The luminal Ca2+ chelator,TPEN, inhibits NAADP-induced Ca2+ release

The regulation of Ca²⁺ release by luminal Ca²⁺ has been well studied for the ryanodine and IP₃ receptors but has been less clear for the NAADP-regulated channel. In view of conflicting reports, we have re-examined the issue by manipulating luminal Ca²⁺ with the membrane-permeant, low affinity Ca²⁺ buffer, TPEN, and monitoring NAADP-induced Ca²⁺ release in sea urchin egg homogenate. NAADP-induced Ca²⁺ release was almost entirely blocked by TPEN (IC₅₀ 17–25 μM) which suppressed the maximal extent of Ca²⁺ release without altering NAADP sensitivity. In contrast, Ca²⁺ release via IP₃ receptors was 3- to 30-fold less sensitive to TPEN whereas that evoked by ionomycin was essentially unaffected. The effect of TPEN on NAADP-induced Ca²⁺ release was not due to an increase in the luminal pH or chelation of trace metals since it could not be mimicked by NH₄Cl or phenanthroline. The fact that TPEN had no effect upon ionophore-induced Ca²⁺ release also argued against a substantial reduction in the driving force for Ca²⁺ efflux. We propose that, in the sea urchin egg, luminal Ca²⁺ is important for gating native NAADP-regulated two-pore channels.     

Ca2+ signalling: a new route to NAADP

The LKB1 serine/threonine kinase is a tumour suppressor responsible for the inherited familial cancer disorder Peutz-Jeghers syndrome and is inactivated in a large percentage of human lung cancers. LKB1 acts a master kinase, directly phosphorylating and activating a family of 14 AMPK (AMP-activated protein kinase)-related kinases which control cell metabolism, cell growth and cell polarity. In this issue of the Biochemical Journal, Hardie and colleagues discover an alternative splice form of LKB1 that alters the C-terminus of the protein containing a few known sites of post-translational regulation. Although widely expressed, the short isoform (LKB1(s)) is the sole splice isoform expressed in testes, and its expression peaks at the time of spermatid maturation. Male mice lacking the LKB1(s) isoform have dramatic defects in spermatozoa, resulting in sterility.     

Pharmacological characterization of NAADP-induced Ca2+ signals in starfish oocytes

The recently discovered second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) is central to the onset of intracellular Ca2+ signals induced by several stimuli, including fertilization. The nature of the Ca2+ pool mobilized by NAADP is still controversial. Depending on the cell type, NAADP may target either an acidic compartment with lysosomal properties or ryanodine receptors (RyRs) on endoplasmic reticulum. In addition, NAADP elicits a robust Ca2+ influx into starfish oocytes by activating a Ca2+-mediated current across the plasma membrane. In the present study, we employed the single-electrode intracellular recording technique to assess the involvement of either acidic organelles or RyRs in NAADP-elicited Ca2+ entry. We found that neither drugs which interfere with acidic compartments nor inhibitors of RyRs affected NAADP-induced depolarization. These data further support the hypothesis that a yet unidentified plasma membrane Ca2+ channel is the target of NAADP in starfish oocytes.     

Ca2+ homeostasis,Ca2+ signalling and somatodendritic vasopressin release in adult rat supraoptic nucleus neurones

Multiple mechanisms that maintain Ca²⁺ homeostasis and provide for Ca²⁺ signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca²⁺ clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca²⁺ homeostatic molecules on cytosolic Ca²⁺ ([Ca²⁺]_i) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca²⁺]_i dynamics. When bathing the cells in a Na⁺-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca²⁺-ATPase (PMCA), La³⁺, all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca²⁺]_i transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca²⁺ transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca²⁺ homeostatic pathways, the Na⁺/Ca²⁺ exchanger, the endoplasmic reticulum Ca²⁺ pump, the plasmalemmal Ca²⁺ pump and mitochondria, are complementary in actively clearing Ca²⁺ from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca²⁺]_i dynamics; (iii) there is (are) Ca²⁺ clearance mechanism(s) distinct from the four outlined above; and (iv) Ca²⁺ homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals.     

