Presence Of External Ca2 Research Articles

Role of Ca 2+, membrane excitability, and Ca 2+ stores in failing muscle contraction with aging

Excitation-contraction (EC) coupling in a population of skeletal muscle fibers of aged mice becomes dependent on the presence of external Ca 2+ ions (Payne, A.M., Zheng, Z., Gonzalez, E., Wang, Z.M., Messi, M.L., Delbono, O., 2004b. External Ca(2+)-dependent excitation – contraction coupling in a population of aging mouse skeletal muscle fibers. J. Physiol. 560, 137–155.). However, the mechanism(s) underlying this process remain unknown. In this work, we examined the role of (1) extracellular Ca 2+; (2) voltage-induced influx of external Ca 2+ ions; (3) sarcoplasmic reticulum (SR) Ca 2+ depletion during repeated contractions; (4) store-operated Ca 2+ entry (SOCE); (5) SR ultrastructure; (6) SR subdomain localization of the ryanodine receptor; and (7) sarcolemmal excitability in muscle force decline with aging. These experiments show that external Ca 2+, but not Ca 2+ influx, is needed to maintain force upon repetitive fiber electrical stimulation. Decline in fiber force is associated with depressed SR Ca 2+ release. SR Ca 2+ depletion, SOCE, and the putative segregated Ca 2+ release store do not play a significant role in external Ca 2+-dependent contraction. More importantly, a significant number of action potentials fail in senescent mouse muscle fibers subjected to a stimulation frequency. These results indicate that failure to generate action potentials accounts for decreased intracellular Ca 2+ mobilization and tetanic force in aging muscle exposed to a Ca 2+-free medium.

PH and monovalent cations regulate cytosolic free Ca 2+ in E. coli

Abstract The results here show for the first time that pH and monovalent cations can regulate cytosolic free Ca 2+ in E. coli through Ca 2+ influx and efflux, monitored using aequorin. At pH 7.5 the resting cytosolic free Ca 2+ was 0.2–0.5 µM. In the presence of external Ca 2+ (1 mM) at alkaline pH this rose to 4 µM, being reduced to 0.9 µM at acid pH. Removal of external Ca 2+ caused an immediate decrease in cytosolic free Ca 2+ at 50–100nM s − 1 . Efflux rates were the same at pH 5.5, 7.5 and 9.5. Thus, ChaA, a putative Ca 2+/H +exchanger, appeared not to be a major Ca 2+-efflux pathway. In the absence of added Na +, but with 1 mM external Ca 2+, cytosolic free Ca 2+ rose to approximately 10 µM. The addition of Na +(half maximum 60 mM) largely blocked this increase and immediately stimulated Ca 2+ efflux. However, this effect was not specific, since K + also stimulated efflux. In contrast, an increase in osmotic pressure by addition of sucrose did not significantly stimulate Ca 2+ efflux. The results were consistent with H + and monovalent cations competing with Ca 2+ for a non-selective ion influx channel. Ca 2+ entry and efflux in chaA and yrbG knockouts were not significantly different from wild type, confirming that neither ChaA nor YrbG appear to play a major role in regulating cytosolic Ca 2+ in Escherichia coli. The number of Ca 2+ ions calculated to move per cell per second ranged from < 1 to 100, depending on conditions. Yet a single eukaryote Ca 2+ channel, conductance 100 pS, should conduct > 6 million ions per second. This raises fundamental questions about the nature and regulation of Ca 2+ transport in bacteria, and other small living systems such as mitochondria, requiring a new mathematical approach to describe such ion movements. The results have important significance in the adaptation of E. coli to different ionic environments such as the gut, fresh water and in sea water near sewage effluents.

The Calponin Homology Domain of Vav1 Associates with Calmodulin and Is Prerequisite to T Cell Antigen Receptor-induced Calcium Release in Jurkat T Lymphocytes

