Aberrant Ca(2+) signalling through acidic calcium stores in pancreatic acinar cells

Pancreatic acinar cells possess a very large Ca²⁺ store in the endoplasmic reticulum, but also have extensive acidic Ca²⁺ stores. Whereas the endoplasmic reticulum is principally located in the baso-lateral part of the cells, although with extensions into the granular area, the acidic stores are exclusively present in the apical part. The two types of stores can be differentiated pharmacologically because the endoplasmic reticulum accumulates Ca²⁺ via SERCA pumps, whereas the acidic pools require functional vacuolar H⁺ pumps in order to maintain a high intra-organellar Ca²⁺ concentration. The human disease acute pancreatitis is initiated by trypsinogen activation in the apical pole and this is mostly due to either complications arising from gall bladder stones or excessive alcohol consumption. Attention has therefore been focussed on assessing the acute effects of bile acids as well as alcohol metabolites. The evidence accumulated so far indicates that bile acids and fatty acid ethyl esters - the non-oxidative products of alcohol and fatty acids - exert their pathological effects primarily by excessive Ca²⁺ release from the acidic stores. This occurs by opening of the very same release channels that are also responsible for normal stimulus-secretion coupling, namely inositol trisphosphate and ryanodine receptors. The inositol trisphosphate receptors are of particular importance and the results of gene deletion experiments indicate that the fatty acid ethyl esters mainly utilize sub-types 2 and 3.     

Ribosome-free Terminals of Rough ER Allow Formation of STIM1 Puncta and Segregation of STIM1 from IP3 Receptors

Store-operated Ca²⁺ entry is a ubiquitous mechanism that prevents the depletion of endoplasmic reticulum (ER) calcium [1]. A reduction of ER calcium triggers translocation of STIM proteins, which serve as calcium sensors in the ER, to subplasmalemmal puncta where they interact with and activate Orai channels ([2], [3], [4], [5], [6], [7] and [8]; reviewed in [9]). In pancreatic acinar cells, inositol 1,4,5-trisphosphate (IP₃) receptors populate the apical part of the ER. Here, however, we observe that STIM1 translocates exclusively to the lateral and basal regions following ER Ca²⁺ loss. This finding is paradoxical because the basal and lateral regions of the acinar cells contain rough ER (RER); the size of the ribosomes that decorate RER is larger than the distance that can be spanned by a STIM-Orai complex [5] and [10], and STIM1 function should therefore not be possible. We resolve this paradox and characterize ribosome-free terminals of the RER that form junctions between the reticulum and the plasma membrane in the basal and lateral regions of the acinar cells. Our findings indicate that different ER compartments specialize in different calcium-handling functions (Ca²⁺ release and Ca²⁺ reloading) and that any potential interference between Ca²⁺ release and Ca²⁺ influx is minimized by the spatial separation of the two processes.     

Extrusion of calcium from a single isolated neuron of the snailHelix pomatia

Summary Simultaneous optical measurements of extra- and intracellular Ca²⁺ concentrations were carried out on isolated snail neurons injected iontophoretically with Ca²⁺. The fluorescent indicator Fura-2 was used to measure intracellular concentration of free Ca, and the absorbant indicator Antipyrylazo III to measure changes in extracellular calcium concentration in the microchamber containing the cell. The velocity of Ca²⁺ extrusion from a single cell has been shown to be in accordance with the level of free Ca in the neuronal cytoplasm. After an increase in intracellular free Ca by iontophoretic injection from a microeletrode to 0.2–0.5 m, the velocity of Ca²⁺ extrusion from the neuron was approximately 0.3–4.6 m/sec per cell volume. During caffeine-induced calcium-dependent calcium release of Ca²⁺ from intracellular stores a stimulation of calcium extrusion took place, reaching the velocity of 5.0 m/sec per cell volume.     