Ca2+ signalling underlying pancreatitis

In spite of significant scientific progress in recent years, acute pancreatitis (AP) is still a dangerous and in up to 5% of cases deadly disease with no specific cure. It is self-resolved in the majority of cases, but could result in chronic pancreatitis (CP) and increased risk of pancreatic cancer (PC). One of the early events in AP is premature activation of digestive pro-enzymes, including trypsinogen, inside pancreatic acinar cells (PACs) due to an excessive rise in the cytosolic Ca²⁺ concentration, which is the result of Ca²⁺ release from internal stores followed by Ca²⁺ entry through the store operated Ca²⁺ channels in the plasma membrane. The leading causes of AP are high alcohol intake and biliary disease with gallstones obstruction leading to bile reflux into the pancreatic duct. Recently attention in this area of research turned to another cause of AP – Asparaginase based drugs – which have been used quite successfully in treatments of childhood acute lymphoblastic leukaemia (ALL). Unfortunately, Asparaginase is implicated in triggering AP in 5–10% of cases as a side effect of the anti-cancer therapy. The main features of Asparaginase-elicited AP (AAP) were found to be remarkably similar to AP induced by alcohol metabolites and bile acids. Several potential therapeutic avenues in counteracting AAP have been suggested and could also be useful for dealing with AP induced by other causes. Another interesting development in this field includes recent research related to pancreatic stellate cells (PSCs) that are much less studied in their natural environment but nevertheless critically involved in AP, CP and PC. This review will attempt to evaluate developments, approaches and potential therapies for AP and discuss links to other relevant diseases.     

Intracellular sphingosine 1-phosphate production: a novel pathway for Ca2+ release

Sphingolipids such as sphingosine 1-phosphate (SPP) and sphingosylphosphorylcholine have long been recognized to possess Ca2+ mobilizing activity, yet to date little is known about their mechanism of action, or indeed their significance as Ca2+ mobilizing intracellular messengers. The recent discovery of extracellular receptors for the sphingolipids has further complicated the interpretation of many experiments in this field. This paper reviews the current literature in which molecular and pharmacological approaches have begun to uncover the signalling components associated with intracellular SPP production and Ca2+ mobilization. The functional significance of this novel Ca2+ release pathway is also discussed.     

Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. II. Releases involving a Ca2+-induced Ca2+ release channel

Calcium ions that have been preloaded into isolated sarcoplasmic reticulum subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances. We report here that not only caffeine, but also Ca2+ ions, thymol, quercetin, menthol, halothane, chloroform, 1-ethyl-2-methylbenzimidazole, ryanodine, tetraphenylboron, ketoconazole, miconazole, clotrimazole, W-7, doxorubicin, 5,5'-dithiobis-(2-nitrobenzoic acid), p-chloromercuribenzoic acid, and low concentrations of Ag+ induce Ca2+ release from such triadic sarcoplasmic reticulum. All these drugs induce increased undirectional Ca2+ efflux. We believe all these drug-induced Ca2+ releases are mediated by Ca2+ efflux through the same ion channel since these releases are all greatly attenuated when light sarcoplasmic reticulum is substituted for triads and are even more pronounced when transverse tubule-free terminal cisternae are substituted for triads, and all these forms of drug-induced Ca2+ release are inhibited by submicromolar concentrations of ruthenium red, and by submillimolar concentrations of tetracaine, 9-aminoacridine, and Ba2+, yet they are not affected by nifedipine even at a concentration of 50 microM.     

The specificity of Ca2+ signalling

A calcium signal is a sudden increase in concentration of calcium ions (Ca2+) in the cytosol. Such signals are crucial for the control of many important functions of the body. In the brain, for example, Ca2+ signals are responsible for memory, in muscle cells they switch on contraction, whereas in gland cells they are responsible for regulation of secretion. In many cases Ca2+ signals can control several different processes in the same cell. As an example, we shall deal with one particular cell type, namely the pancreatic acinar cell, which is responsible for the secretion of the enzymes essential for the digestion of food. In this cell, Ca2+ signals do not only control the normal enzyme secretion, but also regulate growth (cell division) and programmed cell death (apoptosis). Until recently, it was a mystery how the same type of signal could regulate such diverse functions in one and the same cell. Recent technical advances have shown that different patterns of Ca2+ signals can be created, in space and time, which allow specific cellular responses to be elicited.     

The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape

Members of the vertebrate Wnt family have been subdivided into two functional classes according to their biological activities. Some Wnts signal through the canonical Wnt-1/wingless pathway by stabilizing cytoplasmic beta-catenin. By contrast other Wnts stimulate intracellular Ca2+ release and activate two kinases, CamKII and PKC, in a G-protein-dependent manner. Moreover, putative Wnt receptors belonging to the Frizzled gene family have been identified that preferentially couple to the two prospective pathways in the absence of ectopic Wnt ligand and that might account for the signaling specificity of the Wnt pathways. As Ca2+ release was the first described feature of the noncanonical pathway, and as Ca2+ probably plays a key role in the activation of CamKII and PKC, we have named this Wnt pathway the Wnt/Ca2+ pathway.     