Vav1 is a guanine nucleotide exchange factor that is expressed specifically in hematopoietic cells and plays important roles in T cell development and activation. Vav1 consists of multiple structural domains so as to facilitate both its guanine nucleotide exchange activity and scaffold function following T cell antigen receptor (TCR) engagement. Previous studies demonstrated that the calponin homology (CH) domain of Vav1 is required for TCR-stimulated calcium mobilization and thus downstream activation of nuclear factor of activated T cells. However, it remained obscure how Vav1 functions in regulating calcium flux. In an effort to explore molecules interacting with Vav1, we found that calmodulin bound to Vav1 in a calcium-dependent and TCR activation-independent manner. The binding site was mapped to the CH domain of Vav1. Reconstitution of vav1-null Jurkat T cells (J.Vav1) with CH-deleted Vav1 exhibited a severe deficiency in calcium release to the same extent as that of Jurkat cells treated with the calmodulin inhibitor or J.Vav1 cells. The defect persisted even when phospholipase-Cgamma1 was fully activated, indicating a prerequisite role of Vav1 CH domain in calcium signaling. The results suggest that Vav1 and calmodulin function cooperatively to potentiate TCR-induced calcium release. This study unveiled a mechanism by which the Vav1 CH domain is involved in calcium signaling and provides insight into our understanding of the role of Vav1 in T cell activation.

Different profiles of Ca2+ responses to endothelin‐1 and PDGF in liver myofibroblasts during the process of cell differentiation

Hepatic stellate cells play an important role in liver fibrosis but little is known about liver myofibroblasts located around the central vein and in the portal area. In this study, intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured to assess the response to endothelin-1 (ET-1), platelet derived growth factor (PDGF) and ATP in rat liver myofibroblasts. Rat liver myofibroblasts were compared in 'quiescent' (cultured on Matrigel-coated dishes) and 'activated' (cultured on non-coated plastic dishes) conditions. [Ca(2+)](i) was measured with the fluorescent dye fura-2 and mRNA for ET-1, PDGF and their receptors by RT-PCR. ET-1 increased [Ca(2+)](i) in quiescent cells but not in activated cells, whereas PDGF-BB increased [Ca(2+)](i) in activated cells but not in quiescent cells. However, there was no difference between responses to ATP in quiescent or activated cells. ET-1 (in quiescent cells), PDGF-BB (in activated cells) and ATP (in both cells) all induced transient increases in [Ca(2+)](i) in the absence of extracellular Ca(2+) (with EGTA), indicating the involvement of Ca(2+) release from intracellular Ca(2+) stores. The sustained increase in [Ca(2+)](i) in the presence of external Ca(2+) in activated cells (ATP and PDGF) was significantly reduced by nicardipine, a L-type Ca(2+) channel blocker, but not in quiescent cells (ATP and ET-1). The different pharmacological profiles of [Ca(2+)](i)-response in quiescent and activated myofibroblasts suggest that ET-1 and PDGF contribute differently to myofibroblast activation during the process of liver fibrosis.

Elementary Mechanisms Producing Facilitation of Cav2.1 (P/Q-type) Channels

The regulation of CaV2.1 (P/Q-type) channels by calmodulin (CaM) showcases the powerful Ca2+ decoding capabilities of CaM in complex with the family of CaV1-2 Ca2+ channels. Throughout this family, CaM does not simply exert a binary on/off regulatory effect; rather, Ca2+ binding to either the C- or N-terminal lobe of CaM alone can selectively trigger a distinct form of channel modulation. Additionally, Ca2+ binding to the C-terminal lobe triggers regulation that appears preferentially responsive to local Ca2+ influx through the channel to which CaM is attached (local Ca2+ preference), whereas Ca2+ binding to the N-terminal lobe triggers modulation that favors activation via Ca2+ entry through channels at a distance (global Ca2+ preference). CaV2.1 channels fully exemplify these features; Ca2+ binding to the C-terminal lobe induces Ca2+-dependent facilitation of opening (CDF), whereas the N-terminal lobe yields Ca2+-dependent inactivation of opening (CDI). In mitigation of these interesting indications, support for this local/global Ca2+ selectivity has been based upon indirect inferences from macroscopic recordings of numerous channels. Nagging uncertainty has also remained as to whether CDF represents a relief of basal inhibition of channel open probability (P o) in the presence of external Ca2+, or an actual enhancement of P o over a normal baseline seen with Ba2+ as the charge carrier. To address these issues, we undertake the first extensive single-channel analysis of CaV2.1 channels with Ca2+ as charge carrier. A key outcome is that CDF persists at this level, while CDI is entirely lacking. This result directly upholds the local/global Ca2+ preference of the lobes of CaM, because only a local (but not global) Ca2+ signal is here present. Furthermore, direct single-channel determinations of P o and kinetic simulations demonstrate that CDF represents a genuine enhancement of open probability, without appreciable change of activation kinetics. This enhanced-opening mechanism suggests that the CDF evoked during action-potential trains would produce not only larger, but longer-lasting Ca2+ responses, an outcome with potential ramifications for short-term synaptic plasticity.

