Extracellular calcium sensing and extracellular calcium signaling

The cloning of a G protein-coupled extracellular Ca(2+) (Ca(o)(2+))-sensing receptor (CaR) has elucidated the molecular basis for many of the previously recognized effects of Ca(o)(2+) on tissues that maintain systemic Ca(o)(2+) homeostasis, especially parathyroid chief cells and several cells in the kidney. The availability of the cloned CaR enabled the development of DNA and antibody probes for identifying the CaR's mRNA and protein, respectively, within these and other tissues. It also permitted the identification of human diseases resulting from inactivating or activating mutations of the CaR gene and the subsequent generation of mice with targeted disruption of the CaR gene. The characteristic alterations in parathyroid and renal function in these patients and in the mice with "knockout" of the CaR gene have provided valuable information on the CaR's physiological roles in these tissues participating in mineral ion homeostasis. Nevertheless, relatively little is known about how the CaR regulates other tissues involved in systemic Ca(o)(2+) homeostasis, particularly bone and intestine. Moreover, there is evidence that additional Ca(o)(2+) sensors may exist in bone cells that mediate some or even all of the known effects of Ca(o)(2+) on these cells. Even more remains to be learned about the CaR's function in the rapidly growing list of cells that express it but are uninvolved in systemic Ca(o)(2+) metabolism. Available data suggest that the receptor serves numerous roles outside of systemic mineral ion homeostasis, ranging from the regulation of hormonal secretion and the activities of various ion channels to the longer term control of gene expression, programmed cell death (apoptosis), and cellular proliferation. In some cases, the CaR on these "nonhomeostatic" cells responds to local changes in Ca(o)(2+) taking place within compartments of the extracellular fluid (ECF) that communicate with the outside environment (e.g., the gastrointestinal tract). In others, localized changes in Ca(o)(2+) within the ECF can originate from several mechanisms, including fluxes of calcium ions into or out of cellular or extracellular stores or across epithelium that absorb or secrete Ca(2+). In any event, the CaR and other receptors/sensors for Ca(o)(2+) and probably for other extracellular ions represent versatile regulators of numerous cellular functions and may serve as important therapeutic targets.     

Cytoplasmic calcium oscillations and store-operated calcium influx

Intracellular calcium oscillations have fascinated scientists for decades. They provide an important cellular signal which, unlike most signalling mechanisms, is digitally encoded. While it is generally agreed that oscillations most frequently arise from cyclical release and re-uptake of intracellularly stored calcium, it is becoming increasingly clear that influx of calcium across the plasma membrane also plays a critical role in their maintenance and even in delivering their signal to the correct cellular locus. In this review we will discuss the role played by Ca²⁺ entry mechanisms in Ca²⁺ oscillations, and approaches to understanding the molecular nature of this Ca²⁺ entry pathway.     

Gastrointestinal absorption of calcium from milk and calcium salts

Whether ingested calcium is absorbed more efficiently from freely water-soluble calcium salts than from poorly soluble salts is unclear. It is also unknown whether calcium is absorbed better from dairy products than from calcium salts. Using a method by which the net absorption of calcium can be accurately measured after a single dose, we studied eight healthy fasting subjects after they took a 500-mg dose of calcium from each of five calcium salts with various degrees of water solubility and from milk. The order of administration of the agents given was randomly determined. The mean (+/- SEM) net calcium absorption, in decreasing order of the solubility of the salts, was 32 +/- 4 percent from calcium acetate, 32 +/- 4 percent from calcium lactate, 27 +/- 3 percent from calcium gluconate, 30 +/- 3 percent from calcium citrate, and 39 +/- 3 percent from calcium carbonate. The differences in absorption were not statistically significant according to analysis of variance. On the basis of in vitro solubility experiments in acid mediums, we hypothesize that acid dissolution in the gastrointestinal tract may be responsible for the similar absorption of calcium from salts with widely different water solubilities. Calcium absorption from whole milk (31 +/- 3 percent) was similar to absorption from calcium salts. We conclude that calcium absorption from carbonate, acetate, lactate, gluconate, and citrate salts of calcium, and from whole milk, is similar in fasting healthy young subjects. Further study will be required to determine whether the results would be different in older subjects, with a higher dose of calcium, or if the calcium was ingested with food.     

