Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Sphingosine 1-phosphate receptors modulate intracellular Ca2+ homeostasis

Ligation of sphingosine 1-phosphate (S1P) to a set of specific receptors named S1P receptors (S1PRs) regulates important biological processes. Although the ability of S1P to increase cytosolic Ca2+ in various cell types is well known, the role of the individual S1PRs has not been fully characterized. Here, we provide a complete analysis of S1P-dependent intracellular Ca2+ homeostasis in HeLa cells. Overexpression of S1P2, or S1P3, but not S1P1, leads to a significant increase in cytosolic and mitochondrial [Ca2+] in response to S1P challenge. Moreover, cells ectopically expressing S1P2, or S1P3 exhibited an appreciable decrease of the free Ca2+ concentration in the endoplasmic reticulum, dependent on stimulation of receptors by S1P endogenously present in the culture medium which was accompanied by a reduced susceptibility to C2-ceramide-induced cell death. These results demonstrate a differential contribution of individual S1PRs to Ca2+ homeostasis and its possible implication in the regulation of cell survival.     

TRPA1 ion channel stimulation enhances cardiomyocyte contractile function via a CaMKII-dependent pathway

Rationale: Transient receptor potential channels of the ankyrin subtype-1 (TRPA1) are non-selective cation channels that show high permeability to calcium. Previous studies from our laboratory have demonstrated that TRPA1 ion channels are expressed in adult mouse ventricular cardiomyocytes (CMs) and are localized at the z-disk, costamere and intercalated disk. The functional significance of TRPA1 ion channels in the modulation of CM contractile function have not been explored.

Objective: To identify the extent to which TRPA1 ion channels are involved in modulating CM contractile function and elucidate the cellular mechanism of action.

Methods and Results: Freshly isolated CMs were obtained from murine heart and loaded with Fura-2 AM. Simultaneous measurement of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and contractility was performed in individual CMs paced at 0.3 Hz. Our findings demonstrate that TRPA1 stimulation with AITC results in a dose-dependent increase in peak [Ca²⁺]_i and a concomitant increase in CM fractional shortening. Further analysis revealed a dose-dependent acceleration in time to peak [Ca²⁺]_i and velocity of shortening as well as an acceleration in [Ca²⁺]_i decay and velocity of relengthening. These effects of TRPA1 stimulation were not observed in CMs pre-treated with the TRPA1 antagonist, HC-030031 (10 µmol/L) nor in CMs obtained from TRPA1^?/? mice. Moreover, we observed no significant increase in cAMP levels or PKA activity in response to TRPA1 stimulation and the PKA inhibitor peptide (PKI 14–22; 100 nmol/L) failed to have any effect on the TRPA1-mediated increase in CM contractile function. However, TRPA1 stimulation resulted in a rapid phosphorylation of Ca²⁺/calmodulin-dependent kinase II (CaMKII) (1–5 min) that correlated with increases in CM [Ca²⁺]_i and contractile function. Finally, all aspects of TRPA1-dependent increases in CM [Ca²⁺]_i, contractile function and CaMKII phosphorylation were virtually abolished by the CaMKII inhibitors, KN-93 (10 µmol/L) and autocamtide-2-related peptide (AIP; 20 µmol/L).

Conclusions: These novel findings demonstrate that stimulation of TRPA1 ion channels in CMs results in activation of a CaMKII-dependent signaling pathway resulting in modulation of intracellular Ca²⁺ availability and handling leading to increases in CM contractile function. Cardiac TRPA1 ion channels may represent a novel therapeutic target for increasing the inotropic and lusitropic state of the heart.     

