The Essential Role of Cytosolic Cl− in Ca2+ Regulation of an Amiloride-Sensitive Channel in Fetal Rat Pneumocyte

An amiloride-sensitive, Ca²⁺-activated nonselective cation (NSC) channel in the apical membrane of fetal rat alveolar epithelium plays an important role in stimulation of Na⁺ transport by a beta adrenergic agonist (beta agonist). We studied whether Ca²⁺ has an essential role in the stimulation of the NSC channel by beta agonists. In cell-attached patches formed on the epithelium, terbutaline, a beta agonist, increased the open probability (P _o ) of the NSC channel to 0.62 ± 0.07 from 0.03 ± 0.01 (mean ±se; n= 8) 30 min after application of terbutaline in a solution containing 1 mm Ca²⁺. The P _o of the terbutaline-stimulated NSC channel was diminished in the absence of extracellular Ca²⁺ to 0.26 ± 0.05 (n= 8). The cytosolic Ca²⁺ concentration ([Ca²⁺] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 100 ± 6 and 20 ± 2 nm (n= 7) 30 min after application of terbutaline. The cytosolic Cl⁻ concentration ([Cl⁻] _c ) in the presence and absence of extracellular Ca²⁺ was, respectively, 20 ± 1 and 40 ± 2 mm (n= 7) 30 min after application of terbutaline. The diminution of [Ca²⁺] _c from 100 to 20 nm itself had no significant effects on the P _o if the [Cl⁻] _c was reduced to 20 mm; the P _o was 0.58 ± 0.10 at 100 nm [Ca²⁺] _c and 0.55 ± 0.09 at 20 nm [Ca²⁺] _c (n= 8) with 20 mm [Cl⁻] _c in inside-out patches. On the other hand, the P _o (0.28 ± 0.10) at 20 nm [Ca²⁺] _c with 40 mm [Cl⁻] _c was significantly lower than that (0.58 ± 0.10; P < 0.01; n= 8) at 100 nm [Ca²⁺] _c with 20 mm [Cl⁻] _c , suggesting that reduction of [Cl⁻] _c is an important factor stimulating the NSC channel. These observations indicate that the extracellular Ca²⁺ plays an important role in the stimulatory action of beta agonist on the NSC channel via reduction of [Cl⁻] _c . Received: 11 August 2000/Revised: 4 December 2000     

Diphosphoinositol pentakisphosphate as a novel mediator of insulin exocytosis

The pancreatic β-cell has served as an important model system for the revelation of new physiological roles for inositides. Initially, our studies were restricted to the role of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) in the regulation of cytoplasmic free calcium concentration ([Ca²⁺]_i), but it soon became clear that other inositol phosphates could also regulate β-cell [Ca²⁺]_i. For example, inositol hexakisphosphate (InsP₆) promotes the opening of the key voltage-dependent L-type Ca²⁺ channels, responsible for Ca²⁺ influx and the final release of insulin. Furthermore, InsP₆ and inositol lipids such as phosphatidylinositol 4,5-bisphosphate are intimately involved the regulation of endocytosis and exocytosis. We now review our most recent work, which has focused on the phosphorylation product of InsP₆, diphosphoinositol pentakisphosphate (PP-InsP₅ or InsP₇). We have established that InsP₇ via the activity of the InsP₆ kinase, IP6K1, promotes insulin release from the readily releasable pool of vesicles (RRP). The RRP is thought to be synonymous with the first phase of insulin secretion. This has a direct implication for type 2 diabetes, as it is this initial phase of insulin release that is curtailed in the disease. Hints from human genetic linkage studies suggest that disruption of IP6K1 could be a factor in the development of type 2 diabetes and a recent mouse model, where IP6K1 is universally deleted, exhibits lowered plasma insulin levels. Hence, this is yet another important example demonstrating how the β-cell utilizes inositides in the regulation of function. Although we do not fully understand the underlying molecular mechanisms, it is clear that IP6K1-mediated production of InsP₇ has an essential role in the regulation of the insulin secretory process.     

