Blockade by zinc ions of K+-induced contraction and calcium in guinea pigTaenia coli

Preincubation with 0.3 mM Zn²⁺ markedly inhibited both the tonic response and Ca²⁺ binding at low affinity sites induced by K⁺ (60 mM), with smaller effects on the phasic response and the high affinity Ca²⁺ sites, inTaenia coli. However, when the muscle was kept in Zn²⁺-containing medium following the first stimulation with the K⁺, the phasic response and the high affinity Ca²⁺ sites were more severely inhibited during the second stimulation with K⁺. This probably indicates that Zn²⁺ reduced the tonic tension response to K⁺ mainly by inhibiting Ca²⁺ influx at the cell membranes ofTaenia coli. However, when Zn²⁺ is continuously present, Ca²⁺ is not supplied at the storage sites and is not available for the phasic response to a second stimulation with K⁺.     

Ionic mechanisms in pancreatic β cell signaling

The function and survival of pancreatic β cells critically rely on complex electrical signaling systems composed of a series of ionic events, namely fluxes of K⁺, Na⁺, Ca²⁺ and Cl^? across the β cell membranes. These electrical signaling systems not only sense events occurring in the extracellular space and intracellular milieu of pancreatic islet cells, but also control different β cell activities, most notably glucose-stimulated insulin secretion. Three major ion fluxes including K⁺ efflux through ATP-sensitive K⁺ (K_ATP) channels, the voltage-gated Ca²⁺ (Ca_V) channel-mediated Ca²⁺ influx and K⁺ efflux through voltage-gated K⁺ (K_V) channels operate in the β cell. These ion fluxes set the resting membrane potential and the shape, rate and pattern of firing of action potentials under different metabolic conditions. The K_ATP channel-mediated K⁺ efflux determines the resting membrane potential and keeps the excitability of the β cell at low levels. Ca²⁺ influx through Ca_V1 channels, a major type of β cell Ca_V channels, causes the upstroke or depolarization phase of the action potential and regulates a wide range of β cell functions including the most elementary β cell function, insulin secretion. K⁺ efflux mediated by K_V2.1 delayed rectifier K⁺ channels, a predominant form of β cell K_V channels, brings about the downstroke or repolarization phase of the action potential, which acts as a brake for insulin secretion owing to shutting down the Ca_V channel-mediated Ca²⁺ entry. These three ion channel-mediated ion fluxes are the most important ionic events in β cell signaling. This review concisely discusses various ionic mechanisms in β cell signaling and highlights K_ATP channel-, Ca_V1 channel- and K_V2.1 channel-mediated ion fluxes.     

Na+ mechanism of δ-opioid receptor induced protection from anoxic K+ leakage in the cortex

Activation of δ-opioid receptors (DOR) attenuates anoxic K⁺ leakage and protects cortical neurons from anoxic insults by inhibiting Na⁺ influx. It is unknown, however, which pathway(s) that mediates the Na⁺ influx is the target of DOR signal. In the present work, we found that, in the cortex, (1) DOR protection was largely dependent on the inhibition of anoxic Na⁺ influxes mediated by voltage-gated Na⁺ channels; (2) DOR activation inhibited Na⁺ influx mediated by ionotropic glutamate N-methyl-D-aspartate (NMDA) receptors, but not that by non-NMDA receptors, although both played a role in anoxic K⁺ derangement; and (3) DOR activation had little effect on Na⁺/Ca²⁺ exchanger-based response to anoxia. We conclude that DOR activation attenuates anoxic K⁺ derangement by restricting Na⁺ influx mediated by Na⁺ channels and NMDA receptors, and that non-NMDA receptors and Na⁺/Ca²⁺ exchangers, although involved in anoxic K⁺ derangement in certain degrees, are less likely the targets of DOR signal. Received 26 November 2008; received after revision 26 December 2008; accepted 13 January 2009     

ATP-induced Ca<Superscript>2+</Superscript>-signalling mechanisms in the regulation of mesenchymal stem cell migration