Enhanced Stretch-Induced Myogenic Tone in the Basilar Artery of Spontaneously Hypertensive Rats

We investigated if the magnitude of myogenic tone in the basilar artery of SHR differs from that in WKY and, if so, whether RhoA- or PKC-dependent mechanisms were involved. Myogenic tone was developed in response to stretch. Stretch-induced myogenic contraction was significantly greater in the SHR than WKY in the presence of external Ca²⁺. However, in the absence of external Ca²⁺, stretch did not evoke a myogenic tone. The [Ca²⁺]_i-induced contraction was larger in SHR than WKY and the [Ca²⁺]_i-force curve was significantly shifted to the left in SHR compared to WKY. Y-27632 significantly inhibited stretch-induced myogenic tone, but the inhibitory effect was larger in the SHR than WKY. However, PKC inhibitors had no significant effect on the myogenic tone. RhoA and PKCΕ were expressed at higher levels in the SHR compared to the WKY. RhoA and PKCα translocated from the cytosol to the cell membrane in response to stretch in both animals, but PKCΕ was translocated only in SHR. Our results strongly suggest that stretch-induced myogenic tone is enhanced in SHR, and the activation of RhoA/Rho kinase plays an important role in the enhanced myogenic tone in SHR.

Effects of (1R,9S)-.BETA.-Hydrastine on Intracellular Calcium Concentration in PC12 Cells

(1R,9S)-beta-hydrastine (BHS) decreases the basal intracellular Ca(2+) concentration ([Ca(2+)](i)) in PC12 cells.(5) This study examined the effects of (1R,9S)-BHS on [Ca(2+)](i) in PC12 cells. (1R,9S)-BHS at 10-100 microM in combination with a high extracellular K+ level (56 mM) inhibited the release of dopamine in a concentration-dependent manner with an IC(50) value of 66.5 microM. BHS at 100 microM inhibited the sustained increase in [Ca(2+)](i) induced by a high K+ level (56 mM), and had an inhibitory effect on the 2 microM nifedipine-induced blockage of the K+ -stimulated sustained increase in [Ca(2+)](i). In addition, (1R,9S)-BHS at 100 microM prevented the rapid and sustained increase in [Ca(2+)](i) elicited by 20 mM caffeine, but did not have an effect on the increase induced by 1 microM thapsigargin, in the presence of external Ca(2+). These results suggest that the active sites of (1R,9S)-BHS are mainly L-type Ca(2+) channels and caffeine-sensitive Ca(2+)-permeable channels in PC12 cells.

Close Functional Coupling Between Ca2+Release-Activated Ca2+Channels and Reactive Oxygen Species Production in Murine Macrophages

Aim. To investigate the role of Ca2+ release-activated Ca2+ (CRAC) channels in the ROS production in macrophages. Methods. The intracellular [Ca2+]i was analyzed by confocal laser microscopy. The production of ROS was assayed by flow cytometry. Results. Both LPS and thapsigargin induced an increase in intracellular [Ca2+]i, either in the presence or absence of extracellular Ca2+ in murine macrophages. The Ca2+ signal was sustained in the presence of external Ca2+ and only initiated a mild and transient rise in the absence of external Ca2+. CRAC channel inhibitor 2-APB completely suppressed the Ca2+ entry signal evoked by thapsigargin, and suppressed approximately 93% of the Ca2+ entry signal evoked by LPS. The increase in intracellular [Ca2+]i was associated with increased ROS production, which was completely abolished in the absence of extracellular Ca2+ or in the presence of CRAC channel inhibitors 2-APB and Gd3+. The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and the inhibitor of the electron transport chain, antimycin, evoked a marked increase in ROS production and completely inhibited thapsigargin and LPS-evoked responses. Conclusions. These findings indicate that the LPS-induced intracellular [Ca2+]i increase depends on the Ca2+ entry through CRAC channels, and close functional coupling between CRAC and ROS production in murine macrophages.

Inhibitory effects of pentobarbital on glutamate-induced calcium increase in presynaptic nerve terminals

We investigated the effects of pentobarbital on the intracellular Ca2+concentration ([Ca2+]i) of the presynaptic nerve terminals (boutons). In the presence of external Ca2+, glutamate increased [Ca2+]i both in the neurons and in the presynaptic terminals. Pentobarbital suppressed the increase in neurons and in boutons with greater effects on the boutons. Our results indicated that pentobarbital may modulate the neurotransmission by inhibiting the transmitter release, which is induced by glutamate.

