PA1b,a plant peptide,induces intracellular [Ca<Superscript>2+</Superscript>] increase via Ca<Superscript>2+</Superscript> influx through the L-type Ca<Superscript>2+</Superscript> channel and triggers secretion in pancreatic β cells

Using alginic acid to adsorb polypeptides at pH 2.7, we isolated a peptide pea albumin 1b (PA1b) from pea seeds. The PA1b is a single chain peptide consisting of 37 amino acid residues with 6 cysteines which constitutes the cystine-knot structure. Using microfluorometry and patch clamp techniques, we found that PA1b significantly elevated the intracellular calcium level ([Ca²⁺]_i) and elicited membrane capacitance increase in the primary rat pancreatic β cells. The PA1b effect on [Ca²⁺]_i elevation was abolished in the absence of extracellular Ca²⁺ or in the presence of L-type Ca²⁺ channel blocker, nimodipine. Interestingly, we found that PA1b significantly depolarized membrane potential, which could lead to the opening of voltage-dependent L-type Ca²⁺ channels and influx of extracellular Ca²⁺, and then evoke robust secretion. In this study we identified the plant peptide PA1b which is capable of affecting the excitability and function of mammalian pancreatic β cell.     

A Voltage-Dependent Ca<Superscript>2+</Superscript> Influx Pathway Regulates the Ca<Superscript>2+</Superscript>-Dependent Cl<Superscript>−</Superscript> Conductance of Renal IMCD-3 Cells

We have previously shown that the membrane conductance of mIMCD-3 cells at a holding potential of 0 mV is dominated by a Ca²⁺-dependent Cl⁻ current (I_CLCA). Here we report that I_CLCA activity is also voltage dependent and that this dependence on voltage is linked to the opening of a novel Al³⁺-sensitive, voltage-dependent, Ca²⁺ influx pathway. Using whole-cell patch-clamp recordings at a physiological holding potential (−60 mV), I_CLCA was found to be inactive and resting currents were predominantly K⁺ selective. However, membrane depolarization to 0 mV resulted in a slow, sigmoidal, activation of I_CLCA (T _0.5 ~ 500 s), while repolarization in turn resulted in a monoexponential decay in I_CLCA (T _0.5 ~ 100 s). The activation of I_CLCA by depolarization was reduced by lowering extracellular Ca²⁺ and completely inhibited by buffering cytosolic Ca²⁺ with EGTA, suggesting a role for Ca²⁺ influx in the activation of I_CLCA. However, raising bulk cytosolic Ca²⁺ at −60 mV did not produce sustained I_CLCA activity. Therefore I_CLCA is dependent on both an increase in intracellular Ca²⁺ and depolarization to be active. We further show that membrane depolarization is coupled to opening of a Ca²⁺ influx pathway that displays equal permeability to Ca²⁺ and Ba²⁺ ions and that is blocked by extracellular Al³⁺ and La³⁺. Furthermore, Al³⁺ completely and reversibly inhibited depolarization-induced activation of I_CLCA, thereby directly linking Ca²⁺ influx to activation of I_CLCA. We speculate that during sustained membrane depolarization, calcium influx activates I_CLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells.     

β<Subscript>2</Subscript>-Integrin-Mediated Adhesion and Intracellular Ca<Superscript>2+</Superscript> Release in Human Eosinophils

Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added activators. In the present study a method was developed for characterizing eosinophil adhesion by measuring changes in impedance. Impedance measurements were performed in HCO₃-buffered HybriCare medium maintained in a humidified 5% CO₂ incubator at 37°C. Impedance increased by more than 1 kΩ within minutes after eosinophils made contact with the substrate, reaching a peak within 20 min. Blocking mobilization of intracellular [Ca²⁺] that precedes adhesion with BAPTA-AM (10 μM) completely inhibited the rise in impedance as well as the changes in cell shape typically observed in adherent cells. However, lowering the extracellular [Ca²⁺] with 2.5 mM EGTA did not inhibit the increase in impedance. Pretreatment with anti-CD18 antibody to block substrate interactions with β₂-integrins, or jasplakinolide (2 μM) to block actin reorganization, abolished the increase in impedance and adherent morphology of the cells. Exposure of eosinophils to the phosphatidylinositol 3 kinase inhibitor LY294002 (5 μM) or treatment with protein kinase C zeta pseudosubstrate to competitively inhibit activity of the enzyme significantly reduced the increase in impedance and inhibited the cell spreading associated with adhesion. These results demonstrate a novel method for measuring eosinophil adhesion and showed that, following formation of a tethered attachment, a rapid increase in intracellular [Ca²⁺] precedes the cytoskeletal rearrangements required for cell shape changes and plasma membrane-substrate interactions associated with adhesion.     

Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> antiport as a possible mechanism of the Ca<Superscript>2+</Superscript>-translocating ATPase functioning in vesicles of bean root nodule’s symbiosome membrane

Using vesicles of symbiosome membrane (SM), it was shown that the Ca²⁺-ATPase can function as an ATP-energized Ca²⁺/H⁺ antiporter. The initial rate of the acidic shift inside the vesicles, as well as the rate of the ITP-dependent alkalization of the medium inside them markedly increased in the presence of valinomycin. This process was rapidly stopped by eosin Y, a known inhibitor of the type IIB Ca²⁺-ATPase. ITP-dependent uptake of Ca²⁺ was blocked after the addition to the reaction mixture of nigericin in the presence of K⁺. Under these conditions, the alkaline shift of pH inside the vesicles occurred, leading to the inhibition of operation of the calcium pump in SM. Evaluation of the pH shifts inside the vesicles by using pH-indicator pyranine confirmed the ion-exchange mechanism of the Ca²⁺-ATPase functioning in the SM.     