The ability of cells to migrate to the destined tissues or lesions is crucial for physiological processes from tissue morphogenesis, homeostasis and immune responses, and also for stem cell-based regenerative medicines. Cytosolic Ca²⁺ is a primary second messenger in the control and regulation of a wide range of cell functions including cell migration. Extracellular ATP, together with the cognate receptors on the cell surface, ligand-gated ion channel P2X receptors and a subset of G-protein-coupled P2Y receptors, represents common autocrine and/or paracrine Ca²⁺ signalling mechanisms. The P2X receptor ion channels mediate extracellular Ca²⁺ influx, whereas stimulation of the P2Y receptors triggers intracellular Ca²⁺ release from the endoplasmic reticulum (ER), and activation of both type of receptors thus can elevate the cytosolic Ca²⁺ concentration ([Ca²⁺]_c), albeit with different kinetics and capacity. Reduction in the ER Ca²⁺ level following the P2Y receptor activation can further induce store-operated Ca²⁺ entry as a distinct Ca²⁺ influx pathway that contributes in ATP-induced increase in the [Ca²⁺]_c. Mesenchymal stem cells (MSC) are a group of multipotent stem cells that grow from adult tissues and hold promising applications in tissue engineering and cell-based therapies treating a great and diverse number of diseases. There is increasing evidence to show constitutive or evoked ATP release from stem cells themselves or mature cells in the close vicinity. In this review, we discuss the mechanisms for ATP release and clearance, the receptors and ion channels participating in ATP-induced Ca²⁺ signalling and the roles of such signalling mechanisms in mediating ATP-induced regulation of MSC migration.     

Changes in calpastatin localization and expression during calpain activation: a new mechanism for the regulation of intracellular Ca<Superscript>2+</Superscript>-dependent proteolysis

The amount of calpastatin directly available in cytosol is under the control of [Ca²⁺] and [cyclic AMP]. Prolonged calpain activation also promotes degradation of calpastatin. The fluctuation of calpastatin concentration in cell soluble fraction is accompanied by an initial decrease in calpastatin gene expression, followed by a fivefold increase in its expression when the inhibitor protein is degraded. This process can be conceptualized as a mechanism to regulate calpastatin availability in the cell. This conclusion is supported by the fact that calpain, the other component of this proteolytic system, undergoes changes in its levels of expression in a much more limited manner. Furthermore, this process can be observed both in cells exposed to different natural stimuli, or in other cell lines. Modification of calpastatin gene expression might represent a new tool for the in vivo control of the regulatory machinery required for the modulation of Ca²⁺-dependent proteolysis.Received 18 July 2003; received after revision 3 September 2003; accepted 23 September 2003     

The Ca2+-transport ATPases in smooth muscle

Summary A calmodulin stimulated Ca²⁺-transport ATPase which has many of the characteristics of the erythrocyte type Ca²⁺-transport ATPase has been purified from smooth muscle. In particular, the effect of calmodulin on these transport enzymes is mimiced by partial proteolysis and antibodies against erythrocyte Ca²⁺-transport ATPase also bind to the smooth muscle (Ca²⁺+Mg²⁺)ATPase. A correlation between the distribution of the calmodulin stimulated (Ca²⁺+Mg²⁺)ATPase and (Na⁺+K⁺)ATPase activities in smooth muscle membranes separated by density gradient centrifugation suggests a plasmalemmal distribution of this (Ca²⁺+Mg²⁺)ATPase. A phosphoprotein intermediate in smooth muscle which strongly resembles the corresponding phosphoprotein in sarcoplasmic reticulum of skeletal muscle may indicate the presence in smooth muscle of a similar type of Ca²⁺-transport ATPase.     

The effect of a phorbol ester upon the cholinergic regulation of potassium permeability in the rat submandibular gland

Acetylcholine releases calcium from cytoplasmic stores and permits an influx of calcium in salivary acinar cells. The resultant rise in [Ca²⁺]_i causes an increase in potassium permeability which is an important part of the secretory response. We have investigated the effects of 12-0-tetradecanoyl phorbol-13-acetate, a potent activator of protein kinase C, upon this regulation of potassium permeability in superfused pieces of rat submandibular salivary gland. This compound inhibited the initial [Ca²⁺]_o-independent component of the response of acetylcholine but had no effect upon the subsequent [Ca²⁺]_o-dependent phase. This compound does not, therefore, appear to inhibit receptor-regulated calcium influx.     