Volatile Anesthetic Effects on Glutamate versus GABA Release from Isolated Rat Cortical Nerve Terminals: Basal Release

The effects of three volatile anesthetics (isoflurane, enflurane, and halothane) on basal release of glutamate and GABA from isolated rat cerebrocortical nerve terminals (synaptosomes) were compared using a dual isotope superfusion method. Concentration-dependent effects on basal release differed between anesthetics and transmitters. Over a range of clinical concentrations (0.5-2x minimum alveolar concentration), basal glutamate release was inhibited by all three anesthetics, whereas basal GABA release was enhanced (isoflurane) or unaffected (enflurane and halothane). These effects may represent a balance of stimulatory and inhibitory mechanisms between transmitters and anesthetics. There were no significant differences between anesthetic effects on basal release in the absence or presence of external Ca(2+), whereas intracellular Ca(2+) buffering limited volatile anesthetic inhibition of basal glutamate release. Although these results demonstrate fundamental differences in anesthetic effects on basal release between glutamatergic and GABAergic nerve terminals, all three volatile anesthetics at clinical concentrations consistently reduced the ratio of basal glutamate to GABA release. These actions may contribute to the net depression of glutamatergic excitation and potentiation of GABAergic inhibition characteristic of general anesthesia.

Biophysical properties of menthol-activated cold receptor TRPM8 channels

The temperature-sensitive transient receptor potential channel, TRPM8, was recently cloned and found to be activated by cold and menthol. Whole-cell recordings show that TRPM8 is permeable to multiple cations and exhibits a strong outward rectification. Here, we examine the mechanism underlying menthol-evoked current rectification of TRPM8 transiently expressed in tsA-201 cells at room temperature (∼25 °C). Whole-cell currents (ruptured, bath: Na +, K +, Ca 2+, or Ba 2+; pipette: KCl) exhibited a strong outward rectification in the presence of menthol, consistent with previous studies. The outward K + current was reduced in the presence of external Ca 2+ or Ba 2+. Single-channel recordings (cell-attached) showed that menthol induced brief channel openings with two conducting states in the voltage range between −80 and +60 mV. The small current ( i S) conducted both monovalent and divalent ions, and the large one ( i L) predominantly monovalent ions. The i– V plot for Ca 2+ was weakly outward rectifying, whereas those for monovalent ions were linear. The i S may result in the divalent ion-induced reduction of the whole-cell outward current. The open probability ( P o) in all ion conditions tested was low at negative voltages and increased with depolarization, accounting for the small inward currents observed at the whole-cell level. In conclusion, our results indicate that menthol induced steep outward rectification of TRPM8 results from the voltage-dependent open channel probability and the permeating ion-dependent modulation of the unitary channel conductance.

Deoxycholic acid activates protein kinase C and phospholipase C via increased Ca 2+ entry at plasma membrane

Secondary bile acids like deoxycholic acid (DCA) are well-established tumor promoters that may exert their pathologic actions by interfering with intracellular signaling cascades. We evaluated the effects of DCA on Ca2+ signaling in BHK-21 fibroblasts using fura-2 and mag-fura-2 to measure cytoplasmic and intraluminal internal stores [Ca2+], respectively. Furthermore, green fluorescent protein (GFP)-based probes were used to monitor time courses of phospholipase C (PLC) activation (pleckstrin-homology [PH]-PLCdelta-GFP), and translocation of protein kinase C (PKC) and a major PKC substrate, myristolated alanine-rich C-kinase substrate (MARCKS). DCA (50-250 micromol/L) caused profound Ca2+ release from intracellular stores of intact or permeabilized cells. Correspondingly, DCA increased cytoplasmic Ca2+ to levels that were approximately 120% of those stimulated by Ca2+-mobilizing agonists in the presence of external Ca2+, and approximately 60% of control in Ca2+-free solutions. DCA also caused dramatic translocation of PH-PLCdelta-GFP, and conventional, Ca2+/diacylglycerol (DAG)-dependent isoforms of PKC (PKC-betaI and PKC-alpha), and MARCKS-GFP, but only in Ca2+-containing solutions. DCA had no effect on localization of a novel (PKCdelta) or an atypical (PKCzeta) PKC isoform. Data are consistent with a model in which DCA directly induces both Ca2+ release from internal stores and persistent Ca2+ entry at the plasma membrane. The resulting microdomains of high Ca2+ levels beneath the plasma membrane appear to directly activate PLC, resulting in modest InsP 3 and DAG production. Furthermore, the increased Ca2+ entry stimulates vigorous recruitment of conventional PKC isoforms to the plasma membrane.

