G protein-coupled, extracellular Ca2+ (Ca o 2+ )-sensing receptor enables Ca o 2+ to function as a versatile extracellular first messenger

The cloning of a G protein-coupled, extracellular Ca²⁺ (Ca _o ²⁺ )-sensing receptor (CaR) has afforded a molecular basis for a number of the known effects of Ca _o ²⁺ on tissues involved in maintaining systemic calcium homeostasis, especially parathyroid gland and kidney. In addition to providing molecular tools for showing that CaR messenger RNA and protein are present within these tissues, the cloned CaR has permitted documentation that several human diseases are the result of inactivating or activating mutations of this receptor as well as generation of mice that have targeted disruption of the CaR gene. Characteristic changes in the functions of parathyroid and kidney in these patients as well as in the CaR “knockout” mice have elucidated considerably the CaR’s physiological roles in mineral ion homeostasis. Nevertheless, a great deal remains to be learned about how this receptor regulates the functioning of other tissues involved in Ca _o ²⁺ metabolism, such as bone and intestine. Further study of these human diseases and of the mouse models will doubtless be useful in gaining additional understanding of the CaR’s roles in these latter tissues. Furthermore, we understand little of the CaR’s functions in tissues that are not directly involved in systemic mineral ion metabolism, where the receptor probably serves diverse other roles. Some of these functions may be related to the control of intra- and local extracellular concentrations of Ca²⁺, while others may be unrelated to either systemic or local ionic homeostasis. In any case, the CaR and conceivably additional receptors/sensors for Ca²⁺ or other extracellular ions represent versatile regulators of a wide variety of cellular functions and represent important targets for novel classes of therapeutics.     

Deprivation of Na+, Ca2+ and Mg2+ from the extracellular solution increases cytosolic Ca2+ and stimulates catecholamine secretion from cultured bovine adrenal chromaffin cells

We report here that exposing cultured chromaffin cells to a low ionic strength medium (with sucrose in place of NaCl to maintain osmolarity) can induce a marked elevation in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine (CA) release. To determine the underlying mechanism, we first studied the effects of low [Na+]o on single cell [Ca2+]i (using fluo-3 as Ca2+ indicator) and CA release from many cells. In a Mg2+ and Ca2+-deficient medium, lowering the external concentration of Na2+ ([Na+]o) evoked CA secretion preceded by a transitory [Ca2+]i rise, the amplitude of which was inversely related to [Na+]o. By contrast, in the presence of either [Ca2+]o (2 mM) and [Mg2+]o (1.4 mM) or [Mg2+]o alone (3.4 mM), lowering the ionic strength was without effect. Furthermore, in a physiologic [Na+]o, [Ca2+]o and [Mg2+]o medium, two or three consecutive applications of the cholinergic agonist oxotremorine-M (oxo-M) consistently evoked a substantial [Ca2+]i rise. By contrast, consecutive applications of oxo-M in a Ca2+-deficient medium failed to evoke a rise in [Ca2+]i after the first exposure to the agonist. To clarify the underlying mechanism, we measured and compared the effects of low [Na+]o and the cholinergic agonists nicotine and oxo-M on changes in [Ca2+]i; we studied the effects of these agonists on both membrane potential, Vm (under current clamp conditions), and [Ca2+]i by single cell microfluorimetry (indo-1 as Ca2+ indicator). We observed that, in the presence of [Ca2+]o and [Mg2+]o, lowering [Na+]o had no effect on Vm. In a Ca2+-deficient medium, lowering [Na+]o depolarized the membrane from ca. –60 to –10 mV. As expected, we found that nicotine (10 M) depolarized the membrane (from ca. –60 to –20 mV) and simultaneously evoked a substantial [Ca2+]i rise that was [Ca2+]o-dependent. However, contrary to our expectations, we found that the muscarinic agonist oxo-M (50 M) also depolarized the membrane and induced an elevation in [Ca2+]i. Furthermore, both signals were blocked by D-tubocurarine, insinuating the nicotinic character of oxo-M in adrenal chromaffin cells from bovine. These results suggest that both nicotine and oxo-M stimulate Ca2+ entry, probably through voltage-gated Ca2+-channels. We also show here that oxo-M (and not low [Na+]o) stimulates phosphoinositide turnover.     

Rapid spatiotemporal patterning of cytosolic Ca2+ underlies flagellar excision in Chlamydomonas reinhardtii.