Phorbol esters and diacylglycerol inhibit vasopressin-induced increases in cytoplasmic-free Ca and Ca efflux in Swiss 3T3 cells

Vasopressin increased intracellular free calcium concentration [Ca²⁺]_i in quin-2-loaded quiescent Swiss 3T3 cells. This effect of vasopressin was rapidly inhibited by biologically active tumour promoters including phorbol dibutyrate (PBt₂) and by the synthetic diacylglycerol 1-oleoyl-2-acetyl-glycerol (OAG). Prolonged pretreatment of Swiss 3T3 cells with PBt₂ causes a loss of protein kinase C activity (Rodriguez-Pena & Rozengurt, Biochem biophys res commun 120 (1984) 1053) [28]. This pretreatment abolished the inhibition by PBt₂ or OAG of vasopressin-mediated increases in Ca²⁺]_i. Vasopressin also stimulated ⁴⁵Ca²⁺ efflux from cells pre-loaded with the isotope. This effect of the hormone was also inhibited by PBt₂. Prolonged pretreatment with PBt₂ prevented the inhibition of vasopressin-stimulated ⁴⁵Ca²⁺ release by PBt₂. Thus, protein kinase C stimulation inhibits vasopressin-mediated increases in [Ca²⁺]_i and ⁴⁵Ca²⁺ efflux apparently by blocking the increased release of Ca²⁺ from an intracellular store caused by the hormone. These findings suggest that activation of protein kinase C may act as a feedback inhibitor to modulate ligand-mediated increases in [Ca²⁺]_i.     

Carbamylcholine-induced morphological changes and spatial dynamics of [Ca2+]c in Harderian glands of guinea pigs: calcium-dependent lipid secretion and contraction of myoepithelial cells

To determine whether lipid-secreting cells have cytosolic Ca²⁺ concentration ([Ca²⁺]_c)-related secretory mechanisms, morphological changes and intracellular calcium dynamics of Harderian glands of guinea pigs stimulated by secretagogs were studied by electron microspy and Fura-2/AM digital image analysis. Control glandular cells contained large lipid vacuoles that were bordered by multi-layered membranes. Rough-surfaced endoplasmic reticulum, mitochondria, and smooth-surfaced endoplasmic reticulum may be involved in lipid vacuole formation. Myoepithelial cells surrounded alveoli. After carbamylcholine (CCh, 10^–6, 10^–5, and 10^–3 M) stimulation, lipid materials within the membranous structures were frequently discharged by an exocytotic mechanism. Conspicuous deformation of glandular cells caused by vigorous contraction of myoepithelial cells was observed in isolated alveoli after 10^–6M CCh stimulation, whereas the deformaties of glandular tissues perfused via vessels were small even after 10^–3M CCh stimulation. Connective tissue between glandular alveoli inhibited unbridled myoepithelial-cell contraction. Fura-2/AM digital imaging analysis revealed that CCh stimulation caused an increase in [Ca²⁺]_c in isolated alveoli. The morphological reactions and changes in [Ca²⁺]_c were prevented by atropine. When extracellular calcium ions were absent, enhanced extrusion of lipid vacuoles, myoepithelial-cell contraction, and a rise in [Ca²⁺]_c after CCh stimulation were not observed. Nicotine and catecholamines had no effect on the secretion or on the dynamics of [Ca²⁺]_c. It can be concluded that acetylcholine elicits exocytosis in glandular cells and contraction of the myoepithelial cells of Harderian glands, accompanied by an increase in [Ca²⁺]_c. The dynamics of [Ca²⁺]_c of the gland alveoli are mostly dependent on extracellular Ca²⁺.     

Vasopressin induced production of inositol trisphosphate and calcium efflux in a smooth muscle cell line

Phosphatidylinositol metabolism and ⁴⁵Ca²⁺ efflux were examined in a vascular smooth muscle cell line (A₇r₅). [Arg⁸]Vasopressin stimulated the rapid formation (measurable at 1 sec) of inositol phosphates in a concentration-dependent manner. The time course for formation of inositol phosphates was similar to that for ⁴⁵Ca²⁺ efflux from preloaded cells. The efflux of ⁴⁵Ca²⁺ in response to [Arg⁸]vasopressin could be inhibited by a vasopressin antagonist. This supports the hypothesis that inositol 1,4,5-trisphosphate plays a role in vasopressin stimulated calcium mobilisation from an intracellular source in cultured vascular smooth muscle cells.     