Elevation of cytosolic [Ca2+] due to intracellular Ca2+ release retards carbachol stimulation of divalent cation entry in rat parotid gland acinar cells

This study examines the activation of divalent cation entry into rat parotid gland acinar cells by using Mn2+ as a Ca2+ surrogate cation. Following muscarinic-cholinergic stimulation of dispersed parotid acini with carbachol (10 microM), the onset of internal Ca2+ release (cytosolic [Ca2+], [Ca2+]i, increase) and the stimulation of Mn2+ entry (increase in fura2 quenching) are not simultaneously detected. [Ca2+]i elevation, due to intracellular release, is detected almost immediately following carbachol addition and peak [Ca2+]i increase occurs at 6.0 +/- 0.8 sec. However, there is an interval (apparent lag) between carbachol addition and the detection of stimulated Mn2+ entry. This apparent lag is decreased from 26 +/- 3.1 sec to 9.2 +/- 1.5 sec when external Mn2+ ([Mn2+]0) is increased from 12.5 to 500 microM. It is not decreased further with increase in [Mn2+]0 from 500 microM to 1 mM (9.8 +/- 2.1 sec), although both intracellular free Mn2+ and [Mn2+-fura2]/[fura2] increase. Thus, at [Mn2+]0 < 500 microM, the observed lag time is partially due to a limitation in the magnitude of Mn2+ entry. Furthermore, neither peak [Ca2+]i nor the time required to reach peak [Ca2+]i is significantly altered by [Mn2+]0 (12.5 microM to 1 mM). At every [Mn2+]0 tested (i.e., 12.5 microM-1 mM), the apparent lag is significantly greater than the time required to reach peak [Ca2+]i. However, when carbachol stimulation of the [Ca2+]i increase is attenuated by loading the acini with the Ca2+ chelator, 2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), there is no detectable lag in carbachol stimulation of Mn2+ entry (with 1 mM [Mn2+]0). Importantly, in BAPTA-loaded acini, carbachol stimulates Mn2+ entry via depletion of the internal Ca2+ pool and not via direct activation of other divalent cation entry mechanisms. Based on these results, we suggest that the apparent lag in the detection of carbachol stimulation of Mn2+ entry into parotid acinar cells is due to a retardation of Mn2+ entry by the initial increase in [Ca2+]i, due to internal release, which most likely occurs proximate to the site of divalent cation entry.     

Purinergic (ATP) signaling stimulates JNK1 but not JNK2 MAPK in osteoblast-like cells: contribution of intracellular Ca2+ release, stress activated and L-voltage-dependent calcium influx, PKC and Src kinases

This work shows that ATP activates JNK1, but not JNK2, in rat osteoblasts and ROS-A 17/2.8 osteoblast-like cells. In ROS-A 17/2.8 cells ATP induced JNK1 phosphorylation in a dose- and time-dependent manner. JNK1 phosphorylation also increased after osteoblast stimulation with ATPγS and UTP, but not with ADPβS. RT-PCR studies supported the expression of P2Y₂ receptor subtype. ATP-induced JNK1 activation was reduced by PI-PLC, IP₃ receptor, PKC and Src inhibitors and by gadolinium, nifedipine and verapamil or a Ca²⁺-free medium. ERK 1/2 or p38 MAPK inhibitors diminished JNK1 activation by ATP, suggesting a cross-talk between these pathways. ATP stimulated osteoblast-like cell proliferation consistent with the participation of P2Y₂ receptors. These results show that P2Y₂ receptor stimulation by ATP induces JNK1 phosphorylation in ROS-A 17/2.8 cells in a way dependent on PI-PLC/IP₃/intracellular Ca²⁺ release and Ca²⁺ influx through stress activated and L-type voltage-dependent calcium channels and involves PKC and Src kinases.     

