Extent Of Ca2 Research Articles

Locusta migratoria flight muscle troponin partially activates thin filament in a calcium-dependent manner.

The troponin (Tn) complex, the sensor for Ca2+ to regulate contraction of striated muscle, is composed of three subunits, that is, TnT, TnI and TnC. Different isoforms of TnI and TnC are expressed in the thorax dorsal longitudinal muscle (flight muscle) and the hind leg extensor tibiae muscle (jump muscle) of the migratory locust, Locusta migratoria. The major Tn complexes in the flight muscle and the jump muscle are Tn-171 (TnT1/TnI7/TnC1) and Tn-153 (TnT1/TnI5/TnC3), respectively. Here, we prepared a number of recombinant Tn complexes and the reconstituted thin filaments, and investigated their regulation on thin filament. Although both Tn-171 and Tn-153 regulate thin filament in a Ca2+ -dependent manner, the extent of Ca2+ activation mediated by Tn-171 was significantly lower than that by Tn-153. We demonstrated that TnC1 and TnC3, rather than TnI5 and TnI7, are responsible for the different levels of the thin filament activation. Mutagenesis of TnC1 and TnC3 shows that the low level of TnC1-mediated thin filament activation can be attributed to the noncanonical residue Leu60 in the EF-hand-II of TnC1. We therefore propose that, in addition to Ca2+ , other regulatory mechanism(s) is required for the full activation of locust flight muscle.

SARAF and EFHB Modulate Store-Operated Ca2+ Entry and Are Required for Cell Proliferation, Migration and Viability in Breast Cancer Cells.

Simple SummaryBreast cancer accounts as the most extended disease among women worldwide. Store-operated calcium entry (SOCE), a major mechanism that allows calcium entry from the extracellular region through the plasma membrane, is required for several physiological processes. In recent years, it has been revealed that several breast cancer types present dysregulated calcium homeostasis, which contribute to their malignancy. Here we show the role of two important regulators of SOCE, SARAF and EFHB, which are necessary for cell viability, proliferation, and migration in breast cancer and pre-neoplastic cells, respectively, thus suggesting that these regulators play a key function in breast cancer development and progression.Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Store-operated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells.

Submicron Emitters Enable Reliable Quantification of Weak Protein-Glycan Interactions by ESI-MS.

Interactions between carbohydrates (glycans) and glycan-binding proteins (GBPs) regulate a wide variety of important biological processes. However, the affinities of most monovalent glycan-GBP complexes are typically weak (dissociation constant (Kd) > μM) and difficult to reliably measure with conventional assays; consequently, the glycan specificities of most GBPs are not well established. Here, we demonstrate how electrospray ionization mass spectrometry (ESI-MS), implemented with nanoflow ESI emitters with inner diameters of ∼50 nm, allows for the facile quantification of low-affinity glycan-GBP interactions. The small size of the droplets produced from these submicron emitters effectively eliminates the formation of nonspecific glycan-GBP binding (false positives) during the ESI process up to ∼mM glycan concentrations. Thus, interactions with affinities as low as ∼5 mM can be measured directly from the mass spectrum. The general suppression of nonspecific adducts (including nonvolatile buffers and salts) achieved with these tips enables ESI-MS glycan affinity measurements to be performed on C-type lectins, a class of GBPs that bind glycans in a calcium-dependent manner and are important regulators of immune response. At physiologically relevant calcium ion concentrations (2-3 mM), the extent of Ca2+ nonspecific adduct formation observed using the submicron emitters is dramatically suppressed, allowing glycan affinities, and the influence of Ca2+ thereon, to be measured. Finally, we show how the use of submicron emitters and suppression of nonspecific binding enable the quantification of labile (prone to in-source dissociation) glycan-GBP interactions.