ADH Action on Whole-Cell Currents by Cytosolic Ca2+-dependent Pathways in Aldosterone-treated A6 Cells

We studied the characteristics of the basal and antidiuretic hormone (arginine vasotocin, AVT)-activated whole cell currents of an aldosterone-treated distal nephron cell line (A6) at two different cytosolic Ca²⁺ concentrations ([Ca²⁺] _c , 2 and 30 nm). A6 cells were cultured on a permeable support filter for 10 ∼ 14 days in media with supplemental aldosterone (1 μm). At 30 nm [Ca²⁺] _c , basal conductances mainly consisted of Cl⁻ conductances, which were sensitive to 5-nitro-2-(3-phenylpropylamino)-benzoate. Reduction of [Ca²⁺] _c to 2 nm abolished the basal Cl⁻ conductance. AVT evoked Cl⁻ conductances at 2 as well as 30 nm [Ca²⁺] _c . In addition to Cl⁻ conductances, AVT induced benzamil-insensitive nonselective cation (NSC) conductances. This action on NSC conductances was observed at 30 nm [Ca²⁺] _c but not at 2 nm [Ca²⁺] _c . Thus, cytosolic Ca²⁺ regulates NSC and Cl⁻ conductances in a distal nephron cell line (A6) in response to AVT. Keeping [Ca²⁺] _c at an adequate level seems likely to be an important requirement for AVT regulation of ion conductances in aldosterone-treated A6 cells. Received: 6 May 1996/Revised: 28 June 1996     

Calcium Buffering and Free Ca2+ in Rat Brain Synaptosomes

Abstract: The effects of K⁺ depolarization and of stimulation by veratridine on apparent cytosolic free Ca²⁺ ([Ca²⁺]_cyt) and net Ca²⁺ accumulation were measured in isolated rat brain presynaptic nerve terminals (synaptosomes). [Ca²⁺]_cyt was determined with fura-2, and Ca²⁺ accumulation was measured with tracer ⁴⁵Ca. [Ca²⁺]_cyt was ～ 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K⁺]₀, was increased from the normal 5 mM to 30 or 50 mM, ⁴⁵Ca uptake and [Ca²⁺]_cyt both increased within 1 s. Both increases were directly related to [Ca²⁺]₀ for [Ca²⁺]₀= 0.02–1.2 mM; however, the increase in ⁴⁵Ca uptake greatly exceeded the increase in [Ca²⁺]_cyt. With small Ca²⁺ loads ≤100 μmol/L of cell water, equivalent to the Ca²⁺ entry during a train of ≤60 impulses), the ⁴⁵Ca uptake exceeded the increase in [Ca²⁺]_cyt by a factor of nearly 1,000. This indicates that ～99.9% of the entering Ca²⁺ was buffered and/or sequestered within ～ 1 s. With larger Ca²⁺ loads, a larger fraction of the entering Ca²⁺ was buffered; ～99.97% of the load was buffered with loads of 250–425 μmol/L of cell water. The ratio between the total Ca²⁺ entry and the increase in [Ca²⁺]_cyt, the “calcium buffer ratio”β, was therefore ～ 3,500:1. This ratio was somewhat lower than the ratio of total intraterminal calcium: [Ca²⁺]_cyt, which ranged between ～7,300:1 and 12,800:1. When the synaptosomes were activated with 10 μM veratridine ([Ca²⁺]₀= 0.2–0.6 mM), ⁴⁵Ca influx and [Ca²⁺]_cyt increased progressively for ～10 s (β= 2,700:13,050:1) and then leveled off. Application of 10 μM tetrodotoxin before the introduction of veratridine prevented the increases in ⁴⁵Ca influx and [Ca²⁺]_cyt. Application of 10 μM tetrodotoxin after 5–10 s of exposure to veratridine caused both the [Ca²⁺]_cyt and the veratridine-stimulated ⁴⁵Ca within the terminals to decline, thereby demonstrating that the Ca²⁺ loading is reversible in the presence of extracellular Ca²⁺. These data show that synaptosomes are capable of buffering and metabolizing Ca²⁺ in a manner expected for intact neurons.     