Intracellular calcium buffering shapes calcium oscillations in Xenopus melanotropes

The pituitary melanotrope cell of Xenopus laevis displays cytosolic Ca2+ oscillations that arise for the interplay between the burst-like openings of voltage-operated Ca2+ channels and Ca2+-extrusion mechanisms. We have previously shown that Ca2+-extrusion rates increase with increases in [Ca2+]i, suggesting that Ca2+ itself plays a role in shaping the Ca2+ oscillations. The purpose of the present study was to test this hypothesis by manipulating the intracellular Ca2+ buffering capacity of the cell and determining the consequences of such manipulations for the shape of the Ca2+ oscillations. We manipulated the cytosolic buffering capacity by loading the fast Ca2+ chelator BAPTA into cells. During loading the [Ca2+]i was dynamically imaged with confocal laser scanning microscopy. The basal [Ca2+]i was reduced with BAPTA loading and this reduction was associated with lower Ca2+-extrusion rates, a broadening of the Ca2+ oscillations and declined oscillation frequencies. Short loading periods of the buffer led to new, stable patterns of Ca2+ signaling and to reduced but stable levels of peptide secretion. We propose that the cytosolic Ca2+ buffer capacity, and thus by inference the profile of intracellular Ca2+ buffering proteins, is an important factor in setting the frequency and shape of Ca2+ oscillations.     

Effects of calcium chelators on intracellular calcium and excitotoxicity

In an attempt to probe the relationship between excitotoxicity and increases in intracellular calcium ([Ca²⁺]_i), BAPTA-AM and its analogs were applied to cultured hippocampal neurons. Chelation of [Ca²⁺]_i depressed and prolonged transient responses to glutamate and did not effect elevation of [Ca²⁺]_i by prolonged exposure. This explains the inability of the chelators to prevent glutamate-induced toxicity.     

Sodium/calcium exchange regulates cytoplasmic calcium in smooth muscle

The sodium/calcium (Na⁺/Ca²⁺) exchanger is often considered to be a key regulator of the cytoplasmic calcium concentration ([Ca²⁺]) in smooth muscle but neither its precise role in Ca²⁺ homeostasis nor even its existence in smooth muscle are generally agreed upon. Here we directly assessed the role Na⁺/Ca²⁺ exchange plays in regulating [Ca²⁺] in single voltage-clamped smooth muscle cells. Following an elevation of [Ca²⁺], its decline was found to have both voltage-dependent and voltage-independent components. The voltage-dependent component was abolished when Na⁺ was removed from the external bathing solution. During the fall of [Ca²⁺] a small and declining Na⁺-dependent inward current was observed of a magnitude predicted by 31 Na⁺/Ca²⁺ exchange stoichiometry. At [Ca²⁺] above 400 nM the principal efflux of Ca²⁺ above rest was attributed to this Na⁺-dependent removal mechanism. These results establish that a Na⁺/Ca²⁺ exchanger exists in smooth muscle and argue that it can regulate [Ca²⁺] at physiological Ca²⁺ concentrations.     

B-lymphocyte calcium inFlux

Summary:  Dynamic changes in cytoplasmic calcium concentration dictate the immunological fate and functions of lymphocytes. During the past few years, important details have been revealed about the mechanism of store-operated calcium entry in lymphocytes, including the molecular identity of calcium release-activated calcium (CRAC) channels and the endoplasmic reticulum (ER) calcium sensor (STIM1) responsible for CRAC channel activation following calcium depletion of stores. However, details of the potential fine regulation of CRAC channel activation that may be imposed on lymphocytes following physiologic stimulation within an inflammatory environment have not been fully addressed. In this review, we discuss several underexplored aspects of store-operated (CRAC-mediated) and store-independent calcium signaling in B lymphocytes. First, we discuss results suggesting that coupling between stores and CRAC channels may be regulated, allowing for fine tuning of CRAC channel activation following depletion of ER stores. Second, we discuss mechanisms that sustain the duration of calcium entry via CRAC channels. Finally, we discuss distinct calcium permeant non-selective cation channels (NSCCs) that are activated by innate stimuli in B cells, the potential means by which these innate calcium signaling pathways and CRAC channels crossregulate one another, and the mechanistic basis and physiologic consequences of innate calcium signaling.     