Effect of reduced extracellular level of sodium ions on the intracellular level of calcium ions in the cytoplasm of cultured rat cardiomyocytes

Cytosolic free calcium [( Ca2+]in) was measured using fura-2 in isolated cultured ventricular myocytes of neonatal rat. Exposure of the cardiomyocyte to a solution in which all Na+ have been replaced by impermeable cations results in a 400-600 nmol/l increase of [Ca2+]in. This increase is followed by a slow decrease to the initial level. A decrease of the extracellular calcium concentration from 2.5 to 0.5 mmol./l or increase to 10 mmol/l produced, respectively, decrease and increase of the amplitude of [Ca2+]in rise in response to low-Na+ superfusion. Exposure of cardiomyocytes to low-Na+ solutions also led to a 2-3 fold increase of caffeine++-dependent Ca2+ release from intracellular stores. Changes in [Ca2+]in can be attributed to the operation of a sodium-calcium exchanger in heart cells.     

Energetic aspects of CO oxidation in desulfovibrio desulfuricans

In cell suspension of Desulfovibrio desulfuricans B-1388, oxidation of CO as the only energy source is associated with reduction of SO42-. After a 2-h incubation of cells in 8% CO, 81% of the gas is converted. Oxidation of 1 mole CO results in formation of 0.23 mole H2S. Intracellular ATP content increases from 2.5 (control) to 8.3 nmoles/mg (during CO conversion). Dinitrophenol inhibits sulfate reduction and CO oxidation. CO dehydrogenase was detected in cytoplasmic and membrane cell fractions (59 and 34%, respectively).     

Traffic of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway

The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)-dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.     

Feedback equilibrium control during human standing

Equilibrium maintenance during standing in humans was investigated with a 3-joint (ankle, knee and hip) sagittal model of body movement. The experimental paradigm consisted of sudden perturbations of humans in quiet stance by backward displacements of the support platform. Data analysis was performed using eigenvectors of motion equation. The results supported three conclusions. First, independent feedback control of movements along eigenvectors (eigenmovements) can adequately describe human postural responses to stance perturbations. This conclusion is consistent with previous observations (Alexandrov et al., 2001b) that these same eigenmovements are also independently controlled in a feed-forward manner during voluntary upper-trunk bending. Second, independent feedback control of each eigenmovement is sufficient to provide its stability. Third, the feedback loop in each eigenmovement can be modeled as a linear visco-elastic spring with delay. Visco-elastic parameters and time-delay values result from the combined contribution of passive visco-elastic mechanisms and sensory systems of different modalities     

Transformation of local Ca2+ spikes to global Ca2+ transients: the combinatorial roles of multiple Ca2+ releasing messengers

In pancreatic acinar cells, low, threshold concentrations of acetylcholine (ACh) or cholecystokinin (CCK) induce repetitive local cytosolic Ca2+ spikes in the apical pole, while higher concentrations elicit global signals. We have investigated the process that transforms local Ca2+ spikes to global Ca2+ transients, focusing on the interactions of multiple intracellular messengers. ACh-elicited local Ca2+ spikes were transformed into a global sustained Ca2+ response by cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate (NAADP), whereas inositol 1,4,5-trisphosphate (IP3) had a much weaker effect. In contrast, the response elicited by a low CCK concentration was strongly potentiated by IP3, whereas cADPR and NAADP had little effect. Experiments with messenger mixtures revealed a local interaction between IP3 and NAADP and a stronger global potentiating interaction between cADPR and NAADP. NAADP strongly amplified the local Ca2+ release evoked by a cADPR/IP3 mixture eliciting a vigorous global Ca2+ response. Different combinations of Ca2+ releasing messengers can shape the spatio-temporal patterns of cytosolic Ca2+ signals. NAADP and cADPR are emerging as key messengers in the globalization of Ca2+ signals.     