Differential effect of PKA on the Ca2+ release kinetics of the type I and III InsP3 receptors

The effects of protein kinase A (PKA) on the inositol 1,4,5-trisphosphate (InsP(3)) receptor isoforms type I and type III were studied. The effects of PKA on the extent and rate constants for InsP(3)-induced Ca(2+) release (IICR) were different for the two isoforms. The effects of PKA on the type I isoform showed a biphasic relationship dependent upon the concentration of PKA used. At low concentrations of PKA (<50U/ml), both the extent and rate constants for IICR increased, while at higher concentrations (>200U/ml) the extent and rate constants decreased. The type III isoform showed only an increase in the extent of IICR and not in the rate constants. The effects of PKA on the type I InsP(3) receptor using single channel electrophysiological studies were also investigated. The stimulatory effect of PKA is due to an increase in conductance levels and not to a change in the mean open time of the channel.     

Endothelin contraction in pig coronary: Receptor types and Ca2+-mobilization

Endothelin is one of the most potent vasoconstrictors known. It plays an important role in the regulation of vascular tone and in the development of many cardiovascular diseases. This study focuses on the receptor types and the Ca²⁺ mobilization responsible for endothelin-1 (ET-1) contraction in de-endothelialized pig coronary artery rings. ET-1 contracted the artery rings with an EC₅₀ = 6.5 ± 1 nM and a maximum contraction which was 98.6 ± 9% of the contraction produced by 60 mM KCl. BQ123 (5 µM), an ET_A antagonist, reversed 78 ± 3% of the ET-1 contraction (50 nM). IRL1620, a selective ET_B agonist, produced 23 ± 3% of the total ET-1 contraction with an EC₅₀ = 12.7 ± 2 nM. More than 85% of the contraction due to 100 nM IRL 1620 was inhibited by 200 nMBQ788, an ET_B antagonist. Therefore, approximately 80% of the ET-1 contraction in this artery occurred via ET_A receptors, and the other 20% was mediated by ET_B receptors. To assess the Ca²⁺ pools utilized during the ET-1 response, ET-1 contraction was also examined in medium containing an L-type Ca²⁺ channel blocker nitrendipine, and in Ca²⁺ free medium containing 0.2 mM EGTA. In Ca²⁺ containing medium the contraction elicited by ET-1 was 98.6 ± 9% of the KCl contraction, however, in the presence 10 µM nitrendipine the ET-1 induced contraction was 54 ± 7% of the KCl contraction, and in Ca²⁺-free medium it was 13 ± 2%. Similarly, the IRL 1620 contractions in Ca²⁺ containing medium, in the presence of nitrendipine and in Ca²⁺-free medium were 22.4 ± 3%, 12 ± 3% and 11 ± 2% of the KCl response respectively. Thus, both ET_A and ET_B contractions utilize extracellular Ca²⁺ pools via L-type Ca²⁺ channels and other undefined route(s), as well as intracellular Ca²⁺ pools. In the pig coronary artery smooth muscle, ET-1 contractions occur predominantly via ET_A receptors, with ET_B receptors using similar Ca²⁺ mobilization pathways, but the ET_B receptors appear to use the intracellular Ca²⁺ stores to a greater extent.     

Stimulant-evoked depolarization and increase in [Ca2+] i in insulin-secreting cells is dependent on external Na+

Summary The patch-clamp technique and measurements of single cell [Ca²⁺] _i have been used to investigate the importance of extracellular Na⁺ for carbohydrate-induced stimulation of RINm5F insulin-secreting cells. Using patch-clamp whole-cell (current-clamp) recordings the average cellular transmembrane potential was estimated to be –60±1 mV (n=83) and the average basal [Ca²⁺] _i 102±6nm (n=37). When challenged with either glucose (2.5–10mm) ord-glyceraldehyde (10mm) the cells depolarized, which led to the initiation of Ca²⁺ spike potentials and a sharp rise in [Ca²⁺] _i . Similar effects were also observed with the sulphonylurea compound tolbutamide (0.01–0.1mm). Both the generation of the spike potentials and the increase in [Ca²⁺] _i were abolished when Ca²⁺ was removed from the bathing media. When all external Na⁺ was replaced with N-methyl-d-glucamine, in the continued presence of either glucose,d-glyceraldehyde or tolbutamide, a membrane repolarization resulted, which terminated Ca²⁺ spike potentials and attenuated the rise in [Ca²⁺] _i . Tetrodotoxin (TTX) (1–2 m) was also found to both repolarize the membrane and abolish secretagogue-induced rises in [Ca²⁺] _i .     