Amplification of Ca2+ Signaling by Diacylglycerol-mediated Inositol 1,4,5-Trisphosphate Production

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP(3) produced is particularly critical to determining the spatio-temporally coordinated Ca(2+)-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca(2+)-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity.

Direct Voltage Control of Signaling via P2Y1 and Other Gαq-coupled Receptors

Emerging evidence suggests that Ca2+ release evoked by certain G-protein-coupled receptors can be voltage-dependent; however, the relative contribution of different components of the signaling cascade to this response remains unclear. Using the electrically inexcitable megakaryocyte as a model system, we demonstrate that inositol 1,4,5-trisphosphate-dependent Ca2+ mobilization stimulated by several agonists acting via Galphaq-coupled receptors is potentiated by depolarization and that this effect is most pronounced for ADP. Voltage-dependent Ca2+ release was not induced by direct elevation of inositol 1,4,5-trisphosphate, by agents mimicking diacylglycerol actions, or by activation of phospholipase Cgamma-coupled receptors. The response to voltage did not require voltage-gated Ca2+ channels as it persisted in the presence of nifedipine and was only weakly affected by the holding potential. Strong predepolarizations failed to affect the voltage-dependent Ca2+ increase; thus, an alteration of G-protein betagamma subunit binding is also not involved. Megakaryocytes from P2Y1(-/-) mice lacked voltage-dependent Ca2+ release during the application of ADP but retained this response after stimulation of other Galphaq-coupled receptors. Although depolarization enhanced Ca2+ mobilization resulting from GTPgammaS dialysis and to a lesser extent during AlF4- or thimerosal, these effects all required the presence of P2Y1 receptors. Taken together, the voltage dependence to Ca2+ release via Galphaq-coupled receptors is not due to control of G-proteins or down-stream signals but, rather, can be explained by a voltage sensitivity at the level of the receptor itself. This effect, which is particularly robust for P2Y1 receptors, has wide-spread implications for cell signaling.

The blockade of cyclopiazonic acid-induced store-operated Ca 2+ entry pathway by YC-1 in neutrophils

In the presence of external Ca 2+, pretreatment of neutrophils with 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) inhibited the cyclopiazonic acid (CPA)-induced [Ca 2+] i elevation in a concentration- but not a time-dependent manner, while YC-1 had no effect on the Ca 2+ signals in a Ca 2+-free medium. YC-1 failed to inhibit ATP- and interleukin-8 (IL-8)-induced [Ca 2+] i changes. Addition of YC-1 after cell activation strongly inhibited the CPA-induced [Ca 2+] i changes. In a classical Ca 2+ readdition protocol, a similar extent inhibition of Ca 2+ spike by YC-1 introduced either prior to or after CPA stimulation was obtained. In rat neutrophils, mRNA for endothelial differentiation gene (edg)1, edg5, edg6 and edg8, the putative targets for sphingosine 1-phosphate (S1P), could be detected. However, S1P was found to have little effect on Ca 2+ signals. YC-1 did not inhibit but enhanced the sphingosine-induced [Ca 2+] i changes. Inhibition by YC-1 of CPA-induced [Ca 2+] i changes was not prevented by 7-nitroindazole and N-(3-aminomethyl)benzylacetamidine (1400W), two nitric oxide synthase (NOS) inhibitors, by aristolochic acid, a phospholipase A 2 inhibitor, or by suspension in a Na +-deprived medium. YC-1 did not affect the mitochondrial membrane potential. Moreover, YC-1 did not alter [Ca 2+] i changes in response to ionomycin after CPA and formyl-Met-Leu-Phe (fMLP) stimulation in a Ca 2+-free medium. YC-1 had no effect on the basal [Ca 2+] i level, the pharmacologically isolated plasma membrane Ca 2+-ATPase activity, and Ba 2+ entry into CPA-activated cells. YC-1 alone resulted in the accumulation of actin filaments in neutrophils, while significantly reduced the intensity of actin filament staining in the subsequent activation with CPA. These results indicate that YC-1 inhibited CPA-activated store-operated Ca 2+ entry (SOCE) probably through the direct blockade of channel activation and/or the disruption of the integrity of the actin cytoskeleton necessary for supporting Ca 2+ entry pathway in neutrophils.

