Calcium signalling and secretory epithelia

Ca²⁺ is now firmly established as the most important intracellular regulator of physiological and pathological events in a vast number of different cell types, including secretory epithelia. In these tissues, Ca²⁺ signalling is crucially important for the control of both fluid secretion and electrolyte secretion as well as the regulation of macromolecule secretion. In this overview article, I shall attempt to give some general background to the concepts underlying our current thinking about Ca²⁺ signalling in epithelia and its roles in regulating secretion. It is outside the scope of this review to provide a comprehensive account of Ca²⁺ signalling and the many different processes in the many different secretory epithelia that are controlled by Ca²⁺ signals. It is my aim to draw attention to some general features of Ca²⁺ signalling processes in secretory epithelia, which are rather different from those in, for example, endocrine glands. The principal examples will be taken from studies of exocrine cells and, in particular, pancreatic acinar cells, as they are the pioneer cells with regard to investigations of Ca²⁺ signalling due to primary intracellular Ca²⁺ release. They also represent the cell type which has been characterized in most detail with regard to Ca²⁺ transport events and mechanisms.     

STIM1 and STIM2 are located in the acidic Ca2+ stores and associates with Orai1 upon depletion of the acidic stores in human platelets

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.     

Calcium signaling and the secretory activity of bile duct epithelia

Cytosolic calcium (Ca_i²⁺) is a second messenger that is important for the regulation of secretion in many types of tissues. Bile duct epithelial cells, or cholangiocytes, are polarized epithelia that line the biliary tree in liver and are responsible for secretion of bicarbonate and other solutes into bile. Ca_i²⁺ signaling plays an important role in the regulation of secretion by cholangiocytes, and this review discusses the machinery involved in the formation of Ca²⁺ signals in cholangiocytes, along with the evidence that these signals regulate ductular secretion. Finally, this review discusses the evidence that impairments in cholangiocyte Ca²⁺ signaling play a primary role in the pathogenesis of cholestatic disorders, in which hepatic bile secretion is impaired.     

cAMP and Ca signaling in secretory epithelia: Crosstalk and synergism

The Ca²⁺ and cAMP/PKA pathways are the primary signaling systems in secretory epithelia that control virtually all secretory gland functions. Interaction and crosstalk in Ca²⁺ and cAMP signaling occur at multiple levels to control and tune the activity of each other. Physiologically, Ca²⁺ and cAMP signaling operate at 5–10% of maximal strength, but synergize to generate the maximal response. Although synergistic action of the Ca²⁺ and cAMP signaling is the common mode of signaling and has been known for many years, we know very little of the molecular mechanism and mediators of the synergism. In this review, we discuss crosstalk between the Ca²⁺ and cAMP signaling and the function of IRBIT (IP₃ receptors binding protein release with IP₃) as a third messenger that mediates the synergistic action of the Ca²⁺ and cAMP signaling.     

Calmodulin and acidic compounds alter basic protein phosphorylation by the protein kinase from human platelets

A cyclic nucleotide-independent protein kinase of human platelets, which phosphorylated histones, myelin basic protein and protamine and did not catalyze the phosphorylation of acidic proteins such as casein, phosvitin and myosin light chain, has been purified approx. 1,500-fold from the crude extract by steps of DEAE-cellulose, Sephadex G-200, hydroxylapatite and phosphoryl cellulose column chromatography. The substrate phosphorylation by this kinase was markedly enhanced by calmodulin even in the absence of Ca²⁺, when mixed histone was used as a substrate. The interaction of the kinase with mixed histone resulted in an irreversible inactivation of the enzyme. Calmodulin prevented this inactivation, and this compound produced an apparent increase in histone phosphorylation by the kinase. It should be noted that acidic polypeptides such as troponin-C, phospholipids and nucleic acids have a similar ability. The addition of Ca²⁺ reduced the effect of calmodulin more than the effects of other acidic compounds.     

Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca(2+), which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca(2+) from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca(2+) was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 microM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20-40% of sperm, induced hyperactivation. Addition of 1 mM Ni(2+) diminished the response. This suggests that intracellular Ca(2+) is abnormally low in the null sperm flagella. When intracellular Ca(2+) was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca(2+) is required to supplement store-released Ca(2+) to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca(2+) entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility.     