The Role of Calcium in the Regulation of Mitochondrial Respiration under Various Substrate Combinations in the Heart and Kidney

It has been recognized that the efficiency of mitochondrial oxidative phosphorylation (OxPhos) and energy metabolism depends on the choice of respiratory substrates, which feed electrons to the mitochondrial electron transport chain. In addition, mitochondrial enzymes, such as pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (ICDH), and alpha‐ketoglutarate dehydrogenase (AKGDH), have been shown to be stimulated by calcium ions (Ca2+) in isolated enzyme experimental systems. However, the extent of Ca2+ stimulation of these enzymes and the resulting integrated stimulatory effect on the efficiency of mitochondrial OxPhos and energy metabolism in in‐situ and in‐vivo states in the heart and kidney under different substrate combinations are not well‐understood. In this study, we investigated the effect of a variety of substrate combinations and Ca2+ alternations that optimize mitochondrial respiration and ATP synthesis. We hypothesized that Ca2+ stimulation of mitochondrial respiration and ATP synthesis is both substrate dependent and tissue specific. To study mitochondrial function under a specific substrate combination and Ca2+, cardiac and renal mitochondria were isolated from Sprague‐Dawley rats, and oxygen consumption rates (JO2) were measured in the presence and absence of ADP that corresponded to leak (state 2) and ADP‐stimulated (state 3) states, respectively. We first measured the time courses of JO2 at various ADP concentrations to find the saturated ADP‐stimulated JO2. This saturated ADP concentration was then used to measure the time‐courses of JO2 under different [Ca2+]. The substrate combinations used include pyruvate + malate (PM), glutamate + malate (GM), alpha‐ketoglutarate + malate (AM), palmitoyl‐L‐carnitine + malate (PCM), and succinate + rotenone (SUC + ROT). Different substrates led to different levels of the saturated state 3 JO2. On average, it was 9 times higher in the heart compared to the kidney with the PM substrate. Also, the alterations in the respiratory substrates led to dramatically different respiratory rates/dynamics in the isolated mitochondria of the chosen organs. The results also demonstrated that Ca2+ significantly increased mitochondrial state 3 JO2 with GM and AM substrates compared to the other substrates in both organs (Table 1). Moreover, Ca2+ effects on mitochondrial respiration were found to be biphasic, i.e. a small increase in [Ca2+] had a stimulatory effect while large increases had inhibitory effects (Figure 1). These results suggest that energized isolated mitochondria in the presence of Ca2+ do not respond the same extent when different substrates are used. We conclude that isolated mitochondria require a suitable substrate combination, proper [Ca2+], and specific tissue source of mitochondria to achieve an optimal level of OxPhos and energy metabolism comparable to in‐vivo conditions.Support or Funding InformationThis work was supported by the NIH grants P01‐GM066730, P01‐HL116264, R01‐HL‐122662, and U01‐HL122199.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calcium Stimulation of Mitochondrial Respiration is Substrate Dependent and Tissue Specific

Mitochondrial enzymes, such as pyruvate dehydrogenase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase, have been shown to be stimulated by calcium ion in isolated enzyme experimental systems. However, the extent of Ca2+ stimulation of these enzymes and the resulting integrated stimulatory effect on mitochondrial energy metabolism in in-situ and in-vivo states in the heart and kidney remains to be clarified. In this study, we hypothesized that Ca2+ stimulation of mitochondrial respiration and ATP synthesis is substrate dependent and tissue-specific. To test this hypothesis, we isolated mitochondria from heart and kidney outer medulla from Sprague-Dawley rats. Oxygen consumption rates were determined under different respiratory states: leak state (energized mitochondria in absence of ADP) and the ADP-stimulated state (energized mitochondria in presence of saturating concentration of ADP) with/without bolus injection of different concentrations of CaCl2 using an Oroboros Oxygraph-2k Instrument with various respiratory substrates, including pyruvate+malate (PM), glutamate+malate (GM), alpha-ketoglutarate+malate (AM), palmitoyl-L-carnitine+malate (PCM), and succinate (SUC). The results demonstrated that the various respiratory substrates yielded differences in ADP consumption rates and hence, led to dramatically different respiratory rates in isolated mitochondria from the heart and kidney. Importantly, it was found that Ca2+ significantly increased mitochondrial state III respiration with GM and AM substrates compared to the other substrates in both organs. Moreover, Ca2+ effects on mitochondrial respiration were found to be biphasic, i.e. a small increases in [Ca2+] had a stimulatory effect while large increases had inhibitory effect. These results suggest that energized isolated mitochondria in presence of Ca2+ do not respond the same extent when different substrates are used. We conclude that isolated mitochondria require a suitable substrate combination, optimal [Ca2+], and specific tissue source of mitochondria to achieve a level of energy metabolism and ATP production comparable to in-vivo conditions.

