(2+) release from both the ER and an acidic pool in the granular area. Inhibition of ryanodine receptors (RyRs) by ruthenium red (RR) also reduced TLC-S induced liberation from both stores. Combined inhibition of IP(3)Rs and RyRs abolished Ca(2+) release. RyR activation depends on receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), because inactivation by a high NAADP concentration inhibited release from both stores, whereas a cyclic ADPR-ribose antagonist had no effect. Bile acid-elicited intracellular Ca(2+) liberation from both the ER and the apical acidic stores depends on both RyRs and IP(3)Rs.     

Nicotinic acid adenine dinucleotide phosphate triggers Ca2+ release from brain microsomes.

Mobilization of Ca2+ from intracellular stores is an important mechanism for generating cytoplasmic Ca2+ signals [1]. Two families of intracellular Ca(2+)-release channels - the inositol-1,4, 5-trisphosphate (IP3) receptors and the ryanodine receptors (RyRs) - have been described in mammalian tissues [2]. Recently, nicotinic acid adenine dinucleotide phosphate (NAADP), a molecule derived from NADP+, has been shown to trigger Ca2+ release from intracellular stores in invertebrate eggs [3] [4] [5] [6] and pancreatic acinar cells [7]. The nature of NAADP-induced Ca2+ release is unknown but it is clearly distinct from the IP3- and cyclic ADP ribose (cADPR)-sensitive mechanisms in eggs (reviewed in [8] [9]). Furthermore, mammalian cells can synthesize and degrade NAADP, suggesting that NAADP-induced Ca2+ release may be widespread and thus contribute to the complexity of Ca2+ signalling [10] [11]. Here, we show for the first time that NAADP evokes Ca2+ release from rat brain microsomes by a mechanism that is distinct from those sensitive to IP3 or cADPR, and has a remarkably similar pharmacology to the action of NAADP in sea urchin eggs [12]. Membranes prepared from the same rat brain tissues are able to support the synthesis and degradation of NAADP. We therefore suggest that NAADP-mediated Ca2+ signalling could play an important role in neuronal Ca2+ signalling.     

NAADP mobilizes Ca2+ from a thapsigargin-sensitive store in the nuclear envelope by activating ryanodine receptors

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.     

Long distance communication between muscarinic receptors and Ca2+ release channels revealed by carbachol uncaging in cell-attached patch pipette

We have investigated the characteristics of cytosolic Ca2+ signals induced by muscarinic receptor activation of pancreatic acinar cells that reside within intact pancreatic tissue. We show that these cells exhibit global Ca2+ waves and local apical Ca2+ spikes. This is the first evidence for local Ca2+ signaling in undissociated pancreatic tissue. The mechanism of formation of localized Ca2+ signals was examined using a novel approach involving photolysis of caged carbachol inside a patch pipette attached to the basal surface of an acinar unit. This local activation of basal muscarinic receptors elicited local cytosolic Ca2+ spikes in the apical pole more than 15 microm away from the site of stimulation. In some experiments, local basal receptor activation elicited a Ca2+ wave that started in the apical pole and then spread toward the base. Currently, there are two competing hypotheses for preferential apical Ca2+ signaling. One invokes the need for structural proximity of the cholinergic receptors and the Ca2+ release channels in the apical pole, whereas the other postulates long distance communication between basal receptors and the channels. Our intrapipette uncaging experiments provide definitive evidence for long distance communication between basal muscarinic receptors and apical Ca2+ release channels.     

IP(3)and cyclic ADP-ribose induced Ca(2+) release from intracellular stores of pancreatic acinar cells from rat in primary culture