Ca(2+)-dependent signalling processes are implicated in many aspects of flagella function in the green alga, Chlamydomonas. In this study, we examine the spatiotemporal dynamics of cytosolic Ca2+ ([Ca2+](cyt)) in single Chlamydomonas cells during the process of flagellar excision, using biolistically loaded calcium-responsive dyes. Acid-induced deflagellation occurred in parallel with a single transient elevation in whole-cell [Ca2+](cyt), which was absent in the acid deflagellation-deficient adf1 mutant. Deflagellation could also be induced by elevated external Ca2+ ([Ca2+](ext)), which promoted very rapid spiking of [Ca2+](cyt) across the whole cell and in the flagella. We also detected very rapid apically localised Ca2+ signalling events with an approximate duration of 500 msec. Ninety-seven per cent of deflagellation events coincided with a rapid elevation in [Ca2+](cyt) in the apical region of the cell, either in the form of a whole cell or an apically localised increase, indicating that [Ca2+](cyt) elevations in the apical region play an underlying role in deflagellation. Our data indicate that elevated [Ca2+](ext) acts to disrupt Ca2+ homeostasis which induces deflagellation by both Adf1-dependent and Adf1-independent mechanisms. Elevated [Ca2+](ext) also results in further [Ca2+](cyt) elevations after the main period of whole cell spiking which are very strongly associated with deflagellation, exhibit a high degree of apical localisation and are largely absent in the adf1 mutant. We propose that these later elevations may act as specific signals for deflagellation.     

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca²⁺ influx and mitochondrial Ca²⁺ overload. A substantial change in intracellular Ca²⁺ homeostasis or remodeling of intracellular Ca²⁺ homeostasis may favor neuron damage in old neurons. For investigating Ca²⁺ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca²⁺ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca²⁺ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca²⁺ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca²⁺ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.     

Ca2+ signals in plant immunity

Calcium ions function as a key second messenger ion in eukaryotes. Spatially and temporally defined cytoplasmic Ca²⁺ signals are shaped through the concerted activity of ion channels, exchangers, and pumps in response to diverse stimuli; these signals are then decoded through the activity of Ca²⁺‐binding sensor proteins. In plants, Ca²⁺ signaling is central to both pattern‐ and effector‐triggered immunity, with the generation of characteristic cytoplasmic Ca²⁺ elevations in response to potential pathogens being common to both. However, despite their importance, and a long history of scientific interest, the transport proteins that shape Ca²⁺ signals and their integration remain poorly characterized. Here, we discuss recent work that has both shed light on and deepened the mysteries of Ca²⁺ signaling in plant immunity.     

植物体内Ca2+信号转导过程的研究进展

Ca2+是高等植物细胞内普遍存在的一种信使分子,在植物体内起着非常广泛的作用,参与了植物体内多种刺激-反应的藕联过程。本文介绍了植物体内Ca2+转移系统,Ca2+信号的产生、终止和传递途径,Ca2+信号编码的多样性的最近研究进展。     

Calcium-pH crosstalks in the human mast cell line HMC-1: intracellular alkalinization activates calcium extrusion through the plasma membrane Ca2+-ATPase

The human mast cell line (HMC-1) has been used to study the relationship between intracellular pH and cytosolic calcium (Ca2+) in mast cells. Thapsigargin (TG) caused store-operated Ca2+ entry, that is enhanced by the PKC activator PMA. NH4Cl-induced alkalinization showed an inhibitory effect on TG-sensitive stores depletion (not on TG-insensitive stores), and also on final cytosolic Ca2+ levels reached in response to both TG and the ionophore ionomycin. Loperamide, a positive modulator of store-operated channels, induced a slight Ca2+ entry by itself, and also increased TG-induced Ca2+ entry. This enhancement was not enough to reverse the inhibitory effect of NH4Cl-induced alkalinization. When comparing the effect of NH4Cl-induced alkalinization on Ca2+ levels, with those observed using Ca2+ channel blockers (namely Ni2+ and SKF-96365), cytosolic profiles for this ion are different, either in modified saline solution or in HCO3(-)-free medium. Thus, it seems unlikely that the inhibitory effect of NH4Cl-induced alkalinization on Ca2+ is taking place by blockage of Ca2+ entry. Furthermore, inhibition of the plasma membrane Ca2+-ATPase (an important mechanism for Ca2+ efflux) with sodium orthovanadate (SO) matches with the inhibition of the negative effect on Ca2+ levels elicited by NH4Cl. Data indicate that NH4Cl-induced alkalinization might be activating Ca2+ efflux from the cell, by stimulation of the plasma membrane Ca2+-ATPase, and also confirm our previous finding that Ca2+ is a secondary signal to activate HMC-1 cells.     