Dialysate calcium and plasma calcium fractions during and after haemodialysis

Summary Dialysate Calcium and Plasma Calcium Fractions during and after Haemodialysis:The effect of different dialysate Ca concentrations on the plasma Ca fractions was examined in 28 patients. In 10 patients dialysed with a dialysate Ca concentration of 3.0 mEq/l the Ca fractions were determined at the start and end of dialysis. 8 patients were dialysed with dialysate Ca of 3.5 mEq/l. In this group the Ca fractions were also estimated in the dialysis-free interval. The third group was dialysed with a dialysate Ca of 4.5 mEq/l. Total calcium and protein-bound calcium rose significantly in all groups. Ionised calcium in the first group was significantly reduced, in the second group it remained constant and in the third group it was significantly raised. Since parathyroid function depends on the plasma ionised calcium it is concluded that a dialysate concentration of 3.0 mEq/l is partly responsible for the pathogenesis of secondary hyperparathyroidism and of renal osteodystrophy. In normocalcaemic patients a dialysate Ca concentration of 3.5 to 4.0 mEq/l is optimal. In patients entering long-term haemodialysis treatment with pronounced calcium deficiency symptoms a dialysate Ca of up to 4.5 mEq/l may be indicated for a short period after having normalized the inorganic phosphate levels in order to prevent extraosseous calcification.     

Store-operated calcium channels

In electrically nonexcitable cells, Ca(2+) influx is essential for regulating a host of kinetically distinct processes involving exocytosis, enzyme control, gene regulation, cell growth and proliferation, and apoptosis. The major Ca(2+) entry pathway in these cells is the store-operated one, in which the emptying of intracellular Ca(2+) stores activates Ca(2+) influx (store-operated Ca(2+) entry, or capacitative Ca(2+) entry). Several biophysically distinct store-operated currents have been reported, but the best characterized is the Ca(2+) release-activated Ca(2+) current, I(CRAC). Although it was initially considered to function only in nonexcitable cells, growing evidence now points towards a central role for I(CRAC)-like currents in excitable cells too. In spite of intense research, the signal that relays the store Ca(2+) content to CRAC channels in the plasma membrane, as well as the molecular identity of the Ca(2+) sensor within the stores, remains elusive. Resolution of these issues would be greatly helped by the identification of the CRAC channel gene. In some systems, evidence suggests that store-operated channels might be related to TRP homologs, although no consensus has yet been reached. Better understood are mechanisms that inactivate store-operated entry and hence control the overall duration of Ca(2+) entry. Recent work has revealed a central role for mitochondria in the regulation of I(CRAC), and this is particularly prominent under physiological conditions. I(CRAC) therefore represents a dynamic interplay between endoplasmic reticulum, mitochondria, and plasma membrane. In this review, we describe the key electrophysiological features of I(CRAC) and other store-operated Ca(2+) currents and how they are regulated, and we consider recent advances that have shed insight into the molecular mechanisms involved in this ubiquitous and vital Ca(2+) entry pathway.     

Structurally delineating stromal interaction molecules as the endoplasmic reticulum calcium sensors and regulators of calcium release-activated calcium entry

Summary:  The endoplasmic reticulum (ER) lumen stores a crucial source of calcium (Ca²⁺) maintained orders of magnitude higher than the cytosol for the activation of a plethora of cellular responses transmitted in health and disease by a mutually efficient and communicative exchange of Ca²⁺ between compartments. A coordination of the Ca²⁺ signal is evident in the development of Ca²⁺ release-activated Ca²⁺ (CRAC) entry, vital to lymphocyte activation and replenishing of the ER Ca²⁺ stores, where modest decreases in ER luminal Ca²⁺ induce sustained increases in cytosolic Ca²⁺ sourced from steadfast extracellular Ca²⁺ supplies. While protein sensors that transduce Ca²⁺ signals in the cytosol such as calmodulin are succinctly understood, comparative data on the ER luminal Ca²⁺ sensors is only recently coming to light with the discovery that stromal interaction molecules (STIMs) sense variations in ER stored Ca²⁺ levels in the functional regulation of plasma membrane Orai proteins, the major component of CRAC channel pores. Drawing from data on the role of STIMs in the modulation of CRAC entry, this review illustrates the structural features that delimit the functional characteristics of ER Ca²⁺ sensors relative to well known cytoplasmic Ca²⁺ sensors.     