Structure‐dynamic basis of splicing‐dependent regulation in tissue‐specific variants of the sodium‐calcium exchanger

Tissue-specific splice variants of Na(+)/Ca(2+) exchangers contain 2 Ca(2+)-binding regulatory domains (CBDs), CBD1 and CBD2. Ca(2+) interaction with CBD1 activates sodium-calcium exchangers (NCXs), and Ca(2+) binding to CBD2 alleviates Na(+)-dependent inactivation. A combination of mutually exclusive (A, B) and cassette (C-F) exons in CBD2 raises functionally diverse splice variants through unknown mechanisms. Here, the effect of exons on CBDs backbone dynamics were investigated in the 2-domain tandem (CBD12) of the brain, kidney, and cardiac splice variants by using hydrogen-deuterium exchange mass spectrometry and stopped-flow techniques. Mutually exclusive exons stabilize interdomain interactions in the apoprotein, which primarily predefines the extent of responses to Ca(2+) binding. Deuterium uptake levels were up to 20% lower in the cardiac vs. the brain CBD12, reveling that elongation of the CBD2 FG loop by cassette exons rigidifies the interdomain Ca(2+) salt bridge at the 2-domain interface, which secondarily modulates the Ca(2+)-bound states. In matching splice variants, the extent of Ca(2+)-induced rigidification correlates with decreased (up to 10-fold) Ca(2+) off rates, where the cardiac CBD12 exhibits the slowest Ca(2+) off rates. Collectively, structurally disordered/dynamic segments at mutually exclusive and cassette exons have local and distant effects on the folded structures nearby the Ca(2+) binding sites, which may serve as a structure-dynamic basis for splicing-dependent regulation of NCX.

Ligand-Dependent Conformational Changes in the Cardiac Ryanodine Receptor

Global conformational changes in the three-dimensional (3D) structure of the calcium release channel/ryanodine receptor (RyR) occur upon ligand activation. Of several ligands that activate RyR, Ca2+ is the primary activator. Although RyR Ca2+ activation is well characterized functionally, little is known about the conformational changes in RyR induced by Ca2+. Here we generated three fluorescence resonance energy transfer (FRET)-based conformational probes. Each of these probes was constructed by inserting a CFP into one domain and a YFP into a neighboring domain in the cardiac RyR (RyR2) to yield a CFP- and YFP-dual labeled RyR2 (CFP/YFP FRET pair). These CFP/YFP FRET pairs were located in the “clamp” region (RyR2S2367-CFP/Y2801-YFP), the calmodulin binding region (RyR2R3595-CFP/K4269-YFP), and the “bridge” region (RyR2S437-YFP/S2367-CFP), respectively. We monitored the conformational changes in these regions by recording the FRET signals, and the extent of Ca2+ release by measuring store Ca2+ depletion in HEK293 cells expressing each of the CFP/YFP FRET pairs upon activation by Ca2+, caffeine, and ATP. Surprisingly, we found that different ligands induced different conformational changes in different regions of RyR2. For instance, we detected conformational changes in the clamp region for caffeine and ATP, but not for Ca2+, although they all induced Ca2+ release. Considering Ca2+ as the primary activator of RyR2, we determined the impact of cytosolic Ca2+ sensing mutation E3987A on conformational changes. Interestingly, this single mutation abolishes caffeine-induced conformational changes, but not caffeine-induced Ca2+ release. These observations demonstrate that conformational changes in RyR2 are ligand-dependent, and that E3987, which is critical for cytosolic Ca2+ sensing, is essential for ligand-induced conformational changes (Supported by CFI, CIHR, HSFC, NIH, and LCIA).