TPCs: Endolysosomal channels for Ca mobilization from acidic organelles triggered by NAADP

Two-pore channels (TPCs or TPCNs) are novel members of the large superfamily of voltage-gated cation channels with slightly higher sequence homology to the pore-forming subunits of voltage-gated Ca²⁺ and Na⁺ channels than most other members. Recent studies demonstrate that TPCs locate to endosomes and lysosomes and form Ca²⁺ release channels that respond to activation by the Ca²⁺ mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). With multiple endolysosomal targeted NAADP receptors now identified, important new insights into the regulation of endolysosomal function in health and disease will therefore be unveiled.     

Anoctamin 1 in secretory epithelia

Fluid and electrolyte releasing from secretory epithelia are elaborately regulated by orchestrated activity of ion channels. The activity of chloride channel at the apical membrane decides on the direction and the rate of secretory fluid and electrolyte. Chloride-dependent secretion is conventionally associated with intracellular increases in two second messengers, cAMP and Ca²⁺, responding to luminal purinergic and basolateral adrenergic or cholinergic stimulation. While it is broadly regarded that cAMP-dependent Cl⁻ secretion is regulated by cystic fibrosis transmembrane conductance regulator (CFTR), Ca²⁺-activated Cl⁻ channel (CaCC) had been veiled for quite some time. Now, Anoctamin 1 (ANO1 or TMEM16A) confers Ca²⁺-activated Cl⁻ currents. Ano 1 and its paralogs have been actively investigated for multiple functions underlying Ca²⁺-activated Cl⁻ efflux and fluid secretion in a variety of secretory epithelial cells. In this review, we will discuss recent advances in the secretory function and signaling of ANO1 in the secretory epithelia, such as airways, intestines, and salivary glands.     

Role of cations in stability of acidic protein Desulfovibrio desulfuricans apoflavodoxin

Apoflavodoxin from the sulfate reducing bacteria Desulfovibrio desulfuricans is a small, acidic protein with a net charge of -19 at neutral pH. Here, we show that monovalent cations in biologically relevant amounts have dramatic effects on apoflavodoxin stability. The effect is largest for Gdm(+) and decreases as a function of increased cation charge density (Gdm(+)>NH(4)(+)K(+) approximately Cs(+) approximately Na(+)>Li(+)). A linear correlation of stabilizing effects with cation hydration properties suggests an important role of dehydration in efficient cation interaction with the protein. The effects on stability are due to preferential binding of one cation to native apoflavodoxin and results in an increase in thermal midpoint of 20 degrees C and the free energy of unfolding (at 20 degrees C) increases fivefold. Tuning of biophysical properties (such as folding and ligand/cofactor binding) of acidic proteins by cation binding may be important in vivo.     

Evidence for a Role of Calmodulin in Calcium-Induced Noradrenaline Release from Permeated Synaptosomes: Effects of Calmodulin Antibodies and Antagonists

Abstract: The nervous tissue-specific protein B-50 (GAP-43), which has been implicated in the regulation of neurotransmitter release, is a member of a family of atypical calmodulin-binding proteins. To investigate to what extent calmodulin and the interaction between B-50 and calmodulin are involved in the mechanism of Ca²⁺-induced noradrenaline release, we introduced polyclonal anti-calmodulin antibodies, calmodulin, and the calmodulin antagonists trifluoperazine, W-7, calmidazolium, and polymyxin B into streptolysin-O-permeated synaptosomes prepared from rat cerebral cortex. Anti-calmodulin antibodies, which inhibited Ca²⁺/calmodulin-dependent protein kinase II autophosphorylation and calcineurin phosphatase activity, decreased Ca²⁺-induced noradrenaline release from permeated synaptosomes. Exogenous calmodulin failed to modulate release, indicating that if calmodulin is required for vesicle fusion it is still present in sufficient amounts in permeated synaptosomes. Although trifluoperazine, W-7, and calmidazolium inhibited Ca²⁺-induced release, they also strongly increased basal release. Polymyxin B potently inhibited Ca²⁺-induced noradrenaline release without affecting basal release. It is interesting that polymyxin B was also the only antagonist affecting the interaction between B-50 and calmodulin, thus lending further support to the hypothesis that B-50 serves as a local Ca²⁺-sensitive calmodulin store underneath the plasma membrane in the mechanism of neurotransmitter release. We conclude that calmodulin plays an important role in vesicular noradrenaline release, probably by activating Ca²⁺/calmodulin-dependent enzymes involved in the regulation of one or more steps in the release mechanism.     