Rab44, a novel large Rab GTPase,negatively regulates osteoclast differentiation by modulating intracellular calcium levels followed by NFATc1 activation

Rab44 is an atypical Rab GTPase that contains some additional domains such as the EF-hand and coiled-coil domains as well as Rab-GTPase domain. Although Rab44 genes have been found in mammalian genomes, no studies concerning Rab44 have been reported yet. Here, we identified Rab44 as an upregulated protein during osteoclast differentiation. Knockdown of Rab44 by small interfering RNA promotes RANKL-induced osteoclast differentiation of the murine monocytic cell line, RAW-D or of bone marrow-derived macrophages (BMMs). In contrast, overexpression of Rab44 prevents osteoclast differentiation. Rab44 was localized in the Golgi complex and lysosomes, and Rab44 overexpression caused an enlargement of early endosomes. A series of deletion mutant studies of Rab44 showed that the coiled-coil domain and lipidation sites of Rab44 is important for regulation of osteoclast differentiation. Mechanistically, Rab44 affects nuclear factor of activated T-cells c1 (NFATc1) signaling in RANKL-stimulated macrophages. Moreover, Rab44 depletion caused an elevation in intracellular Ca²⁺ transients upon RANKL stimulation, and particularly regulated lysosomal Ca²⁺ influx. Taken together, these results suggest that Rab44 negatively regulates osteoclast differentiation by modulating intracellular Ca²⁺ levels followed by NFATc1 activation.     

Molecular regulation of CRAC channels and their role in lymphocyte function

Calcium (Ca²⁺) influx is required for the activation and function of all cells in the immune system. It is mediated mainly by store-operated Ca²⁺ entry (SOCE) through Ca²⁺ release-activated Ca²⁺ (CRAC) channels located in the plasma membrane. CRAC channels are composed of ORAI proteins that form the channel pore and are activated by stromal interaction molecules (STIM) 1 and 2. Located in the membrane of the endoplasmic reticulum, STIM1 and STIM2 have the dual function of sensing the intraluminal Ca²⁺ concentration in the ER and to activate CRAC channels. A decrease in the ER’s Ca²⁺ concentration induces STIM multimerization and translocation into puncta close to the plasma membrane where they bind to and activate ORAI channels. Since the identification of ORAI and STIM genes as the principal mediators of CRAC channel function, substantial advances have been achieved in understanding the molecular regulation and physiological role of CRAC channels in cells of the immune system and other organs. In this review, we discuss the mechanisms that regulate CRAC channel function and SOCE, the role of recently identified proteins and mechanisms that modulate the activation of ORAI/STIM proteins and the consequences of CRAC channel dysregulation for lymphocyte function and immunity.     

Direct inhibition of phospholipid scrambling activity in erythrocytes by potassium ions

The exposure of phosphatidylserine (PS) at the cell surface plays a critical role in blood coagulation and serves as a macrophage recognition moiety for the engulfment of apoptotic cells. Previous observations have shown that a high extracellular [K⁺] and selective K⁺ channel blockers inhibit PS exposure in platelets and erythrocytes. Here we show that the rate of PS exposure in erythrocytes decreases by ~50% when the intracellular [K⁺] increases from 0 to physiological concentrations. Using resealed erythrocyte membranes, we further show that lipid scrambling is inducible by raising the intracellular [Ca²⁺] and that K⁺ ions have a direct inhibitory effect on this process. Lipid scrambling in resealed ghosts occurs in the absence of cell shrinkage and microvesicle formation, processes that are generally attributed to Ca²⁺-induced lipid scrambling in intact erythrocytes. Thus, opening of Ca²⁺-sensitive K⁺ channels causes loss of intracellular K⁺ that results in reduced intrinsic inhibitory effect of these ions on scramblase activity. Received 11 September 2008; received after revision 17 October 2008; accepted 27 October 2008     

Interaction of sulfonylurea with the pancreatic B-cell

Conclusions Sulfonylureas have a variety of effects on pancreatic B-cells. In the present review an attempt has been made to identify those that appear fundamental from a mechanistic point of view and in that sense common to all hypoglycemic drugs tested. On several points the available able experimental information is limited. With this reservation in mind, the following general hypothesis is presented for the insulin-releasing action of this class of drugs. Hypoglycemic sulfonylureas and related [(acylamino)alkyl]benzoic acids bind to the B-cell plasma membrane, a step in which hydrophobic anchoring is essential. Dissociated acidic COOH or SO₂NHCO groups in the drugs are thus presented to an ion-gating protein in the plasma membrane, possibly in the vicinity of a pair of sulfur atoms. The reduced state of these sulfurs is promoted, preventing the formation of a disulfide bridge. K⁺ permeability is thereby decreased, favoring depolarization of the B-cell and Ca²⁺ influx through voltage-dependent channels. Finally, Ca²⁺ triggers the physiological apparatus for discharge of the insulin secretory granules. The effect of this insulinreleasing signal chain is amplified by cyclic AMP which increases in the B-cell as a consequence of depolarization and Ca²⁺ influx. This hypothesis does not attribute an ionophoretic role to the sulfonylureas per sebecause various experiments with cells and artificial membrane systems render such an idea apparently less tenable.     