Calcium influx determines the muscular response to electrotransfer

Cell membrane permeabilization by electric pulses (electropermeabilization), results in free exchange of ions across the cell membrane. The role of electrotransfer-mediated Ca(2+)-influx on muscle signaling pathways involved in degeneration (β-actin and MurF), inflammation (IL-6 and TNF-α), and regeneration (MyoD1, myogenin, and Myf5) was investigated, using pulse parameters of both electrochemotherapy (8 HV) and DNA delivery (HVLV). Three pulsing conditions were used: 8 high-voltage pulses (8 HV), resulting in large permeabilization and ion flux, and a combination of one high-voltage pulse and one low-voltage pulse (HVLV), either alone or in combination with injection of DNA. Mice and rats were anesthetized before pulsing. At the times given, animals were killed, and intact tibialis cranialis muscles were excised for analysis. Uptake of Ca(2+) was assessed using (45)Ca as a tracer. Using gene expression analyses and histology, we showed a clear association between Ca(2+) influx and muscular response. Moderate Ca(2+) influx induced by HVLV pulses results in activation of pathways involved in immediate repair and hypertrophy. This response could be attenuated by intramuscular injection of EGTA reducing Ca(2+) influx. Larger Ca(2+) influx as induced by 8-HV pulses leads to muscle damage and muscle fiber regeneration through recruitment of satellite cells. The extent of Ca(2+) influx determines the muscular response to electrotransfer and, thus, the success of a given application. In the case of electrochemotherapy, in which the objective is cell death, a large influx of Ca(2+) may be beneficial, whereas for DNA electrotransfer, muscle recovery should occur without myofiber loss to ensure preservation of plasmid DNA.

A Red Wine Extract Supplement Protects against Apoptosis and Inhibits Changes in Mitochondrial Permeability and Membrane Potential

The effects of a red wine extract supplement (Super S.O.D) on hypoxia/reoxygenation-induced apoptosis were examined in H9c2 cardiomyocytes. Super S.O.D pretreatment dose-dependently protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes. The cytoprotection was associated with increases in cellular reduced glutathione level and mitochondrial membrane potential, as well as the decrease in the extent of Ca2+-induced mitochondrial permeability transition (MPT) pore opening in both unchallenged and challenged cells, as compared with the unpretreated control. The results indicated that Super S.O.D pretreatment protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes, possibly mediated by the inhibition of MPT triggered by hypoxia/reoxygenation challenge.

Vesicular Calcium Regulates Coat Retention, Fusogenicity, and Size of Pre-Golgi Intermediates

The significance and extent of Ca(2+) regulation of the biosynthetic secretory pathway have been difficult to establish, and our knowledge of regulatory relationships integrating Ca(2+) with vesicle coats and function is rudimentary. Here, we investigated potential roles and mechanisms of luminal Ca(2+) in the early secretory pathway. Specific depletion of luminal Ca(2+) in living normal rat kidney cells using cyclopiazonic acid (CPA) resulted in the extreme expansion of vesicular tubular cluster (VTC) elements. Consistent with this, a suppressive role for vesicle-associated Ca(2+) in COPII vesicle homotypic fusion was demonstrated in vitro using Ca(2+) chelators. The EF-hand-containing protein apoptosis-linked gene 2 (ALG-2), previously implicated in the stabilization of sec31 at endoplasmic reticulum exit sites, inhibited COPII vesicle fusion in a Ca(2+)-requiring manner, suggesting that ALG-2 may be a sensor for the effects of vesicular Ca(2+) on homotypic fusion. Immunoisolation established that Ca(2+) chelation inhibits and ALG-2 specifically favors residual retention of the COPII outer shell protein sec31 on pre-Golgi fusion intermediates. We conclude that vesicle-associated Ca(2+), acting through ALG-2, favors the retention of residual coat molecules that seem to suppress membrane fusion. We propose that in cells, these Ca(2+)-dependent mechanisms temporally regulate COPII vesicle interactions, VTC biogenesis, cargo sorting, and VTC maturation.