Presynaptic calcium channels and the depletion of synaptic cleft calcium ions

The entry of calcium ions (Ca(2+)) through voltage-gated calcium channels is an essential step in the release of neurotransmitter at the presynaptic nerve terminal. Because the calcium channels are clustered at the release sites, the flux of Ca(2+) into the terminal inevitably removes the ion from the adjacent extracellular space, the synaptic cleft. We have used the large calyx-type synapse of the chick ciliary ganglion to test for synaptic cleft Ca(2+) depletion. The terminal was voltage clamped at a holding potential (V(H)) of -80 mV and a depolarizing pulse was applied to a range of potentials (-60 to +60 mV). The voltage pulse activated a sustained inward calcium current and was followed, on return of the membrane potential to V(H), by an inward calcium tail current. The amplitude of the tail current reflects both the number of open calcium channels at the end of the voltage pulse and the Ca(2+) electrochemical gradient. External barium was substituted for calcium as the charge-carrying ion because initial experiments demonstrated calcium-dependent inactivation of the presynaptic calcium channels. Tail current recruitment was compared in calyx nerve terminals that remained attached to the postsynaptic neuron and therefore retained a synaptic cleft, with terminals that had been fully isolated. In isolated terminals, the tail currents exhibited recruitment curves that could be fit by a Boltzmann distribution with a mean V(1/2) of 0.4 mV and a slope factor of 5.4. However, in attached calyces tail current recruitment was skewed to depolarized potentials with a mean V(1/2) of 11.9 mV and a slope factor of 12.0. The degree of skew of the recruitment curve in the attached calyces correlated with the amplitude of the inward current evoked by the step depolarization. The simplest interpretation of these findings is that during the depolarizing pulse Ba(2+) is removed from the synaptic cleft faster than it is replenished, thus reducing the tail current by reducing the driving force for ion entry. Ca(2+) depletion during presynaptic calcium channel activation is likely to be a general property of chemical transmission at fast synapses that sets a functional limit to the duration of sustained secretion. The synapse may have evolved to minimized cleft depletion by developing a calcium-efficient mechanism to gate transmitter release that requires the concurrent opening of only a few low conductance calcium channels.     

Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells

Treatment of human epithelial kidney (HEK293) cells with low concentrations of the muscarinic agonist methacholine results in the activation of complex and repetitive cycling of intracellular calcium ([Ca²⁺]_i), known as [Ca²⁺]_i oscillations. These oscillations occur with a frequency that depends on the concentration of methacholine, whereas the magnitude of the [Ca²⁺]_i spikes does not. The oscillations do not persist in the absence of extracellular Ca²⁺, leading to the conclusion that entry of Ca²⁺ across the plasma membrane plays a significant role in either their initiation or maintenance. However, treatment of cells with high concentrations of GdCl₃, a condition which limits the flux of calcium ions across the plasma membrane in both directions, allows sustained [Ca²⁺]_i oscillations to occur. This suggests that the mechanisms that both initiate and regenerate [Ca²⁺]_i oscillations are intrinsic to the intracellular milieu and do not require entry of extracellular Ca²⁺. This would additionally suggest that, under normal conditions, the role of calcium entry is to sustain [Ca²⁺]_i oscillations. By utilizing relatively specific pharmacological manoeuvres we provide evidence that the Ca²⁺ entry that supports Ca²⁺ oscillations occurs through the store-operated or capacitative calcium entry pathway. However, by artificial introduction of a non-store-operated pathway into the cells (TRPC3 channels), we find that other Ca²⁺ entry mechanisms can influence oscillation frequency in addition to the store-operated channels.     

Regulation of the rat sarcoplasmic reticulum calcium release channel by calcium

The regulation by calcium of the ryanodine receptor/SR calcium release channel (RyR) from rat skeletal muscle was studied under isolated conditions and in situ. RyRs were either solubilized and incorporated into lipid bilayers or single fibres were mounted into a Vaseline gap voltage clamp. Single channel data were compared to parameters determined from the calculated calcium release flux. With K⁺ (250 mM) being the charge carrier the single channel conductance was 529 pS at 50 M Ca²⁺ cis and trans, and decreased with increasing cis [Ca²⁺]. Open probability showed a bell shaped calcium dependence revealing an activatory and an inhibitory Ca²⁺ binding site (Hill coefficients of 1.18 and 1.28, respectively) with half activatory and inhibitory concentrations of 9.4 and 298 M. The parameters of the inhibitory site agreed with the calcium dependence of channel inactivation deduced from the decline in SR calcium release in isolated fibres. Mean open time showed slight [Ca²⁺] dependence following a single exponential at every Ca²⁺ concentration tested. Closed time histograms, at high [Ca²⁺], were fitted with three exponentials, from which the longest was calcium independent, and resembled the recovery time constant of SR inactivation (115 ± 15 ms) obtained in isolated fibres. The data are in agreement with a model where calcium binding to the inhibitory site on RyR would be responsible for the calcium dependent inactivation in situ.     