Hg 2+ signaling in trout hepatoma (RTH-149) cells: involvement of Ca 2+-induced Ca 2+ release

Mercury is a non-essential heavy metal affecting intracellular Ca 2+ dynamics. We studied the effects of Hg 2+ on [Ca 2+] i in trout hepatoma cells (RTH-149). Confocal imaging of fluo-3-loaded cells showed that Hg 2+ induced dose-dependent, sustained [Ca 2+] i transient, triggered intracellular Ca 2+ waves, stimulated Ca 2+-ATPase activity, and promoted InsP 3 production. The effect of Hg 2+ was reduced by the Ca 2+ channel blocker verapamil and totally abolished by extracellular GSH, but was almost unaffected by cell loading with the heavy metal chelator TPEN or esterified GSH. In a Ca 2+-free medium, Hg 2+ induced a smaller [Ca 2+] i transient, that was unaffected by TPEN, but was abolished by U73122, a PLC inhibitor, and by cell loading with GDP-βS, a G protein inhibitor, or heparin, a blocker of intracellular Ca 2+ release. Data indicate that Hg 2+ induces Ca 2+ entry through verapamil-sensitive channels, and intracellular Ca 2+ release via a G protein-PLC-InsP 3 mechanism. However, in cells loaded with heparin and exposed to Hg 2+ in the presence of external Ca 2+, the [Ca 2+] i rise was maximally reduced, indicating that the global effect of Hg 2+ is not a mere sum of Ca 2+ entry plus Ca 2+ release, but involves an amplification of Ca 2+ release operated by Ca 2+ entry through a CICR mechanism.

Characterization of maleimide-activated Ca 2+ entry in neutrophils

N-Ethylmaleimide (NEM), a thio-alkylating agent, concentration-dependently stimulated the elevation of [Ca 2+] i in rat neutrophils in the presence of external Ca 2+. This effect was not observed in Ca 2+-free medium and was abrogated by dithiothreitol pretreatment. The application of NEM after cyclopiazonic acid (CPA) stimulated the store-emptying activation of Ca 2+ entry. Unlike CPA-induced cation entry, NEM showed poor uptake of Ba 2+ and Sr 2+ and did not induce Mn 2+ influx. NEM diminished CPA-induced Mn 2+ influx, an effect that was blocked by dithiothreitol. Both Ni 2+ and La 3+ attenuated the elevation of [Ca 2+] i in response to NEM; however, greater resistance was observed to Ni 2+ inhibition of NEM-induced Ca 2+ influx than inhibition of store-operated Ca 2+ entry. Both cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A) and 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1 H-imidazole (SKF-96365), Ca 2+ channel blockers, and calyculin A, an inhibitor of protein serine/threonine phosphatases 1/2, diminished the NEM-induced Ca 2+ entry. Treatment of cells with genistein, a general tyrosine kinase inhibitor, or with wortmannin and 2-(4-morpholinyl)-8-phenyl-4 H-1-benzopyran-4-one (LY294002), phosphatidylinositol 3-kinase inhibitors, had no appreciable inhibitory effects on the action of NEM. However, 2-aminoethyldiphenyl borate, an inositol trisphosphate receptor antagonist, enhanced rather than inhibited the [Ca 2+] i change in response to NEM. These results indicate that NEM stimulates Ca 2+ entry and regulates Ca 2+ signaling through direct thiol oxidation, bypassing the cellular signal transduction pathway. The NEM-regulated Ca 2+ signal demonstrates characteristics that distinguish it from the store-emptying operation in neutrophils, and therefore represents two distinct modes of Ca 2+ regulation.

Glycerotoxin from Glycera convoluta stimulates neurosecretion by up-regulating N-type Ca2+ channel activity.