Calcineurin activity is required for the completion of cytokinesis

Successful completion of cytokinesis requires the spatio-temporal regulation of protein phosphorylation and the coordinated activity of protein kinases and phosphatases. Many mitotic protein kinases are well characterized while mitotic phosphatases are largely unknown. Here, we show that the Ca²⁺- and calmodulin-dependent phosphatase, calcineurin (CaN), is required for cytokinesis in mammalian cells, functioning specifically at the abscission stage. CaN inhibitors induce multinucleation in HeLa cells and prolong the time cells spend connected via an extended intracellular bridge. Upon Ca²⁺ influx during cytokinesis, CaN is activated, targeting a set of proteins for dephosphorylation, including dynamin II (dynII). At the intracellular bridge, phospho-dynII and CaN are co-localized to dual flanking midbody rings (FMRs) that reside on either side of the central midbody ring. CaN activity and disassembly of the FMRs coincide with abscission. Thus, CaN activity at the midbody plays a key role in regulating the completion of cytokinesis in mammalian cells.     

Evidence for suppression of Na-dependent Ca2+ efflux from rat brain synaptosomes by ovarian steroids in vivo

Summary The possibility that intracellular Ca²⁺, which mediates neurotransmitter release, regulation of membrane permeability, microtubule polymerization and axonal transport, is influenced by gonadal steroids via a Na–Ca exchange mechanism was examined. The resting Ca²⁺ uptake into synaptosomes was measured using crude synaptosomal pellets (P₂ fraction), isolated from the brain stem, mesencephalic reticular formation (MRF), nucleus caudatus (NC) and the hippocampus of intact, long-term ovariectomized (OVX) and OVX plus progesterone (P) or estradiol-17 benzoate (EB) treated adult female rats. Irrespective of the brain structure investigated, the uptake was 1) markedly increased in synaptosomes from OVX animals in comparison to intact controls, and 2) reduced to near control values in synaptosomes from OVX rats treated s.c. with a single dose of 2 mg P or 5 g EB. Since Ca²⁺ influx into synaptosomes was shown earlier to depend on external sodium concentration, which was the same in all experiments described in this work, the results obtained indicate that ovarian steroids modulate basal synaptic activity in the rat brain by suppressing Na-dependent Ca²⁺ efflux from the nerve cell.     

Neuronal calcium signaling: function and dysfunction

Calcium (Ca²⁺) is an universal second messenger that regulates the most important activities of all eukaryotic cells. It is of critical importance to neurons as it participates in the transmission of the depolarizing signal and contributes to synaptic activity. Neurons have thus developed extensive and intricate Ca²⁺ signaling pathways to couple the Ca²⁺ signal to their biochemical machinery. Ca²⁺ influx into neurons occurs through plasma membrane receptors and voltage-dependent ion channels. The release of Ca²⁺ from the intracellular stores, such as the endoplasmic reticulum, by intracellular channels also contributes to the elevation of cytosolic Ca²⁺. Inside the cell, Ca²⁺ is controlled by the buffering action of cytosolic Ca²⁺-binding proteins and by its uptake and release by mitochondria. The uptake of Ca²⁺ in the mitochondrial matrix stimulates the citric acid cycle, thus enhancing ATP production and the removal of Ca²⁺ from the cytosol by the ATP-driven pumps in the endoplasmic reticulum and the plasma membrane. A Na⁺/Ca²⁺ exchanger in the plasma membrane also participates in the control of neuronal Ca²⁺. The impaired ability of neurons to maintain an adequate energy level may impact Ca²⁺ signaling: this occurs during aging and in neurodegenerative disease processes. The focus of this review is on neuronal Ca²⁺ signaling and its involvement in synaptic signaling processes, neuronal energy metabolism, and neurotransmission. The contribution of altered Ca²⁺ signaling in the most important neurological disorders will then be considered.     