Pertussis toxin-sensitive pathway inhibits glucose-stimulated Ca2+ signals of rat islet beta-cells by affecting L-type Ca2+ channels and voltage-dependent K+ channels

A role of pertussis toxin (PTX)-sensitive pathway in regulation of glucose-stimulated Ca2+ signaling in rat islet beta-cells was investigated by using clonidine as a selective agonist to alpha2-adrenoceptors which link to the pathway. An elevation of extracellular glucose concentration from 5.5 to 22.2 mM (glucose stimulation) increased the levels of [Ca2+]i of beta-cells, and clonidine reversibly reduced the elevated levels of [Ca2+]i. This clonidine effect was antagonized by yohimbine, and abolished in beta-cells pre-treated with PTX. Clonidine showed little effect on membrane currents including those through ATP-sensitive K+ channels induced by voltage ramps from -90 to -50 mV. Clonidine showed little effect on the magnitude of whole-cell currents through L-type Ca2+ channels (ICa(L)), but increased the inactivation process of the currents. Clonidine increased the magnitude of the voltage-dependent K+ currents (IVK). These clonidine effects on ICa(L) and IVK were abolished in beta-cells treated with PTX or GDP-betaS. These results suggest that the PTX-sensitive pathway increases IVK activity and decreases ICa(L) activity of islet beta-cells, resulting in a decrease in the levels of [Ca2+]i elevated by depolarization-induced Ca2+ entry. This mechanism seems responsible at least in part for well-known inhibitory action of PTX-sensitive pathway on glucose-stimulated insulin secretion from islet beta-cells.     

Endothelin-1 and insulin activate the steady-state voltage dependent R-type Ca2+ channel in aortic smooth muscle cells via a pertussis toxin and cholera toxin sensitive G-protein

In single rabbit aortic smooth muscle cells, and at a concentration known to induce a maximum sustained increase of intracellular Ca²⁺ via activation of the steady-state voltage dependent R-type Ca²⁺ channels, endothelin-1 (10^-7 M) and insulin (80 U/ml) were found to induce a sustained increase in cytosolic free Ca²⁺ ([Ca]_i) levels that was significantly attenuated by pre-treatment with either pertussis toxin (PTX), cholera toxin (CTX) or removal of extracellular Ca²⁺.However, both PTX and CTX failed to inhibit the sustained depolarization-evoked sustained Ca²⁺ influx and [Ca]_i elevation via activation of the R-type Ca²⁺ channels. Moreover, ET-1 and insulin-evoked sustained increases in Ca²⁺ influx were not attenuated by the selective PKC inhibitor, bisindolylmaleimide (BIS), or the specific L-type Ca²⁺ channel blocker, nifedipine, but were completely reversed by the R-type Ca²⁺ channel blocker, (-) PN 200-110 (isradipine). These data suggest that both insulin and ET-1 activate the nifedipine-insensitive but isradipine-sensitive steady state voltage dependent R-type Ca²⁺ channels present on rabbit VSMCs and these channels are directly coupled to PTX and CTX sensitive G protein(s).     