Ca2+ -induced Ca2+ release through localized Ca2+ uncaging in smooth muscle

Ca(2+)-induced Ca(2+) release (CICR) from the sarcoplasmic reticulum (SR) occurs in smooth muscle as spontaneous SR Ca(2+) release or Ca(2+) sparks and, in some spiking tissues, as Ca(2+) release that is triggered by the activation of sarcolemmal Ca(2+) channels. Both processes display spatial localization in that release occurs at a higher frequency at specific subcellular regions. We have used two-photon flash photolysis (TPFP) of caged Ca(2+) (DMNP-EDTA) in Fluo-4-loaded urinary bladder smooth muscle cells to determine the extent to which spatially localized increases in Ca(2+) activate SR release and to further understand the molecular and biophysical processes underlying CICR. TPFP resulted in localized Ca(2+) release in the form of Ca(2+) sparks and Ca(2+) waves that were distinguishable from increases in Ca(2+) associated with Ca(2+) uncaging, unequivocally demonstrating that Ca(2+) release occurs subsequent to a localized rise in [Ca(2+)](i). TPFP-triggered Ca(2+) release was not constrained to a few discharge regions but could be activated at all areas of the cell, with release usually occurring at or within several microns of the site of photolysis. As expected, the process of CICR was dominated by ryanodine receptor (RYR) activity, as ryanodine abolished individual Ca(2+) sparks and evoked release with different threshold and kinetics in FKBP12.6-null cells. However, TPFP CICR was not completely inhibited by ryanodine; Ca(2+) release with distinct kinetic features occurred with a higher TPFP threshold in the presence of ryanodine. This high threshold release was blocked by xestospongin C, and the pharmacological sensitivity and kinetics were consistent with CICR release at high local [Ca(2+)](i) through inositol trisphosphate (InsP(3)) receptors (InsP(3)Rs). We conclude that CICR activated by localized Ca(2+) release bears essential similarities to those observed by the activation of I(Ca) (i.e., major dependence on the type 2 RYR), that the release is not spatially constrained to a few specific subcellular regions, and that Ca(2+) release through InsP(3)R can occur at high local [Ca(2+)](i).     

Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: Ca2+-dependent passive Ca2+ efflux

Characterization of the putative Ca2+-gated Ca2+ channel of sarcoplasmic reticulum, which is thought to mediate Ca2+-induced Ca2+ release, was carried out in order to elucidate the mechanism of Ca2+-induced Ca2+ release. Heavy and light fractions of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle were loaded passively with Ca2+, and then passive Ca2+ efflux was measured under various conditions. The fast phase of the Ca2+ efflux depended on the extravesicular free Ca2+ concentration and was assigned to the Ca2+ efflux through the Ca2+-gated Ca2+ channel. Vesicles with the Ca2+-gated Ca2+ channels comprised about 85% of the heavy fraction and about 40% of the light fraction. The amount of Ca2+ loaded in FSR was found to be much larger than that estimated on the basis of vesicle inner volume and the equilibration of intravesicular with extravesicular Ca2+, indicating Ca2+ binding inside FSR. Taking this fact into account, the Ca2+ efflux curve was quantitatively analyzed and the dependence of the Ca2+ efflux rate constant on the extravesicular free Ca2+ concentration was determined. The Ca2+ efflux was maximal, with the rate constant of 0.75 s-1, when the extravesicular free Ca2+ was at 3 microM. Caffeine increased the affinity for Ca2+ of Ca2+-binding sites for opening the channel with only a slight change in the maximum rate of Ca2+ efflux. Mg2+ inhibited the Ca2+ binding to the sites for opening the channel while procaine seemed to inhibit the Ca2+ efflux by blocking the ionophore moiety of the channel.     

Silymarin-induced mitochondrial Ca2+ release

The effect of silymarin on different functions of mitochondria isolated from rat kidneys was studied. Addition of silymarin to mitochondria oxidizing succinate, induced stimulation of the respiratory State 4; while in mitochondria oxidizing NAD-dependent substrates, the drug produced inhibition of the oxygen consumption. It is also shown that silymarin induces mitochondrial swelling, a drop in the transmembrane potential, as well as Ca2+ release. It is proposed that due to its hydrophobic character, silymarin produces an alteration in the lipidic milieu of the inner membrane which is conductive to an inhibition of the electron transport in the NAD-CoQ span of the respiratory chain, as well as to the loss of the energy dependent accumulated Ca2+.     