Sr2+ can become incorporated into an agonist-sensitive,cytoplasmic Ca2+ store in a cell line derived from the equine sweat gland epithelium

We have explored the properties of a Ca²⁺-dependent cell-signalling pathway that becomes active when cultured equine sweat gland cells are stimulated with ATP. The ATP-regulated, Ca²⁺-influx pathway allowed Sr²⁺ to enter the cytoplasm but permitted only a minimal influx of Ba²⁺. Experiments in which cells were repeatedly stimulated with ATP suggested that Sr²⁺, but not Ba²⁺, could become incorporated into the agonist-sensitive, cytoplasmic Ca²⁺ store. Further evidence for this was provided by experiments using ionomycin, a Ca²⁺ ionophore which has no affinity for Sr²⁺.     

The action of the peptidoleukotriene LTD4 on intracellular calcium in rat mesangial cells

The dose-dependent effect of CGP 45715A on the LTD₄-induced Ca²⁺ response of glomerular mesangial cells has been studied. Our results demonstrate that the LTD₄-dependent increase in the cytosolic Ca²⁺ concentration primarily involves an InsP₃-mediated release of Ca²⁺ from intracellular storage sites and to a minor extent an enhanced influx of Ca²⁺ through receptor-operated Ca²⁺ channels located in the plasma membrane. The action of CGP 45715A on the Ca²⁺ response is an inhibitory one and is convincingly explained by a displacement of LTD₄ from its receptor site(s). The contractile effect of LTD₄ on pulmonary smooth muscle is proposed to be mainly caused by a receptor-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate.     

The role of calcium in aggregation and development ofDictyostelium

Changes in cytosolic Ca²⁺ play an important role in a wide array of cell types and the control of its concentration depends upon the interplay of many cellular constituents. Resting cells maintain cytosolic calcium ([Ca²⁺]_i) at a low level in the face of steep gradients of extracellular and sequestered Ca²⁺. Many different signals can provoke the opening of calcium channels in the plasma membrane or in intracellular compartments and cause rapid influx of Ca²⁺ into the cytosol and elevation of [Ca²⁺]_i. After such stimulation Ca²⁺ ATPases located in the plasma membrane and in the membranes of intracellular stores rapidly return [Ca²⁺]_i to its basal level. Such responses to elevation of [Ca²⁺]_i are a part of an important signal transduction mechanism that uses calcium (often via the binding protein calmodulin) to mediate a variety of cellular actions responsive to outside influences.     

Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

Summary The role of Ca²⁺ in secretagogue-induced insulin release is documented not only by the measurements of⁴⁵Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca²⁺, [Ca²⁺]_i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca²⁺]_i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca²⁺ homeostasis by organelles from a rat insulinoma, was investigated with a Ca²⁺ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca²⁺ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca²⁺]_i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca²⁺ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

Ca2+ release-activated Ca2+ (CRAC) current, structure, and function

Store-operated Ca²⁺ entry describes the phenomenon that connects a depletion of internal Ca²⁺ stores to an activation of plasma membrane-located Ca²⁺ selective ion channels. Tremendous progress towards the underlying molecular mechanism came with the discovery of the two respective limiting components, STIM and Orai. STIM1 represents the ER-located Ca²⁺ sensor and transmits the signal of store depletion to the plasma membrane. Here it couples to and activates Orai, the highly Ca²⁺-selective pore-forming subunit of Ca²⁺ release-activated Ca²⁺ channels. In this review, we focus on the molecular steps that these two proteins undergo from store-depletion to their coupling, the activation, and regulation of Ca²⁺ currents.     

Darier’s disease: a calcium-signaling perspective

Ca²⁺ influx evoked across the plasma membrane upon internal store depletion is essential for a myriad of cellular functions including gene expression, cell proliferation, differentiation and even apoptosis. Darier’s disease (DD), an autosomal dominant inherited disorder of the skin, arising due to mutations in the isoform 2 of the sarco (endo) plasmic reticulum Ca²⁺ ATPase (SERCA2), exemplifies an anomaly of Ca²⁺ signaling disturbances. Owing to loss of function mutations in SERCA2, keratinocytes in DD patients have a reduced pool of endoplasmic reticulum (ER) Ca²⁺. Importantly, the status of ER Ca²⁺ is critical for the activation of a class of plasma membrane Ca²⁺ channels referred to as store operated Ca²⁺ channels (SOCs). The widely expressed transient receptor potential (TRP) family of channels is proposed to be SOCs. In this review we discuss DD from the viewpoint of Ca²⁺ signaling and present a potential role for TRPC1 in the disease pathogenesis. Received 30 August 2007; received after revision 17 October 2007; accepted 6 November 2007