Contribution of L-type Ca2+ channels to early afterdepolarizations induced by I Kr and I Ks channel suppression in guinea pig ventricular myocytes

Early afterdepolarizations (EADs) induced by suppression of cardiac delayed rectifier I (Kr) and/or I (Ks) channels cause fatal ventricular tachyarrhythmias. In guinea pig ventricular myocytes, partial block of one of the channels with complete block of the other reproducibly induced EADs. Complete block of both I (Kr) and I (Ks) channels depolarized the take-off potential and reduced the amplitude of EADs, which in some cases were not clearly separated from the preceding action potentials. A selective L-type Ca(2+) (I (Ca,L)) channel blocker, nifedipine, effectively suppressed EADs at submicromolar concentrations. As examined with the action potential-clamp method, I (Ca,L) channels mediated inward currents with a spike and dome shape during action potentials. I (Ca,L) currents decayed mainly due to inactivation in phase 2 and deactivation in phase 3 repolarization. When EADs were induced by complete block of I (Kr) channels with partial block of I (Ks) channels, repolarization of the action potential prior to EAD take-off failed to increase I (K1) currents and thus failed to completely deactivate I (Ca,L) channels, which reactivated and mediated inward currents during EADs. When both I (Kr) and I (Ks) channels were completely blocked, I (Ca,L) channels were not deactivated and mediated sustained inward currents until the end of EADs. Under this condition, the recovery and reactivation of I (Ca,L) channels were absent before EADs. Therefore, an essential mechanism underlying EADs caused by suppression of the delayed rectifiers is the failure to completely deactivate I (Ca,L) channels.     

Pharmacological modulation of intracellular Ca2+ channels at the single-channel level

Synaptic signaling, memory formation, neuronal development, and neuronal pathology are strongly influenced by the properties of intracellular Ca²⁺ channels, ryanodine, and inositol 1, 4, 5 trisphosphate receptors. This review will focus on recently developed and discovered pharmacological tools to modulate these channel proteins at the single-channel level. It will allow the readers of Molecular Neurobiology to evaluate the current knowledge on the pharmacological modulation of intracellular Ca²⁺ channels and to direct future research efforts effectively using available experimental tools and concepts.     

Modulation of intracellular Ca(2+) via alpha(1B)-adrenoreceptor signaling molecules,G alpha(h) (transglutaminase II) and phospholipase C-delta 1

We characterized the alpha(1B)-adrenoreceptor (alpha(1B)-AR)-mediated intracellular Ca(2+) signaling involving G alpha(h) (transglutaminase II, TGII) and phospholipase C (PLC)-delta 1 using DDT1-MF2 cell. Expression of wild-type TGII and a TGII mutant lacking transglutaminase activity resulted in significant increases in a rapid peak and a sustained level of intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to activation of the alpha(1B)-AR. Expression of a TGII mutant lacking the interaction with the receptor or PLC-delta 1 substantially reduced both the peak and sustained levels of [Ca(2+)](i). Expression of TGII mutants lacking the interaction with PLC-delta 1 resulted in a reduced capacitative Ca(2+) entry. Reduced expression of PLC-delta 1 displayed a transient elevation of [Ca(2+)](i) and a reduction in capacitative Ca(2+) entry. Expression of the C2-domain of PLC-delta 1, which contains the TGII interaction site, resulted in reduction of the alpha(1B)-AR-evoked peak increase in [Ca(2+)](i), while the sustained elevation in [Ca(2+)](i) and capacitative Ca(2+) entry remained unchanged. These findings demonstrate that stimulation of PLC-delta 1 via coupling of the alpha(1B)-AR with TGII evokes both Ca(2+) release and capacitative Ca(2+) entry and that capacitative Ca(2+) entry is mediated by the interaction of TGII with PLC-delta 1.     

Activation of M3 muscarinic receptors inhibits T-type Ca(2+) channel currents via pertussis toxin-sensitive novel protein kinase C pathway in small dorsal root ganglion neurons