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.     

Separation of Fe from transferrin in endocytosis: Role of the acidic endosome

NH₄Cl and monensin, two agents which neutralize intracellular acidic compartments, block the segregation of iron from transferrin after endocytosis, while neither of these reagents affects internalization of diferric transferrin into the cell. In conclusion the molecular separation of iron from transferrin inside the cell requires a non-lysosomal acidic compartment     

Analysis of ultra acidic proteins by the use of anodic acidic gels

Ultra acidic proteins, generated by posttranslational modifications, are becoming increasingly important due to recent evidence showing their function as regulatory elements or as intermediates in degradation pathways in bacteria. Such proteins are important in neurodegenerative diseases and embryonic development, and they include the Alzheimer-related tau (τ) protein (resulting from posttranslational modifications) and the phosphor-storage embryonic proteins. The ultra acidic proteins are difficult to study because standard two-dimensional gel electrophoresis is inadequate for their analysis. Here we describe a novel electrophoresis system of anodic acidic gels that can replace isoelectric focusing as the first dimension of separation in two-dimensional electrophoresis. The system is based on a sodium acetate buffer (pH 4.6), is compatible with traditional stains (e.g., Coomassie blue) as well as novel fluorescent dyes (e.g., Pro-Q Diamond), and is quantitative for the analysis of ultra acidic proteins. The anodic acidic gels were used for the functional classification of the ultra acidic part of the Bacillus subtilis proteome, showing significant improvement over traditional two-dimensional electrophoresis.     

S100A13-lipid interactions—role in the non-classical release of the acidic fibroblast growth factor

S100A13 is a 98-amino acid, calcium binding protein. It is known to participate in the non-classical secretion of signal peptide-less proteins, such as the acidic fibroblast growth factor. In this study, we investigate the lipid binding properties of S10013 using a number of biophysical techniques, including multidimensional NMR spectroscopy. Isothermal titration calorimetry and steady state fluorescence experiments show that apoS100A13 exhibits preferential binding to small unilamelar vesicles of l-phosphatidyl serine (pS). In comparison, Ca²⁺-bound S100A13 is observed to bind weakly to unilamelar vesicles (SUVs) of pS. Equilibrium thermal unfolding and limited trypsin digestion analysis reveal that apoS100A13 is significantly destabilized upon binding to SUVs of pS. Results of the far UV circular dichroism and ANS (8-anilino-1-napthalene sufonate) binding experiments indicate a subtle conformational change resulting in the increase in the solvent-accessible hydrophobic surface in the protein. Availability of the solvent-exposed hydrophobic surface(s) in apoS10013 facilitates its interaction with the lipid vesicles. Our data suggest that Ca²⁺ binding dictates the membrane binding affinity of S100A13. Based on the results of this study, a model describing the sequence of molecular events that possibly can occur during the non-classical secretion of FGF-1 is presented.     

酸性水和投加铝、钙对鲢鱼早期发育和鳃超微结构的影响

在实验室条件下,研究了酸性水和投加铝、钙对鲢鱼胚胎孵化和鱼苗存活以及幼鱼鳃超微结构的影响。pH4.0引起所有胚胎在24小时内死亡,暴露于pH4.5-6.0的胚胎孵化率和暴露于pH4.0-6.0的5-15日龄鱼苗存活率随pH值上升而增高。投加0.5mgAl³⁺/L使在酸性pH暴露条件下的胚胎孵化率和鱼苗存活率进一步降低。投加3.0mgCa²⁺/L可显著提高暴露于pH4.5和5.0的胚胎孵化率;投加2.0mgCa²⁺/L可在一定程度上提高暴露于pH4.5和5.0的鱼苗存活率。幼苗经pH4.5暴露8小时后出现严重的鳃超微结构损害;投加1.0mgAl³⁺/L使鳃结构损害加剧;投加5.0mgCa²⁺/L可明显缓解酸性水对鳃的损害。     