Signalling specificity in GPCR-dependent Ca2+ signalling

Cells use signalling networks to translate with high fidelity extracellular signals into specific cellular functions. Signalling networks are often composed of multiple signalling pathways that act in concert to regulate a particular cellular function. In the centre of the networks are the receptors that receive and transduce the signals. A versatile family of receptors that detect a remarkable variety of signals are the G protein-coupled receptors (GPCRs). Virtually all cells express several GPCRs that use the same biochemical machinery to transduce their signals. Considering the specificity and fidelity of signal transduction, a central question in cell signalling is how signalling specificity is achieved, in particular among GPCRs that use the same biochemical machinery. Ca(2+) signalling is particularly suitable to address such questions, since [Ca(2+)](i) can be recorded with excellent spatial and temporal resolutions in living cells and tissues and now in living animals. Ca(2+) is a unique second messenger in that both biochemical and biophysical components form the Ca(2+) signalling complex to regulate its concentration. Both components act in concert to generate repetitive [Ca(2+)](i) oscillations that can be either localized or in the form of global, propagating Ca(2+) waves. Most of the key proteins that form Ca(2+) signalling complexes are known and their activities are reasonably well understood on the biochemical and biophysical levels. We review here the information gained from studying Ca(2+) signalling by GPCRs to gain further understanding of the mechanisms used to generate cellular signalling specificity.     

Ca2+ signalling and muscle disease.

Transient elevations of intracellular Ca2+ play a signalling role in such complex cellular functions as contraction, secretion, fertilization, proliferation, metabolism, heartbeat and memory. However, prolonged elevation of Ca2+ above about 10 microM is deleterious to a cell and can activate apoptosis. In muscle, there is a narrow window of Ca2+ dysregulation in which abnormalities in Ca2+ regulatory proteins can lead to disease, rather than apoptosis. Key proteins in the regulation of muscle Ca2+ are the voltage-dependent, dihydropyridine-sensitive, L-type Ca2+ channels located in the transverse tubule and Ca2+ release channels in the junctional terminal cisternae of the sarcoplasmic reticulum. Abnormalities in these proteins play a key role in malignant hyperthermia (MH), a toxic response to anesthetics, and in central core disease (CCD), a muscle myopathy. Sarco(endo)plasmic reticulum Ca2+ ATPases (SERCAs) return sarcoplasmic Ca2+ to the lumen of the sarcoplasmic reticulum. Loss of SERCA1a Ca2+ pump function is one cause of exercise-induced impairment of the relaxation of skeletal muscle, in Brody disease. Phospholamban expressed in cardiac muscle and sarcolipin expressed in skeletal muscle regulate SERCA activity. Studies with knockout and transgenic mice show that gain of inhibitory function of phospholamban alters cardiac contractility and could be a causal feature in some cardiomyopathies. Calsequestrin, calreticulin, and a series of other acidic, lumenal, Ca2+ binding proteins provide a buffer for Ca2+ stored in the sarcoplasmic reticulum. Overexpression of cardiac calsequestrin leads to cardiomyopathy and ablation of calreticulin alters cardiac development.     

Mechanisms of Ca2+ signalling in cells

The review summarizes recent data and current opinions of the Ca2+ signal formation in cells. Mechanisms of Ca2+ mobilization from the intracellular Ca2+ stores are discussed along with the pathways of Ca2+ entry from the external medium.     

Ca2+signalling in stomatal guard cells

Ca(2+) is a ubiquitous second messenger in the signal transduction pathway(s) by which stomatal guard cells respond to external stimuli. Increases in guard-cell cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) have been observed in response to stimuli that cause both stomatal opening and closure. In addition, several important components of Ca(2+)-based signalling pathways have been identified in guard cells, including the cADP-ribose and phospholipase C/Ins(1, 4,5)P(3)-mediated Ca(2+)-mobilizing pathways. The central role of stimulus-induced increases in [Ca(2+)](cyt) in guard-cell signal transduction has been clearly demonstrated in experiments examining the effects of modulating increases in [Ca(2+)](cyt) on alterations in guard-cell turgor or the activity of ion channels that act as effectors in the guard-cell turgor response. In addition, the paradox that Ca(2+) is involved in the transduction of signals that result in opposite end responses (stomatal opening and closure) might be accounted for by the generation of stimulus-specific Ca(2+) signatures, such that increases in [Ca(2+)](cyt) exhibit unique spatial and temporal characteristics.     

Coordination of Ca2+ signalling by NAADP

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes intracellular Ca2+ stores in several cell types. Ample evidence suggests that NAADP activates intracellular Ca2+ channels distinct from those that are sensitive to inositol trisphosphate and ryanodine/cyclic ADP-ribose. Recent studies in intact cells have demonstrated functional coupling ('channel chatter') between Ca2+ release pathways mediated by NAADP, inositol trisphosphate and cyclic ADP-ribose. Thus, NAADP is probably an important determinant in shaping cytosolic Ca2+ signals.