Cobrotoxin (CbT), a short-chain postsynaptic α-neurotoxin, has been reported to play a role in analgesia. However, to date, the detailed mechanisms still remain unknown. In the present study, we identify a novel functional role of CbT in modulating T-type Ca²⁺ channel currents (T-currents) in small dorsal root ganglia (DRG) neurons as well as pain behaviors in mice. We found that CbT inhibited T-currents in a dose-dependent manner. CbT at 1 μM reversibly inhibited T-currents by ~ 26.3%. This inhibitory effect was abolished by the non-selective muscarinic acetylcholine receptor (mAChR) antagonist atropine, or the selective M3 mAChR antagonist 4-DAMP, while naloxone, an opioid receptor antagonist had no effect. Intracellular infusion of GDP-β-S or pretreatment of the cells with pertussis toxin (PTX) completely blocked the inhibitory effects of CbT. Using depolarizing prepulse, we found the absence of direct binding between G-protein βγ subunits and T-type Ca²⁺ channels in CbT-induced T-current inhibition. CbT responses were abolished by the phospholipase C inhibitor U73122 (but not the inactive analog U73343). The classical and novel protein kinase C (nPKC) antagonist chelerythrine chlorid or GF109203X abolished CbT responses, whereas the classical PKC antagonist Ro31-8820 or inhibition of PKA elicited no such effects. Intrathecal administration of CbT (5 μg/kg) produced antinociceptive effects in mechanical, thermal, and inflammatory pain models. Moreover, CbT-induced antinociception could be abrogated by 4-DAMP. Taken together, these results suggest that CbT acting through M3 mAChR inhibits T-currents via a PTX-sensitive nPKC pathway in small DRG neurons, which could contribute to its analgesic effects in mice.     

Comparative study of the effects of cromakalim (BRL 34915) and diazoxide on membrane potential, [Ca2+] i and ATP-sensitive potassium currents in insulin-secreting cells

Summary Patch-clamp and single cell [Ca²⁺] _i measurements have been used to investigate the effects of the potassium channel modulators cromakalim, diazoxide and tolbutamide on the insulin-secreting cell line RINm5F. In intact cells, with an average cellular transmembrane potential of –62±2 mV (n=42) and an average basal [Ca²⁺] _i of 102±6nm (n=37), glucose (2.5–10mm): (i) depolarized the membrane, through a decrease in the outward K_ATP current, (ii) evoked Ca²⁺ spike potentials, and (iii) caused a sharp rise in [Ca²⁺] _i . In the continued presence of glucose both cromakalim (100–200 m) and diazoxide (100 m) repolarized the membrane, terminated Ca²⁺ spike potentials and attenuated the secretagogue-induced rise in [Ca²⁺] _i . In whole cells (voltage-clamp records) and excised outside-out membrane patches, both cromakalim and diazoxide enhanced the current by opening ATP-sensitive K⁺ channels. Diazoxide was consistently found to be more potent than cromakalim. Tolbutamide, a specific inhibitor of ATP-sensitive K⁺ channels, reversed the effects of cromakalim on membrane potential and K_ATP currents.     

Preventive effect of Ninjin-yoei-to,a Kampo medicine,on amyloid β1-42-induced neurodegeneration via intracellular Zn2+ toxicity in the dentate gyrus

Ninjin-yoei-to (NYT), a Kampo medicine, has ameliorative effects on cognitive dysfunction via enhancing cholinergic neuron activity. To explore an efficacy of NYT administration for prevention and cure of Alzheimer’s disease, here we examined the effect of NYT on amyloid β_1-42 (Aβ_1-42)-induced neurodegeneration in the dentate gyrus. A diet containing 3% NYT was administered to mice for 2 weeks and human Aβ_1-42 was intracerebroventricularly injected. Neurodegeneration in the dentate granule cell layer of the hippocampus, which was determined 2 weeks after the injection, was rescued by administration of the diet for 4 weeks. Aβ staining (uptake) was not modified in the dentate granule cell layer by pre-administration of the diet for 2 weeks, while Aβ_1-42-induced increase in intracellular Zn²⁺ was reduced, suggesting that pre-administration of NYT prior to Aβ injection is effective for reducing Aβ_1-42-induced Zn²⁺ toxicity in the dentate gyrus. As a matter of fact, Aβ_1-42-induced neurodegeneration in the dentate gyrus was rescued by pre-administration of NYT. Interestingly, the level of metallothioneins, intracellular Zn²⁺-binding proteins, which can capture Zn²⁺ from Zn-Aβ_1-42 complexes, was elevated in the dentate granule cell layer by pre-administration of NYT. The present study suggests that pre-administration of NYT prevents Aβ_1-42-mediated neurodegeneration in the dentate gyurs by induced synthesis of metallothioneins, which reduces intracellular Zn²⁺ toxicity induced by Aβ_1-42.     