Measurement of Inositol 1,4,5-Trisphosphate in Living Cells Using an Improved Set of Resonance Energy Transfer-Based Biosensors

Improved versions of inositol-1,4,5-trisphosphate (InsP₃) sensors were created to follow intracellular InsP₃ changes in single living cells and in cell populations. Similar to previous InsP₃ sensors the new sensors are based on the ligand binding domain of the human type-I InsP₃ receptor (InsP₃R-LBD), but contain a mutation of either R265K or R269K to lower their InsP₃ binding affinity. Tagging the InsP₃R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP₃ in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP₃ concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP₃ sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP₃ after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP₃ and Ca²⁺ levels in BRET experiments, the Cameleon D3 Ca²⁺ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P₂ resulted in the fall of InsP₃ level, followed by the decrease of the Ca²⁺-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca²⁺-signal preceded the fall of InsP₃ level indicating an InsP₃-, independent, direct regulation of capacitative Ca²⁺ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP₃ sensor can be used to monitor both the increase and decrease of InsP₃ levels in live cells suitable for high-throughput BRET applications.     

Mechanism of Ca2+-dependent Inactivation of L-type Ca2+ Channels in GH3 Cells: Direct Evidence Against Dephosphorylation by Calcineurin

Dephosphorylation of Ca²⁺ channels by the Ca²⁺-activated phosphatase 2B (calcineurin) has been previously suggested as a mechanism of Ca²⁺-dependent inactivation of Ca²⁺ current in rat pituitary tumor (GH₃) cells. Although recent evidence favors an inactivation mechanism involving direct binding of Ca²⁺ to the channel protein, the alternative ``calcineurin hypothesis' has not been critically tested using the specific calcineurin inhibitors cyclosporine A (CsA) or FK506 in GH₃ cells. To determine if calcineurin plays a part in the voltage- and/or Ca²⁺-dependent components of dihydropyridine-sensitive Ca²⁺ current decay, we rapidly altered the intracellular Ca²⁺ buffering capacity of GH₃ cells by flash photolysis of DM-nitrophen, a high affinity Ca²⁺ chelator. Flash photolysis induced a highly reproducible increase in the extent of Ca²⁺ current inactivation in a two-pulse voltage protocol with Ca²⁺ as the charge carrier, but had no effect when Ba²⁺ was substituted for Ca²⁺. Despite confirmation of the abundance of calcineurin in the GH₃ cells by biochemical assays, acute application of CsA or FK506 after photolysis had no effect on Ca²⁺-dependent inactivation of Ca²⁺ current, even when excess cyclophilin or FK binding protein were included in the internal solution. Prolonged preincubation of the cells with FK506 or CsA did not inhibit Ca²⁺-dependent inactivation. Similarly, blocking calmodulin activation with calmidazolium or blocking calcineurin with fenvalerate did not influence the extent of Ca²⁺-dependent inactivation after photolysis. The results provide strong evidence against Ca²⁺-dependent dephosphorylation as the mechanism of Ca²⁺ current inactivation in GH₃ cells, but support the alternative idea that Ca²⁺-dependent inactivation reflects a direct effect of intracellular Ca²⁺ on channel gating. Received: 12 August 1996/Revised: 21 October 1996     

Cold-induced cytosolic free calcium ion concentration changes in wheat

Relatively little is known about changes in the cytosolic free calcium ion concentration ([Ca²⁺]_c) in monocotyledonous plants. Therefore, we produced transgenic winter wheat lines stably expressing the calcium-sensitive photoprotein aequorin constitutively in the cytosol. [Ca²⁺]_c was detected in vivo by luminometry, and [Ca²⁺]_c elevations were imaged at video rate. Experiments with the transgenic seedlings focused on potential changes in [Ca²⁺]_c during cold exposure. Temperature-induced changes in [Ca²⁺]_c were found to be more dependent on the change in temperature (dT dt⁻¹) than on the absolute value of temperature. [Ca²⁺]_c increased only at cooling rates higher than 8°C min⁻¹, indicating that an overall cellular [Ca²⁺]_c increase is of minor relevance as a signal for cold acclimation in wheat under ecological conditions. The results are discussed with regard to the so-called ‘calcium signature hypothesis’.     

Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons

Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca²⁺]_i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5?μM Fura-2/AM and [Ca²⁺]_i transients for stimulation with 30?mM KCl (Hi K⁺) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380?nm, emission was recorded at 510?nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca²⁺]_i transients in a reversible manner. [Ca²⁺]_i increase evoked by Hi K⁺ was significantly reduced to 99.9?±?5.1% (n?=?18) and 62.5?±?4.2% (n?=?15, P?<?0.05) after the application of 5 and 50?µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K⁺-induced [Ca²⁺]_i responses, reduced to 98.7?±?4.8% (n?=?10) and 69.5?±?4.5% (n?=?9, P?<?0.05) inhibition of fluorescence ratio values of Hi K⁺-induced responses at 5 and 50?μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.     

Novel model for “calcium paradox” in sympathetic transmission of smooth muscles: Role of cyclic AMP pathway

It is well established that reduction of Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channel (L-type VDCC), or increase of cytosolic cAMP concentration ([cAMP]c), inhibit contractile activity of smooth muscles in response to transmitters released from sympathetic nerves. Surprisingly, in this work we observed that simultaneous administration of L-type VDCC blocker (verapamil) and [cAMP]c enhancers (rolipram, IBMX and forskolin) potentiated purinergic contractions evoked by electrical field stimulation of rat vas deferens, instead of inhibiting them. These results, including its role in sympathetic transmission, can be considered as a “calcium paradox”. On the other hand, this potentiation was prevented by reduction of [cAMP]c by inhibition of adenylyl cyclase (SQ 22536) or depletion of Ca²⁺ storage of sarco-endoplasmic reticulum by blockade of Ca²⁺ reuptake (thapsigargin). In addition, cytosolic Ca²⁺ concentration ([Ca²⁺]_c) evaluated by fluorescence microscopy in rat adrenal medullary slices was significantly reduced by verapamil or rolipram. In contrast, simultaneous incubation of adrenal slices with these compounds significantly increased [Ca²⁺]_c. This effect was prevented by thapsigargin. Thus, a reduction of [Ca²⁺]_c due to blockade of Ca²⁺ influx through L-type VDCC could stimulate adenylyl cyclase activity increasing [cAMP]c thereby stimulating Ca²⁺ release from endoplasmic reticulum, resulting in augmented transmitter release in sympathetic nerves and contraction.     

Direct inhibitory action of EGTA-Ca complex on reverse-mode Na/Ca exchange inMyxicola giant axons

Summary Giant axons from the marine annelidMyxicola infundibulum were internally dialyzed with solutions containing²²Na ions as tracers of Na efflux. In experiments performed in Li-substituted seawater, Na efflux that is dependent on external Ca ion concentration, [Ca²⁺] _o , was measured using dialysis to maintain [Na⁺] _i at 100mm, which enhances the [Ca²⁺] _o -dependent Na efflux component, (i.e., reverse-mode Na/Ca exchange). When dialysis fluid contained EGTA (1mm) to buffer the internal Ca concentration, [Ca²⁺] _i , to desired levels, Na efflux lost its normal sensitivity to external calcium. The inhibition was not simply due to the Ca-chelating action of EGTA to produce insufficient [Ca²⁺] _i to activate Na/Ca exchange. The addition of EGTA inhibited Ca _o -dependent Na efflux even when a large enough excess of [Ca²⁺] _i was present to saturate the EGTA and still produce elevated values of [Ca²⁺] _i . Control experiments showed that these high values of [Ca²⁺] _i resulted in normal Na/Ca exchange in the absence of EGTA. It is concluded that the presence of EGTA itself interferes with the manifestation of reverse-mode Na/Ca exchange inMyxicola giant axons.     