A comparison of fluorescent Ca2+ indicators for imaging local Ca2+ signals in cultured cells

Localized subcellular changes in Ca²⁺ serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca²⁺ signaling have been greatly facilitated by the availability of fluorescent Ca²⁺ indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca²⁺ levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca²⁺ signals is lacking. Here, we evaluated several fluorescent Ca²⁺ indicators in the context of local Ca²⁺ signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP₃) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca²⁺ dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca²⁺-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca²⁺ puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca²⁺ signals.     

Mechanical stimulation induces Ca2+i transients and membrane depolarization in cultured endothelial cells. Effects on Ca2+i in co-perfused smooth muscle cells

Cytosolic Ca²⁺ concentration and membrane potential were monitored in individual cultured enothelial cells mechanically stimulated with a micropipette attached to the stage of a microscope. Both dimpling and poking of endothelial cells resulted in Ca²⁺_i transients (from 63 ± 12 to 397 ± 52 nM, characterized by a refractory period of approx. 2 min) and cell depolarization. Ca²⁺_i transients of the reduced amplitude (201 ± 41 nM) were evoked by mechanical stimulation of endothelial cells incubated in a Ca²⁺-free medium. Dimpling-induced Ca²⁺_i transients were refractory to the pretreatments with pertussis toxin, colchicine, or cytochalasin B, and were not mimicked by an increase in the hydrodynamic pressure. In a co-perfusion system (endothelium: smooth muscle), both the KCl-induced depolarization and ionomycin-induced increase in Ca²⁺_i in the endothelial cells resulted in the reduction of Ca²⁺_i in the smooth muscle cells. The data reported are consistent with the phenomenon of vascular relaxation in response to the increased blood flow. We hypothesize that the mechanical interaction of the formed elements with the microvascular endothelium can serve as a pacemaker for the sustained relaxation of vascular smooth muscle.     

The oppositely directed Ca2+ and Na+ transmembrane transport in algal cells

Summary The countertransport of Ca²⁺ and Na⁺ across the membranes of the unicellular fresh-water algaChlamydomonas reinhardtii CW-15 and twoDunaliella species differing in salt tolerance was studied. All algae used are devoid of cell walls. The calcium uptake by twoDunaliella species depended markedly on the intracellular sodium concentration. This calcium uptake was accompanied by Na⁺ release. For 15 and 30 s after artificial gradient formation (Na_int ⁺ greater than Na_ext ⁺) the ratio of released Na⁺ to absorbed Ca²⁺ was 31 and 41, respectively. For the extremely halotolerantD. salina, the apparent Michaelis constant of the Ca²⁺ uptake was 33 M, and for the marine halotolerant algaD. maritima, it was equal to 400 M, presuming more efficient Na⁺-for-Ca²⁺ exchange inD. salina cells. Ouabain, an inhibitor of Na⁺/K⁺-ATPase, suppressed Na⁺ transfer by 25%, whereas the agents blocking Ca²⁺-channels did not affect the transport of Ca²⁺ and Na⁺. The oppositely directed transmembrane Ca²⁺ and Na⁺ transfer was shown to depend on the external concentrations of Na⁺ and H⁺. In the fresh-water algaC. reinhardtii CW-15 (Na_ext ⁺ greater than Na_int ⁺), the direction of Ca²⁺ and Na⁺ fluxes across the plasma membrane was opposite to those described for Dunaliella cells. The results obtained point to the ability of the Na⁺-Ca²⁺ exchanger function in plasma membranes of algal cells.     