Role of calcium during Toxoplasma gondii invasion and egress

Calcium is a ubiquitous signalling molecule involved in a large number of cellular processes in eukaryotic cells. In the obligate intracellular parasite, Toxoplasma gondii, for example, a rise in calcium concentration is associated with significant morphological changes, secretion of proteins involved in host cell invasion and rapid egress from the host cell. Recent findings indicate that calcium released from the parasite's intracellular pools is necessary and sufficient to induce some of the events critical for invasion and egress. In addition, ethanol, a powerful inducer of invasion-related events, is shown here to also induce rapid egress from the host cell, indicating that a common mechanism for calcium release might be involved during both invasion and egress.     

酸性水和投加铝、钙对鲢鱼早期发育和鳃超微结构的影响

在实验室条件下,研究了酸性水和投加铝、钙对鲢鱼胚胎孵化和鱼苗存活以及幼鱼鳃超微结构的影响。pH4.0引起所有胚胎在24小时内死亡,暴露于pH4.5—6.0的胚胎孵化率和暴露于pH4.0—6.0的5—15日龄鱼苗存活率随pH值上升而增高。投加0.5mg Al~(3+)/L使在酸性pH暴露条件下的胚胎孵化率和鱼苗存活率进一步降低。投加3.0mg Ca~(2+)/L可显著提高暴露于pH4.5和5.0的胚胎孵化率;投加2.0mg Ca~(2+)/L可在一定程度上提高暴露于pH4.5和5.0的鱼苗存活率。幼苗经pH4.5暴露8小时后出现严重的鳃超微结构损害;投加1.0mg Al~(3+)/L使鳃结构损害加剧;投加5.0mg Ca~(2+)/L可明显缓解酸性水对鳃的损害。     

Cytosolic Calmodulin Is Increased in SK-N-SH Human Neuroblastoma Cells Due to Release of Calcium from Intracellular Stores

Abstract: Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca²⁺ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca²⁺ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca²⁺ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000-g cytosol and membrane fractions. The influx of extracellular Ca²⁺ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca²⁺ channel blocker Ni²⁺. Blocking the influx of extracellular Ca²⁺ had no effect on carbachol-mediated increases in cytosolic CaM (168 ± 18% of control values for carbachol treatment alone vs. 163 ± 28% for Ni²⁺ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca²⁺ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 ± 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca²⁺ by pretreatment with the cell-permeant Ca²⁺ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 ± 37% of control without BAPTA/AM vs. 136 ± 13% with BAPTA/AM). The effect of direct entry of extracellular Ca²⁺ into the cell by K⁺ depolarization was assessed. Incubation of SK-N-SH cells with 60 mM K⁺ elicited an immediate and persistent increase in intracellular free Ca²⁺ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca²⁺ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca²⁺, which releases Ca²⁺ from intracellular stores, induced an increase in cytosolic CaM (203 ± 30% of control). The mechanism for the CaM release may involve activation of the α isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K⁺ depolarization. These data demonstrate that release of Ca²⁺ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells.     

Physical properties of two types of calcium stores and SERCAs in human platelets

The aim of this work was to obtain experimental data depending on the properties of calcium stores and SERCAs, to analyse these data in terms of the models based on simulation of the cellular compartments as communicating vessels, and to relate this way the data to the above properties. The main characteristics of the stores and corresponding SERCAs have been estimated. Calcium content in the DTS is ∼1.5 × 10⁶ ions per cell, that in the acidic stores, ∼0.64 × 10⁶ ions per cell. The rate constant of background calcium leakage from the DTS is ∼0.0033 s⁻¹, that from the acidic stores, ∼0.1 s⁻¹. The background activity of SERCA2b is ∼0.22 × 10⁶ s⁻¹ ions per cell, that of SERCA3, ∼2.5 × 10⁶ s⁻¹ ions per cell. The properties of both calcium stores and the SERCAs and the characteristics found might be related to physiological or pathological state of the cells.