Ammonium uptake and cellular alkalisation in roots of Arabidopsis thaliana: The involvement of cytoplasmic calcium1

Using roots from Arabidopsis thaliana expressing the recombinant calcium indicator aequorin, we show that NH₃ uptake and alkalisation of plant cells act as a stimulus which induces transient elevations of the cytoplasmic free calcium concentration ([Ca²⁺]_c). The magnitudes of these [Ca²⁺]_c elevations are dependent on the concentration of the membrane permeable form, NH₃, and hence, particularly dependent on the pH in the external medium. EGTA and La³⁺ are able to significantly suppress the [Ca²⁺]_c transients showing that Ca²⁺ influx through the plasma membrane is likely to be involved. Verapamil and nifedipine had no inhibitory effects, which suggests that Ca²⁺ release from internal stores might not contribute significantly to the NH₃‐triggered [Ca²⁺]_c response. Pre‐incubation in l ‐methionine‐dl ‐sulphoximine – an inhibitor of the glutamine synthetase – did not alter the NH₃‐induced [Ca²⁺]_c responses at all. These results are consistent with previous studies where NH₃‐induced changes of cytoplasmic and vacuolar pH were investigated in maize roots. Furthermore, the similarity between the kinetics of NH₃‐driven cellular pH changes demonstrated in previous studies and the [Ca²⁺]_c transients shown here suggests a direct relationship between [Ca²⁺]_c and cellular alkalisation (cytoplasmic pH and/or vacuolar pH). However, the mechanism behind this possible causal relation remains to be elucidated.     

Role of cytosolic free calcium in the reorientation of pollen tube growth

Growing pollen tubes of Agapanthus umbellatus exhibited a tip-to-base gradient in cytosolic free calcium ([Ca²⁺]_c). Although this gradient is believed to be involved in pollen tube growth, its role in specifying reorientation is unknown. The direction of pollen tube growth could be modified by iontophoretic micro-injection, electrical fields (EFs) or photolysis of caged Ca²⁺. Iontophoretic injection resulted in a temporary cessation of growth, an increase in [Ca²⁺]_c and, upon recovery, reoriented growth. Weak EFs also elevated [Ca²⁺]_c, reduced growth rates and resulted in the reorientation of pollen tubes towards the cathode. Treatment with very low concentrations of the Ca²⁺-channel blocker lanthanum chloride, prior to exposure to an EF, inhibited both the increase in [Ca²⁺]_c and reorientation whilst only slightly affecting growth rates. The responses of growth inhibition and reorientation were mimicked when [Ca²⁺]_c was artificially elevated by photoactivating caged Ca²⁺ (Nitr-5). Our data suggest that [Ca²⁺]_c is part of a transduction mechanism which enables growing pollen tubes to successfully reorient to directional signals in the style and thus accomplish eventual fertilization of the egg.     

Effects of Phenylalanine and its Metabolites on Cytoplasmic Free Calcium in Cortical Neurons

Classic phenylketonuria (PKU) is characterized by brain lesions. However, its underlying neurotoxic mechanisms remain unknown. Based on our previous studies, we hypothesized that calcium might participate in PKU-associated neuropathy. In cultured cortical neurons, cytoplasmic free calcium concentration ([Ca²⁺]_i) decreased dramatically when treatment with phenylalanine (Phe) and phenyllactic acid, while phenylacetic acid treatment immediately increased [Ca²⁺]_i, which began to decrease after 3 min. Moreover, [Ca²⁺]_i decreased dramatically after Phe treatment in the presence of EGTA suggesting that Phe might increase [Ca²⁺]_i efflux. Phe-induced [Ca²⁺]_i decrease was strongly inhibited by vanadate, a non-specific plasma membrane Ca²⁺-ATPase (PMCA) antagonist, suggesting that Phe might increase [Ca²⁺]_i efflux throught modulating PMCA. These findings were further supported by the facts that Phe could increase membrance ⁴⁵Ca-uptake capability and PMCA activity. In contrast, treatment of KBR7943 or thapsigargin, antagonists to Na/Ca Exchanger (NCX) and Sarco/Endoplasmic reticulum Ca²⁺-ATPase (SERCA), respectively, did not elicit any changes in [Ca²⁺]_i. Specific siRNA against PMCA had an effect similar to vanadate. Since the brain injury induced by phenylalaninemia was thought to be a chronic process, we cultured cortical neurons in the presence of Phe for 2 weeks and measured [Ca²⁺]_i, PMCA activity and ⁴⁵Ca-uptake capability at days 3, 7, 9 and 14, respectively. PMCA activity and ⁴⁵Ca-uptake capability decreased from day 9, at the same time [Ca²⁺]_i increase was observed. In conclusion, PMCA participate in regulating Phe-induced initial rapid decrease in [Ca²⁺]_i and subsequent long-term increase in [Ca²⁺]_i.     