We have measured Ca(2+)concentration changes in intracellular Ca(2+)stores ([Ca(2+)](store)) of rat pancreatic acinar cells in primary culture in response to the Ca(2+)mobilizing substances inositol-1,4,5-trisphosphate (IP(3)) and cyclic ADP-ribose (cADPr) using the Ca(2+)-sensitive dye mag Fura-2. We found that in this cell model IP(3)releases Ca(2+)in a quantal manner. Higher Ca(2+)concentration in the stores allowed a response to lower IP(3)concentrations ([IP(3)]) indicating that the sensitivity of IP(3)receptors to IP(3)is regulated by the Ca(2+)concentration in the stores. Cyclic ADPr, that modifies 'Ca(2+)-induced-Ca(2+)-release' (CICR), was also able to release Ca(2+)from intracellular stores of pancreatic acinar cells in primary culture. In comparison to the Ca(2+)ionophore ionomycin, which induced a maximal decrease (100%) in [Ca(2+)](store), a hypermaximal [IP(3)] (10 microM) dropped [Ca(2+)](store)by 87% and cADPr had no further effect. Cyclic ADPr reduced [Ca(2+)](store)by only 56% and subsequent IP(3)addition caused further maximal decrease in [Ca(2+)](store). Furthermore, a maximal [IP(3)] caused the same decrease in [Ca(2+)](store)in all regions of the cell, whereas cADPr dropped the [Ca(2+)](store)between 20 and 80% in different cell regions. From these data we conclude that in primary cultured rat pancreatic acinar cells at least three types of Ca(2+)stores exist. One type possessing both cADPr receptors and IP(3)receptors, a second type possessing only IP(3)receptors, and a third type whose Ca(2+)can be released by ionomycin but neither by IP(3)nor by cADPr.     

Calmodulin and calcium-release channels

Calmodulin (CaM) is a ubiquitous cytosolic protein that plays a critical role in regulating cellular functions by altering the activity of a large number of ion channels. There are many examples for CaM directly mediating the feedback effects of Ca2+ on Ca2+ channels. Recently the molecular mechanisms by which CaM interacts with voltage-gated Ca2+ channels, Ca(2+)-activated K+ channels and ryanodine receptors have been clarified. CaM plays an important role in regulating these ion channels through lobe-specific Ca2+ detection. CaM seems to behave as a channel subunit. It binds at low [Ca2+] and undergoes conformational changes upon binding of Ca2+, leading to an interaction with another part of the channel to regulate its gating. Here we focus on the mechanism by which CaM regulates the inositol 1,4,5-trisphosphate receptor (IP3R). Although the IP3R is inhibited by CaM and by other CaM-like proteins in the presence of Ca2+, we conclude that CaM does not act as the Ca2+ sensor for IP3R function. Furthermore we discuss a novel Ca(2+)-induced Ca(2+)-release mechanism found in A7r5 (embryonic rat aorta) and 16HBE14o- (human bronchial mucosa) cells for which CaM acts as a Ca2+ sensor.     

Identification of cyclic ADP-ribose-dependent mechanisms in pancreatic muscarinic Ca(2+) signaling using CD38 knockout mice

We showed that muscarinic acetylcholine (ACh)-stimulation increased the cellular content of cADPR in the pancreatic acinar cells from normal mice but not in those from CD38 knockout mice. By monitoring ACh-evoked increases in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) using fura-2 microfluorimetry, we distinguished and characterized the Ca(2+) release mechanisms responsive to cADPR. The Ca(2+) response from the cells of the knockout mice (KO cells) lacked two components of the muscarinic Ca(2+) release present in wild mice. The first component inducible by the low concentration of ACh contributed to regenerative Ca(2+) spikes. This component was abolished by ryanodine treatment in the normal cells and was severely impaired in KO cells, indicating that the low ACh-induced regenerative spike responses were caused by cADPR-dependent Ca(2+) release from a pool regulated by a class of ryanodine receptors. The second component inducible by the high concentration of ACh was involved in the phasic Ca(2+) response, and it was not abolished by ryanodine treatment. Overall, we conclude that muscarinic Ca(2+) signaling in pancreatic acinar cells involves a CD38-dependent pathway responsible for two cADPR-dependent Ca(2+) release mechanisms in which the one sensitive to ryanodine plays a crucial role for the generation of repetitive Ca(2+) spikes.     

Two different but converging messenger pathways to intracellular Ca(2+) release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphosphate

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 specific cytosolic 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 specifically by cyclic ADP-ribose 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.     

Mechanism of Ca2+ wave propagation in pancreatic acinar cells.