Intracellular- and extracellular-derived Ca influence phospholipase A2-mediated fatty acid release from brain phospholipids

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) and Ca²⁺-independent phospholipase A₂ (iPLA₂), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca²⁺, even though iPLA₂ has been thought to be Ca²⁺-independent. The source of Ca²⁺ for activation of cPLA₂ is largely extracellular, whereas Ca²⁺ released from the endoplasmic reticulum can activate iPLA₂ by a number of mechanisms. This review focuses on the role of Ca²⁺ in modulating cPLA₂ and iPLA₂ activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca²⁺ signal.     

GABAergic signaling in primary lens epithelial and lentoid cells and its involvement in intracellular Ca2+ modulation

Primary lens epithelial cell (LEC) cultures derived from newborn (P0) and one-month-old (P30) mouse lenses were used to study GABA (gamma-aminobutyric acid) signaling expression and its effect on the intracellular Ca²⁺ ([Ca²⁺]_i) level. We have found that these cultures express specific cellular markers for lens epithelial and fiber cells, all components of the functional GABA signaling pathway and GABA, thus recapitulating the developmental program of the ocular lens. Activation of both GABA-A and GABA-B receptors (GABA_AR and GABA_BR) with the specific agonists muscimol and baclofen, respectively induces [Ca²⁺]_i transients that could be blocked by the specific antagonists bicuculline and CGP55845 and were dependent on extracellular Ca²⁺. Bicuculline did not change the GABA-evoked Ca²⁺ responses in Ca²-containing buffers, but suppressed them significantly in Ca²⁺-free buffers suggesting the two receptors couple to convergent Ca²⁺ mobilization mechanisms with different extracellular Ca²⁺ sensitivity. Prolonged activation of GABA_BR induced wave propagation of the Ca²⁺ signal and persistent oscillations. The number of cells reacting to GABA or GABA + bicuculline in P30 mouse LEC cultures expressing predominantly the synaptic type GABA_AR did not differ significantly from the number of reacting cells in P0 mouse LEC cultures. The GABA-induced Ca²⁺ transients in P30 (but not P0) mouse LEC could be entirely suppressed by co-application of bicuculline and CGP55845. The GABA-mediated Ca²⁺ signaling may be involved in a variety of Ca²⁺-dependent cellular processes during lens growth and epithelial cell differentiation.     

Regulatory and structural EF-hand motifs of neuronal calcium sensor-1: Mg 2+ modulates Ca 2+ binding, Ca 2+ -induced conformational changes, and equilibrium unfolding transitions

Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca²⁺ signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca²⁺-specific) and structural (Ca²⁺- or Mg²⁺-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca²⁺ binding using NMR and EF-hand mutants. Ca²⁺ titration, as monitored by [¹⁵N,¹H] heteronuclear single quantum coherence, suggests that Ca²⁺ binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg²⁺ binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca²⁺-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca²⁺ and not Mg²⁺. Ca²⁺ binding induces conformational rearrangements in the protein by reversing Mg²⁺-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg²⁺ with Ca²⁺ reduces the Ca²⁺-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca²⁺-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg²⁺-bound NCS-1 are similar, the early unfolding transitions of Ca²⁺-bound NCS-1 are partially influenced in the presence of Mg²⁺. This study demonstrates the importance of Mg²⁺ as a modulator of calcium homeostasis and active-state behavior of NCS-1.