The Calcium/Calcineurin Pathway Promotes Hemidesmosome Stability through Inhibition of β4 Integrin Phosphorylation

Cell migration depends on cells being able to create and disassemble adhesive contacts. Hemidesmosomes are multiprotein structures that attach epithelia to basal lamina and disassemble during migration and carcinoma invasion. Phosphorylation of the β4 integrin, a hemidesmosome component, induces disassembly. Although kinases involved in β4 phosphorylation have been identified, little is known about phosphatases countering kinase action. Here we report that calcineurin, a serine-threonine protein phosphatase, regulates β4 phosphorylation. Calcineurin inhibitor cyclosporin A (CsA) and calcineurin-siRNA increase β4 phosphorylation, induce hemidesmosome disassembly, and increase migration in HaCat keratinocytes, suggesting that calcineurin negatively regulates β4 phosphorylation. We found no direct dephosphorylation of β4 by calcineurin or association between β4 and calcineurin, suggesting indirect regulation of β4 phosphorylation. We therefore assessed calcineurin influence on MAPK and PKC, known to phosphorylate β4. CsA increased MAPK activity, whereas MAPK inhibitors reduced CsA-induced β4 phosphorylation, suggesting that calcineurin restricts β4 phosphorylation by MAPK. Calcineurin is activated by calcium. Increased [Ca²⁺]_i reduces β4 phosphorylation and stabilizes hemidesmosomes, effects that are reversed by CsA, indicating that calcineurin mediates calcium effects on β4. However, MAPK activation is increased when [Ca²⁺]_i is increased, suggesting that calcineurin activates an additional mechanism that counteracts MAPK-induced β4 phosphorylation. Interestingly, in some squamous cell carcinoma cells, which have reduced hemidesmosomes and increased β4 phosphorylation, an increase in [Ca²⁺]_i using thapsigargin, bradykinin, or acetylcholine can increase hemidesmosomes and reduce β4 phosphorylation in a calcineurin-dependent manner. These findings have implications in calcineurin-inhibitor induced carcinoma, a complication of immunosuppressive therapy.     

Temperature-induced labelling of Fluo-3 AM selectively yields brighter nucleus in adherent cells

Fluo-3 is widely used to study cell calcium. Two traditional approaches: (1) direct injection and (2) Fluo-3 acetoxymethyl ester (AM) loading, often bring conflicting results in cytoplasmic calcium ([Ca²⁺]_c) and nuclear calcium ([Ca²⁺]_n) imaging. AM loading usually yields a darker nucleus than in cytoplasm, while direct injection always induces a brighter nucleus which is more responsive to [Ca²⁺]_n detection. In this work, we detailedly investigated the effects of loading and de-esterification temperatures on the fluorescence intensity of Fluo-3 in response to [Ca²⁺]_n and [Ca²⁺]_c in adherent cells, including osteoblast, HeLa and BV2 cells. Interestingly, it showed that fluorescence intensity of nucleus in osteoblast cells was about two times larger than that of cytoplasm when cells were loaded with Fluo-3 AM at 4 °C and allowed a subsequent step for de-esterification at 20 °C. Brighter nuclei were also acquired in HeLa and BV2 cells using the same experimental condition. Furthermore, loading time and adhesion quality of cells had effect on fluorescence intensity. Taken together, cold loading and room temperature de-esterification treatment of Fluo-3 AM selectively yielded brighter nucleus in adherent cells.     

Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction

An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca ²⁺]_i). This study analyzed the [Ca ²⁺]_i wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca ²⁺]_i wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca ²⁺]_i wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca ²⁺]_i wave area in the first bout it was likely to have a large [Ca ²⁺]_i wave area in the second bout (r ² = 0.933). These findings support the idea that individual bone cells have “calcium fingerprints” (i.e., a unique [Ca ²⁺]_i wave profile that is reproducible for repeated exposure to a given stimulus).     

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.