Mitochondrial Ca2+ Uptake Increases Ca2+ Release from Inositol 1,4,5-Trisphosphate Receptor Clusters in Smooth Muscle Cells

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP(3))-mediated increases in cytosolic Ca2+ concentration ([Ca2+](c)). Local Ca2+ release from an InsP(3) receptor (InsP(3)R) cluster present on the sarcoplasmic reticulum is termed a Ca2+ puff. Ca2+ released via InsP(3)R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca2+ rise. In smooth muscle, mitochondrial Ca2+ uptake maintains global InsP(3)-mediated Ca2+ release by preventing a negative feedback effect of high [Ca2+] on InsP(3)R. Mitochondria may regulate InsP(3)-mediated Ca2+ signals by operating between or within InsP(3)R clusters. In the former mitochondria could regulate only global Ca2+ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP(3)-mediated Ca2+ release by operating within (local) or between (global) InsP(3)R clusters has been addressed. Ca2+ puffs evoked by localized photolysis of InsP(3) in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5-4.0 F/F(0), durations of approximately 112 ms at half-maximum amplitude, and were abolished by the InsP(3)R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized DeltaPsi(M) to prevent mitochondrial Ca2+ uptake and attenuated Ca2+ puffs by approximately 66 or approximately 60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca2+ puffs by approximately 62%. The "fast" Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acted like mitochondria to prolong InsP(3)-mediated Ca2+ release suggesting that mitochondrial influence is via their Ca2+ uptake facility. These results indicate Ca2+ uptake occurs quickly enough to influence InsP(3)R communication at the intra-cluster level and that mitochondria regulate both local and global InsP(3)-mediated Ca2+ signals.

Sustained Activation of the Tyrosine Kinase Syk by Antigen in Mast Cells Requires Local Ca2+ Influx through Ca2+ Release-activated Ca2+ Channels

Mast cell activation involves cross-linking of IgE receptors followed by phosphorylation of the non-receptor tyrosine kinase Syk. This results in activation of the plasma membrane-bound enzyme phospholipase Cgamma1, which hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores. This Ca2+ release phase is accompanied by sustained Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. Here, we find that engagement of IgE receptors activates Syk, and this leads to Ca2+ release from stores followed by Ca2+ influx. The Ca2+ influx phase then sustains Syk activity. The Ca2+ influx pathway activated by these receptors was identified as the CRAC channel, because pharmacological block of the channels with either a low concentration of Gd3+ or exposure to the novel CRAC channel blocker 3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide or RNA interference knockdown of Orai1, which encodes the CRAC channel pore, all prevented the increase in Syk activity triggered by Ca2+ entry. CRAC channels and Syk are spatially close together, because increasing cytoplasmic Ca2+ buffering with the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis failed to prevent activation of Syk by Ca2+ entry. Our results reveal a positive feedback step in mast cell activation where receptor-triggered Syk activation and subsequent Ca2+ release opens CRAC channels, and the ensuing local Ca2+ entry then maintains Syk activity. Ca2+ entry through CRAC channels therefore provides a means whereby the Ca2+ and tyrosine kinase signaling pathways can interact with one another.

Phosphate Is Essential for Inhibition of the Mitochondrial Permeability Transition Pore by Cyclosporin A and by Cyclophilin D Ablation

Energized mouse liver mitochondria displayed the same calcium retention capacity (a sensitive measure of the propensity of the permeability transition pore (PTP) to open) irrespective of whether phosphate, arsenate, or vanadate was the permeating anion. Unexpectedly, however, phosphate was specifically required for PTP desensitization by cyclosporin A (CsA) or by genetic inactivation of cyclophilin D (CyP-D). Indeed, when phosphate was replaced by arsenate, vanadate, or bicarbonate, the inhibitory effects of CsA and of CyP-D ablation on the PTP disappeared. After loading with the same amount of Ca(2+) in the presence of arsenate or vanadate but in the absence of phosphate, the sensitivity of the PTP to a variety of inducers was identical in mitochondria from wild-type mice, CyP-D-null mice, and wild-type mice treated with CsA. These findings call for a reassessment of conclusions on the role of the PTP in cell death that are based on the effects of CsA or of CyP-D ablation.