An increase in cytosolic Ca2+ often begins as a Ca2+ wave, and this wave is thought to result from sequential activation of Ca(2+)-sensitive Ca2+ stores across the cell. We tested that hypothesis in pancreatic acinar cells, and since Ca2+ waves may regulate acinar Cl- secretion, we examined whether such waves also are important for amylase secretion. Ca2+ wave speed and direction was determined in individual cells within rat pancreatic acini using confocal line scanning microscopy. Both acetylcholine (ACh) and cholecystokinin-8 induced rapid Ca2+ waves which usually travelled in an apical-to-basal direction. Both caffeine and ryanodine, at concentrations that inhibit Ca(2+)-induced Ca2+ release (CICR), markedly slowed the speed of these waves. Amylase secretion was increased over 3-fold in response to ACh stimulation, and this increase was preserved in the presence of ryanodine. These results indicate that 1) stimulation of either muscarinic or cholecystokinin-8 receptors induces apical-to-basal Ca2+ waves in pancreatic acinar cells, 2) the speed of such waves is dependent upon mobilization of caffeine- and ryanodine-sensitive Ca2+ stores, and 3) ACh-induced amylase secretion is not inhibited by ryanodine. These observations provide direct evidence that Ca(2+)-induced Ca2+ release is important for propagation of cytosolic Ca2+ waves in pancreatic acinar cells.     

GTP-sensitivity of the energy-dependent Ca2+ storage pool in permeabilized pancreatic acinar cells

Isolated rabbit pancreatic acinar cells, permeabilized by saponin treatment and incubated in the presence of 0.1 microM free Ca2+, accumulated 3.3 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Part of this energy-dependent pool could be released by GTP in a polyethylene glycol-dependent manner. The kinetics of GTP-induced release of Ca2+ showed a biphasic pattern with an initial rapid phase followed by a sustained slower phase. In contrast, IP3-induced release of Ca2+ was completed within 30 s following addition of IP3. No reuptake of Ca2+ was observed following GTP- or IP3-induced release of Ca2+. The GTP effect was independent of IP3 and not inhibited by Ca2+, indicating that the IP3-operated Ca2+ channel is not involved in GTP-induced release of Ca2+. The size of the IP3-releasable pool was not affected by GTP, indicating that GTP, when added to permeabilized acinar cells, does not promote the coupling between IP3-insensitive and IP3-sensitive Ca2+ accumulating organelles. Thus, in permeabilized acinar cells, GTP and IP3 act on different Ca2+ sequestering pools. Interestingly, however, comparison of the size of the GTP-releasable pool with that of the IP3-releasable pool for the cell preparations used in the present study, revealed an inversed relationship, indicating that at the time of permeabilization the GTP-releasable pool can be coupled to a greater or lesser extent to the IP3-releasable pool. This suggests that, in the intact cell, a GTP-dependent mechanism may exist that controls the size of the IP3-releasable pool by coupling IP3-insensitive to IP3-sensitive organelles. Moreover, this suggests that the extent of coupling is preserved during permeabilization.     

Effects of cholecystokinin on cytosolic calcium in human pancreatic cancer cells.

Cholecystokinin (CCK) has been shown to increase cytosolic calcium and stimulate enzyme release from pancreatic acinar cells and a rat acinar cell line, AR42J. CCK is also trophic to normal pancreas and pancreatic cancer; however, the cellular mechanisms which regulate CCK-stimulated growth are unknown. The effect of CCK on intracellular calcium was evaluated in four human pancreatic cancer cell lines known to grow in response to CCK but not secrete enzymes (SW-1990, MIA PaCa-2, BXPC-3 and PANC-1) and a rat acinar cell line (AR42J) shown to secrete enzymes but not grow with CCK. By using single cell fluorescence microscopy in fura-2 loaded cells, intracellular calcium [Ca2+]i was measured. After obtaining baseline fluorescent cell images, synthetic CCK-octapeptide (CCK8) was added to the cells and images of cell fluorescence captured. [Ca2+]i of the rat acinar cells increased (603%) over the baseline within the first minute after the addition of CCK (4.10(-13) M to 4.10(-10) M) in 77% of cells tested. In contrast [Ca2+]i failed to significantly change in the human cancer cells treated with CCK. To further localize the defect in hormone signal transduction in cancer cells, cells were suspended in low calcium media and the plasma membranes were selectively permeabilized with digitonin. Media free calcium concentration was continuously monitored by fura-2 fluorescence. Addition of inositol 1,4,5-trisphosphate (IP3) resulted in a marked increase in medium calcium concentration indicating IP3 was capable of releasing calcium from intracellular stores in both the AR42J rat acinar cell line and in the human pancreas cancer cell lines. In conclusion, CCK does not increase cytosolic calcium in human pancreatic cancer cells in contrast to rat acinar cells although all contain IP3-sensitive intracellular Ca2+ pools. Our results suggest that growth promoting and secretory effects of CCK on pancreatic cells may occur via two independent signalling pathways.