Effects of secretagogues and bile acids on mitochondrial membrane potential of pancreatic acinar cells: comparison of different modes of evaluating DeltaPsim

In this study, we investigated the effects of secretagogues and bile acids on the mitochondrial membrane potential of pancreatic acinar cells. We measured the mitochondrial membrane potential using the tetramethylrhodamine-based probes tetramethylrhodamine ethyl ester and tetramethylrhodamine methyl ester. At low levels of loading, these indicators appeared to have a low sensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and no response was observed to even high doses of cholecystokinin. When loaded at high concentrations, tetramethylrhodamine methyl ester and tetramethylrhodamine ethyl ester undergo quenching and can be dequenched by mitochondrial depolarization. We found the dequench mode to be 2 orders of magnitude more sensitive than the low concentration mode. Using the dequench mode, we resolved mitochondrial depolarizations produced by supramaximal and by physiological concentrations of cholecystokinin. Other calcium-releasing agonists, acetylcholine, JMV-180, and bombesin, also produced mitochondrial depolarization. Secretin, which employs the cAMP pathway, had no effect on the mitochondrial potential; dibutyryl cAMP was also ineffective. The cholecystokinin-induced mitochondrial depolarizations were abolished by buffering cytosolic calcium. A non-agonist-dependent calcium elevation induced by thapsigargin depolarized the mitochondria. These experiments suggest that a cytosolic calcium concentration rise is sufficient for mitochondrial depolarization and that the depolarizing effect of cholecystokinin is mediated by a cytosolic calcium rise. Bile acids are considered possible triggers of acute pancreatitis. The bile acids taurolithocholic acid 3-sulfate, taurodeoxycholic acid, and taurochenodeoxycholic acid, at low submillimolar concentrations, induced mitochondrial depolarization, resolved by the dequench mode. Our experiments demonstrate that physiological concentrations of secretagogues and pathologically relevant concentrations of bile acids trigger mitochondrial depolarization in pancreatic acinar cells.     

Calcium-binding protein 1 is an inhibitor of agonist-evoked, inositol 1,4,5-trisphosphate-mediated calcium signaling

Intracellular calcium signals are responsible for initiating a spectrum of physiological responses. The caldendrins/calcium-binding proteins (CaBPs) represent mammal-specific members of the CaM superfamily. CaBPs display a restricted pattern of expression in neuronal/retinal tissues, suggesting a specialized role in Ca2+ signaling in these cell types. Recently, it was reported that a splice variant of CaBP1 functionally interacts with inositol 1,4,5-trisphosphate (InsP3) receptors to elicit channel activation in the absence of InsP3 (Yang, J., McBride, S., Mak, D.-O. D., Vardi, N., Palczewski, K., Haeseleer, F., and Foskett, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711-7716). These data indicate a new mode of InsP3 receptor modulation and hence control of intracellular Ca2+ concentration ([Ca2+]i) in neuronal tissues. We have analyzed the biochemistry of the long form splice variant of CaBP1 (L-CaBP1) and show that, in vitro, a recombinant form of the protein is able to bind Ca2+ with high affinity and undergo a conformational change. We also describe the localization of endogenous and overexpressed L-CaBP1 in the model neuroendocrine PC12 cell system, where it was associated with the plasma membrane and Golgi complex in a myristoylation-dependent manner. Furthermore, we show that overexpressed L-CaBP1 is able to substantially suppress rises in [Ca2+]i in response to physiological agonists acting on purinergic receptors and that this inhibition is due in large part to blockade of release from intracellular Ca2+ stores. The related protein neuronal calcium sensor-1 was without effect on the [Ca2+]i responses to agonist stimulation. Measurement of [Ca2+] within the ER of permeabilized PC12 cells demonstrated that LCaBP1 directly inhibited InsP3-mediated Ca2+ release. Expression of L-CaBP1 also inhibited histamine-induced [Ca2+]i oscillations in HeLa cells. Together, these data suggest that L-CaBP1 is able to specifically regulate InsP3 receptor-mediated alterations in [Ca2+]i during agonist stimulation.