Alteration of cytosolic free calcium homeostasis by SIN-1: high sensitivity of L-type Ca2+ channels to extracellular oxidative/nitrosative stress in cerebellar granule cells

Exposure of cerebellar granule neurones in 25 mm KCl HEPES-containing Locke's buffer (pH 7.4) to 50-100 microm SIN-1 during 2 h decreased the steady-state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 +/- 33 nm to 60 +/- 10 nm, whereas exposure to > or = 0.3 mm SIN-1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 +/- 10 nm (due to inactivation of L-type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN-1-induced necrotic cell death. Both effects of SIN-1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4-benzoic acid)porphyrin chloride. Supplementation of Locke's buffer with catalase before addition of 0.5-1 mm SIN-1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60-120 min exposure to SIN-1 and also protected against SIN-1-induced necrotic cell death. alpha-Tocopherol, the potent NMDA receptor antagonist (+)-MK-801 and the N- and P-type Ca2+ channels blocker omega-conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN-1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60-120 min exposure to 0.5-1 mm SIN-1. It is concluded that L-type Ca2+ channels are a primary target of SIN-1-induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5-1 microm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.     

Modulation of the voltage sensor of L-type Ca2+ channels by intracellular Ca2+

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (>/=100 microM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from approximately 0.1 to 100-300 microM sped up the conversion of the gating charge into the negatively distributed mode 10-100-fold. Since the "IQ-AA" mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the "IQ" motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Delta1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation.     

A novel role of Ca2+ and Zn2+: Protection of cells against membrane damage

Certain cytotoxic agents damage cells by the induction of pores across their plasma membrane. Ca²⁺ and Zn²⁺ protect against such damage by promoting pore closure. Zn²⁺ may play a beneficial role in this regard in certain disease states.     

AKAP complex regulates Ca2+ re-uptake into heart sarcoplasmic reticulum

The beta-adrenergic receptor/cyclic AMP/protein kinase A (PKA) signalling pathway regulates heart rate and contractility. Here, we identified a supramolecular complex consisting of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2), its negative regulator phospholamban (PLN), the A-kinase anchoring protein AKAP18delta and PKA. We show that AKAP18delta acts as a scaffold that coordinates PKA phosphorylation of PLN and the adrenergic effect on Ca(2+) re-uptake. Inhibition of the compartmentalization of this cAMP signalling complex by specific molecular disruptors interferes with the phosphorylation of PLN. This prevents the subsequent release of PLN from SERCA2, thereby affecting the Ca(2+) re-uptake into the sarcoplasmic reticulum induced by adrenergic stimuli.     

Effect of Extracellular Calmodulin on the Cytosolic Ca2+ Concentration in Lily Pollen Grains

Using Lilium davidii Duchartre pollen as material, the calcium ion-fluorescence indicator fluo-3AM was loaded successfully into the pollen grains by low temperature loading method. Laser confocal scanning microscopy was used to study the effect of extracellular calmodulin on intracellular calcium. It is found that the purified exogenous calmodulin could elevate the intracellular calcium ion concentration, and the effect was correlated with the concentration of exogenous calmodulin to a certain extent. Cell membrane nonpermeable inhibitor of calmodulin, W 7-agarose, and the anti-serum of calmodulin could decrease the cytosolic calcium level. The results show that the endogenous extracellular calmodulin may play an important role in maintaining and increasing the cytosolic calcium level in pollen grain cell.     

Ca2+-dependent and Ca2+-independent somatic release from trigeminal neurons

Besides the nerve endings, the soma of trigeminal neurons also respond to membrane depolarizations with the release of neurotransmitters and neuromodulators in the extracellular space within the ganglion, a process potentially important for the cross-communication between neighboring sensory neurons. In this study, we addressed the dependence of somatic release on Ca²⁺ influx in trigeminal neurons and the involvement of the different types of voltage-gated Ca²⁺ (Cav) channels in the process. Similar to the closely related dorsal root ganglion neurons, we found two kinetically distinct components of somatic release, a faster component stimulated by voltage but independent of the Ca²⁺ influx, and a slower component triggered by Ca²⁺ influx. The Ca²⁺-dependent component was inhibited 80% by ω-conotoxin-MVIIC, an inhibitor of both N- and P/Q-type Cav channels, and 55% by the P/Q-type selective inhibitor ω-agatoxin-IVA. The selective L-type Ca²⁺ channel inhibitor nimodipine was instead without effect. These results suggest a major involvement of N- and P/Q-, but not L-type Cav channels in the somatic release of trigeminal neurons. Thus antinociceptive Cav channel antagonists acting on the N- and P/Q-type channels may exert their function by also modulating the somatic release and cross-communication between sensory neurons.     

Local Resting Ca2+ Controls the Scale of Astroglial Ca2+ Signals

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