10.1007/s11827-008-1003-x

Snake venoms contain a vast array of toxic polypeptide components interacting with a variety of cell targets. Thus, Elapidae snake venoms contain α-neurotoxins with very high affinity for nicotinic acetylcholine receptors (nAChRs) and a few toxins able to suppress the activity of Ca2+ and K+ channels. Experimental evidence for the presence of nAChR antagonists and voltage-gated ionic channel blockers in venoms of Viperidae snakes is very scarce. In this study, effects of crude venoms of seven snake species (Vipera nikolskii, Echis multisquamatus, Gloydius saxatilis, Bitis arietans, Vipera renardi, Vipera lebetina, and Naja kaouthia) on nAChRs and voltage-gated Ca2+ channels were studied for the first time. The experiments were carried out on isolated identified neurons of the fresh-water mollusc Lymnaea stagnalis using voltage-clamp and intracellular perfusion techniques. All Viperidae snake venoms under study blocked nAChRs and voltage-gated Ca2+ channels. The potency of these venoms against nAChRs was significantly lower in comparison with N. kaouthia venom which is rich of α-neurotoxins; however, the extent of Ca2+ channel block by venoms of Viperidae snakes and N. kaouthia was similar. The data obtained suggest that Viperidae snake venoms tested in this study contain peptides with affinity both for nAChRs and for voltage-gated Ca2+ channels.

MGluR-Mediated Calcium Waves that Invade the Soma Regulate Firing in Layer V Medial Prefrontal Cortical Pyramidal Neurons

Factors that influence the activity of prefrontal cortex (PFC) pyramidal neurons are likely to play an important role in working memory function. One such factor may be the release of Ca2+ from intracellular stores. Here we investigate the hypothesis that metabotropic glutamate receptors (mGluRs)-mediated waves of internally released Ca2+ can regulate the intrinsic excitability and firing patterns of PFC pyramidal neurons. Synaptic or focal pharmacological activation of mGluRs triggered Ca2+ waves in the dendrites and somata of layer V medial PFC pyramidal neurons. These Ca2+ waves often evoked a transient SK-mediated hyperpolarization followed by a prolonged depolarization that respectively decreased and increased neuronal excitability. Generation of the hyperpolarization depended on whether the Ca2+ wave invaded or came near to the soma. The depolarization also depended on the extent of Ca2+ wave propagation. We tested factors that influence the propagation of Ca2+ waves into the soma. Stimulating more synapses, increasing inositol trisphosphate concentration near the soma, and priming with physiological trains of action potentials all enhanced the amplitude and likelihood of evoking somatic Ca2+ waves. These results suggest that mGluR-mediated Ca2+ waves may regulate firing patterns of PFC pyramidal neurons engaged by working memory, particularly under conditions that favor the propagation of Ca2+ waves into the soma.

Third Activity of Bordetella Adenylate Cyclase (AC) Toxin-Hemolysin: MEMBRANE TRANSLOCATION OF AC DOMAIN POLYPEPTIDE PROMOTES CALCIUM INFLUX INTO CD11b+MONOCYTES INDEPENDENTLY OF THE CATALYTIC AND HEMOLYTIC ACTIVITIES

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.

Measurement of Ca2+signaling dynamics in exocrine cells with total internal reflection microscopy

In nonexcitable cells, such as exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca2+ signals consist of both Ca2+ release and Ca2+ influx. We have investigated the contribution of these processes to membrane-localized Ca2+ signals in pancreatic and parotid acinar cells using total internal reflection fluorescence (TIRF) microscopy (TIRFM). This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and the coverslip. In TIRFM mode, physiological agonist stimulation resulted in Ca2+ oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional wide-field microscopy (WFM). Because local Ca2+ release in the TIRF zone would be expected to saturate the Ca2+ indicator (Fluo-4), these data suggest that Ca2+ release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca2+ release, followed by a substantial, maintained plateau phase indicative of Ca2+ entry, was observed. The contribution of Ca2+ influx and Ca2+ release in isolation to these near-plasma membrane Ca2+ signals was investigated by using a Ca2+ readmission protocol. In the absence of extracellular Ca2+, the profile and magnitude of the initial Ca2+ release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition were similar to those obtained with WFM in both pancreas and parotid acini. In contrast, when Ca2+ influx was isolated by subsequent Ca2+ readmission, the Ca2+ signals evoked were more robust than those measured with WFM. Furthermore, in parotid acinar cells, Ca2+ readdition often resulted in the apparent saturation of Fluo-4 but not of the low-affinity dye Fluo-4-FF. Interestingly, Ca2+ influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca2+ influx was observed in parotid acinar cells even at low physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near-membrane Ca2+ signals mediated by Ca2+ influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+ influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.