The effects of calcium and calcium channel blockers on sodium pump

The effects of 10 mM Ca²⁺ and Ca²⁺ channel blockers verapamil, diltiazem and flunarizine on the ouabain-sensitive electrogenic Na⁺, K⁺ pump activity of mouse diaphragm muscle fibres enriched with Na⁺ were compared with the changes in cytosolic [Ca²⁺]. The electrogenic Na⁺ pump activity produced by adding K⁺ to muscles previously bathed for 4 h in a K⁺-free, 2-mM [Ca²⁺] solution increased the resting membrane potential by about 18 mV. This hyperpolarization was completely inhibited after 10 min incubation in 10 mM Ca²⁺. Verapamil 10^–5M, 10^–5M diltiazem and 10^–7 M flunarizine effectively prevented the effect of elevated [Ca²⁺]. At these concentrations, these drugs did not affect the K⁺-induced hyperpolarization. In mouse diaphragm, the basal cytosolic [Ca²⁺] measured by the fluorescent indicator 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]2-(2-amino 5-methylphenoxy) ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (fura-2/AM) was 261±6 nM. After 4 h in a Liley K⁺-free, 2 mM [Ca²⁺] solution, the cytosolic [Ca²⁺] increased to 314±28 nM. Increase in [Ca²⁺] from 2 to 10 mM caused a twofold increase of cytosolic [Ca²⁺] to 637±26 nM. This rise was, like the Ca²⁺-induced inhibition of electrogenic pump, prevented by 10^–5 M verapamil, 10^–5M diltiazem and 10^–7 M flunarizine. The results suggest that substances which block Ca²⁺ entry into the cell prevent the Ca²⁺ induced inhibition of the Na⁺ pump.     

人工骨材料磷酸钙及硫酸钙在腱-骨愈合中的作用

背景：有关促进腱-骨愈合的方法文献报道很多,主要方法就是给腱-骨间隙添加一些刺激物质,以此促进腱骨的愈合。磷酸钙盐作为生物活性材料具有骨传导性,已广泛应用于临床骨缺损的替代和填充。而硫酸钙作为人工材料,具有潜在的骨诱导活性。 目的：在自体腘绳肌肌腱移植重建膝关节前交叉韧带过程中,观察人工骨材料磷酸钙及硫酸钙促进腱-骨愈合的效应。 方法：选用36条雄性成熟比格犬,先行切断双侧膝关节前交叉韧带,取同侧后肢趾长屈肌腱作为移植物,采用悬吊式固定重建前交叉韧带。按随机数字表法分为3组,磷酸钙组于股骨腱骨隧道中注入磷酸钙,硫酸钙组注入硫酸钙,空白组韧带重建结束后不添加任何填充物。分别于重建后1,2,3,4,6个月取材行大体观察、组织学和生物力学观测。 结果与结论：前交叉韧带重建后1,2,3,4个月时,磷酸钙组及硫酸钙组腱骨界面纤维连接明显强于空白组,而磷酸钙组、硫酸钙组差异无显著性意义。6个月时,各组愈合程度相似。生物力学方面,重建后1个月时,磷酸钙组及硫酸钙组腱骨界面的抗拉脱强度均高于空白组(P < 0.05),而磷酸钙组、硫酸钙组差异无显著性意义(P > 0.05)。提示磷酸钙及硫酸钙均能促进腱-骨愈合,两者之间无明显差异。     

How calcium from calcium carbonate and milk benefit peptic ulcer patients

Calcium from calcium containing antacids and milk enhance the integrity of gastrointestinal mucosa and mucus, as it is the natural linker agent of these structures, which strengthens their defense function.     

Presynaptic calcium stores underlie large-amplitude miniature IPSCs and spontaneous calcium transients

The cellular mechanisms responsible for large miniature currents in some brain synapses remain undefined. In Purkinje cells, we found that large-amplitude miniature inhibitory postsynaptic currents (mIPSCs) were inhibited by ryanodine or by long-term removal of extracellular Ca2+. Two-photon Ca2+ imaging revealed random, ryanodine-sensitive intracellular Ca2+ transients, spatially constrained at putative presynaptic terminals. At high concentration, ryanodine decreased action-potential-evoked rises in intracellular Ca2+. Immuno-localization showed ryanodine receptors in these terminals. Our data suggest that large mIPSCs are multivesicular events regulated by Ca2+ release from ryanodine-sensitive presynaptic Ca2+ stores.