We report here the purification of glycerotoxin from the venom of Glycera convoluta, a novel 320 kDa protein capable of reversibly stimulating spontaneous and evoked neurotransmitter release at the frog neuromuscular junction. However, glycerotoxin is ineffective at the murine neuromuscular junction, which displays a different subtype of voltage- dependent Ca(2+) channels. By sequential and selective inhibition of various types of Ca(2+) channels, we found that glycerotoxin was acting via Ca(v)2.2 (N-type). In neuroendocrine cells, it elicits a robust, albeit transient, influx of Ca(2+) sensitive to the Ca(v)2.2 blockers omega-conotoxin GVIA and MVIIA. Moreover, glycerotoxin triggers a Ca(2+) transient in human embryonic kidney (HEK) cells over-expressing Ca(v)2.2 but not Ca(v)2.1 (P/Q-type). Whole-cell patch-clamp analysis of Ca(v)2.2 expressing HEK cells revealed an up-regulation of Ca(2+) currents due to a leftward shift of the activation peak upon glycerotoxin addition. A direct interaction between Ca(v)2.2 and this neurotoxin was revealed by co-immunoprecipitation experiments. Therefore, glycerotoxin is a unique addition to the arsenal of tools available to unravel the mechanism controlling Ca(2+)-regulated exocytosis via the specific activation of Ca(v)2.2.

Yessotoxin, a novel phycotoxin, activates phosphodiesterase activity: Effect of yessotoxin on cAMP levels in human lymphocytes

Yessotoxin (YTX) is a novel phycotoxin with an unknown mechanism of action that has been reported as cardiotoxic, when injected, but non-toxic if ingested orally. In this paper, we studied the effect of YTX on adenosine 3′,5′-cyclic monophosphate (cAMP) pathway, since this pathway can be a cellular target to this toxin as happens in other diarrhetic toxins. We determined cAMP levels by enzymeimmunoassay and by using the cAMP dye recombinant fluorescein- and rhodamine-labeled protein kinase A, which increases their fluorescence when cAMP levels are increased. In the presence of YTX, and after a transient small increase, cAMP levels were decreased. This effect was Ca 2+ dependent since in a Ca 2+-free medium YTX increased cAMP levels, but this event was reverted after addition of external calcium. YTX also reverted the increase of cAMP induced by the adenylyl cyclase activator forskolin. These variations in fluorescence units were confirmed when cAMP levels were measured by enzymeimmunoassay, YTX decreases cAMP from 52.81±3.66 to 44.53±4.5 fmol. Phosphodiesterase (PDE) IV inhibitors, rolipram or etazolate, did not modify the effect of YTX, however, when PDE IV was first inhibited no effect of YTX was observed. On the other hand, the PDE III inhibitor milrinone counteracted the effect of YTX, and a similar effect was observed with the unspecific PDE I inhibitor chlorpromazine. These results point to an effect of YTX on PDE activity. In the presence of YTX, the fluorescent PDE substrate Mant-cAMP, increased its rate of hydrolysis, the same as the PDE from bovine brain increased the hydrolysis of cAMP substrate. In addition, YTX increased interleukin-2 production, which indirectly confirms a decrease in cAMP. Although results show a very complex pattern of responses, due to the interactions and crosstalks between many systems, results suggest that YTX is a PDE activator in the presence of external Ca 2+.

Biphasic modulation of synaptic transmission by hypertonicity at the embryonic Drosophila neuromuscular junction.

Puff-application of hypertonic saline (sucrose added to external saline) causes a transient increase in the frequency of spontaneous miniature synaptic currents (mSCs) at the neuromuscular junctions of Drosophila embryos. The frequency gradually returns to pre-application levels. External Ca(2+) is not needed for this response, but it may modify it. At 50 mM added sucrose, for example, enhanced spontaneous release was observed only in the presence of external Ca(2+), suggesting that Ca(2+) augments the response. In a high-K(+) solution, in which the basal mSC frequency was elevated, higher sucrose concentrations produced an increase in mSC frequency that was followed (during and after the hypertonic exposure) by depression, with the magnitude of both effects increasing with hypertonicity between 100 and 500 mM. Evoked release by nerve stimulation showed only depression in response to hypertonicity. We do not believe that the depression of spontaneous or evoked release can be explained by the depletion of releasable quanta, however, since the frequency of quantal release did not reach levels compatible with this explanation and the enhancement and depression could be obtained independent of one another. In a mutant lacking neuronal synaptobrevin, only the depression of mSC frequency was induced by hypertonicity. Conversely, only the enhancing effect was observed in wild-type embryos when the mSC frequency was elevated with forskolin in Ca(2+)-free saline. In cultured embryonic Drosophila neurons, Ca(2+) signals that were induced by high K(+) and detected by Fura-2, were reduced by hypertonicity, suggesting that the depressing response is due to a direct effect of hypertonicity on Ca(2+) influx.