Influence of viscosity and uronic acid composition of alginates on the properties of alginate films and microspheres produced by emulsification

This study investigated the influence of viscosity and uronic acid composition of alginates on the properties of alginate films and microspheres produced by emulsification. Tensile properties of films were determined while the yield, size, drug contents and release characteristics of the microspheres were examined. Tensile properties of calcium alginate matrix were significantly affected by the orientation and arrangement of the polymer chains. High viscosity alginates gave rise to higher yields and bigger microspheres. Generally, microspheres with high drug content and slower rate of drug release had high Ca2+ contents and were produced from alginates of higher viscosity. Within an alginate microsphere batch, small sized microsphere fractions had higher drug contents but showed faster drug release rates. Microspheres having a defined size range revealed great dependence of encapsulation efficiency and drug release rates on viscosity and extent of Ca2+-alginate interaction. Viscosity appeared to exert a predominant influence on the microsphere properties.

Absorption/desorption of calcium ions on polypyrrole-loaded reticulated vitreous carbon

Polypyrrole/polystyrene sulfonate-loaded reticulated vitreous carbon electrodes RVC/PPy/PSS were studied as electrochemical cation exchangers for the absorption of Ca2+ ions from aqueous solutions. Two grades of RVC were used mainly with 20 and 45 PPI. The preparation and characterization of the PPy/PSS layers on RVC are described. The structure and morphology of the porous electrodes were characterized using cyclic voltammetry (CV) and scanning electron microscopy (SEM). The potential dependent absorption and desorption of Ca2+ on RVC/PPy/PSS electrodes was studied in 0.1 M CaCl2 solution using chronoamperometry and atomic adsorption spectroscopy (AAS). The extent of Ca2+ ions absorbed into RVC/PPy/PSS was found to increase with decreasing absorption potential, the specific surface area and the extent of polymer loading. Recovery of the RVC/PPy/PSS electrode was performed by anodic polarization at potentials at which Ca2+ ions are desorbed. The coulomb efficiency for the Ca2+ desorption process was found to be higher than that for the Ca2+ absorption process. From these results the feasibility of using RVC/PPy/PSS for water softening was determined under the prevailing experimental conditions.

Microfluidic glucose stimulation reveals limited coordination of intracellular Ca2+ activity oscillations in pancreatic islets.

The pancreatic islet is a functional microorgan involved in maintaining normoglycemia through regulated secretion of insulin and other hormones. Extracellular glucose stimulates insulin secretion from islet beta cells through an increase in redox state, which can be measured by NAD(P)H autofluorescence. Glucose concentrations over approximately 7 mM generate synchronous oscillations in beta cell intracellular Ca2+ concentration ([Ca2+]i), which lead to pulsatile insulin secretion. Prevailing models assume that the pancreatic islet acts as a functional syncytium, and the whole islet [Ca2+]i response has been modeled in terms of islet bursting and pacemaker models. To test these models, we developed a microfluidic device capable of partially stimulating an islet, while allowing observation of the NAD(P)H and [Ca2+]i responses. We show that beta cell [Ca2+]i oscillations occur only within regions stimulated with more than approximately 6.6 mM glucose. Furthermore, we show that tolbutamide, an antagonist of the ATP-sensitive K+ channel, allows these oscillations to travel farther into the nonstimulated regions of the islet. Our approach shows that the extent of Ca2+ propagation across the islet depends on a delicate interaction between the degree of coupling and the extent of ATP-sensitive K+-channel activation and illustrates an experimental paradigm that will have utility for many other biological systems.