Investigation of the encapsulation of metal cations (Cu<Superscript>2+</Superscript>, Zn<Superscript>2+</Superscript>, Ca<Superscript>2+</Superscript> and Ba<Superscript>2+</Superscript>) by the dipeptide Phe–Phe using natural bond orbital theory and molecular dynamics simulation

Complexes of the dipeptide phenylalanine–phenylalanine (Phe–Phe) with divalent metal cations (Cu²⁺, Zn²⁺, Ca²⁺ and Ba²⁺) were studied at the B3LYP and MP2 levels of theory with the basis sets 6-311++G(d,p) and 6-31 + G(d) in the gas phase. The relative energies of these complexes indicated that cation–π bidentate/tridentate conformations are more favourable than other conformations with uncoordinated rings. These findings were confirmed by the calculated values of thermodynamic parameters such as the Gibbs free energy. Natural bond orbital (NBO) analysis was carried out to explore the metal–ligand coordination in Phe–Phe–Cu²⁺/Zn²⁺ complexes. Possible orbital transitions, types of orbitals and their occupancies were determined for a range of Phe–Phe–Cu²⁺/Zn²⁺ complexes. The charge transfer involved in various orbital transitions was explored by considering the second-order perturbation energy. NBO analysis revealed that the change transfer is stronger when the metal cation uses both the 4s + 4p subshells rather than just its 4p subshell. We also performed molecular dynamics (MD) simulations to check the stability and consistency of the most favourable binding motifs of Cu²⁺, Zn²⁺, Ca²⁺ and Ba²⁺ with Phe–Phe over time. The structures of the Phe–Phe–Cu²⁺/Zn²⁺/Ca²⁺/Ba²⁺ complexes obtained using MD simulation were found to be in good agreement with those obtained in the DFT-based calculations.

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Graphical Abstract Conformational search on encapsulation of divalent metal cations (Ca²⁺, Zn²⁺, Ca²⁺, Ba²⁺) by the Phe-Phe dipeptide

     

γ<Subscript>1</Subscript>-Dependent Down-regulation of Recombinant Voltage-gated Ca<Superscript>2+</Superscript> Channels

(1) Voltage-gated Ca²⁺ (Ca_V) channels are multi-subunit membrane complexes that allow depolarization-induced Ca²⁺ influx into cells. The skeletal muscle L-type Ca_V channels consist of an ion-conducting Ca_V1.1 subunit and auxiliary α₂δ−1, β₁ and γ₁ subunits. This complex serves both as a Ca_V channel and as a voltage sensor for excitation–contraction coupling. (2) Though much is known about the mechanisms by which the α₂δ−1 and β₁ subunits regulate Ca_V channel function, there is far less information on the γ₁ subunit. Previously, we characterized the interaction of γ₁ with the other components of the skeletal Ca_V channel complex, and showed that heterologous expression of this auxiliary subunit decreases Ca²⁺ current density in myotubes from γ₁ null mice. (3) In the current report, using Western blotting we show that the expression of the Ca_V1.1 protein is significantly lower when it is heterologously co-expressed with γ₁. Consistent with this, patch-clamp recordings showed that transient transfection of γ₁ drastically inhibited macroscopic currents through recombinant N-type (Ca_V2.2/α₂δ−1/β₃) channels expressed in HEK-293 cells. (4) These findings provide evidence that co-expression of the auxiliary γ₁ subunit results in a decreased expression of the ion-conducting subunit, which may help to explain the reduction in Ca²⁺ current density following γ₁ transfection.     

The role of Ca<Superscript>2+</Superscript>, H<Superscript>+</Superscript>, and Cl<Superscript>−</Superscript> ions in generation of variation potential in pumpkin plants

The contributions of Ca²⁺, H⁺, and Cl⁻ in generation of variation potentials (VP) in 3- to 4-week-old pumpkin (Cucurbita pepo L., cv. Mozoleevskaya) plants were assessed. During VP generation, transient alkalinization of the medium around the stem was recorded with a potentiometric method. The pH changes were kinetically similar to the electric potential changes and were apparently due to temporal suppression of the plasma-membrane electrogenic H⁺ pump. These data and the observed inhibition of VP in the stem zone treated locally with a metabolic inhibitor (NaN₃) indicate that the VP generation is related to the reversible suppression of the H⁺-pump. The anion channel blocker (ethacrynic acid) decelerated significantly the front slope of VP and reduced the VP amplitude. A short-term increase in external Cl⁻ concentration around the stem was observed during potential transients representing the VP front slope and the pulses integrated into VP. The removal of Ca²⁺ from extracellular medium inhibited the VP generation. It is proposed that Ca²⁺ plays a role in activation of anion channels and in the H⁺-pump inactivation. The VP generation is probably determined by a complex mechanism, with contributions from passive ion fluxes (Ca²⁺, Cl⁻) moving along the electrochemical gradients and from changes in the electrogenic pump activity.     

Ca<Superscript><Emphasis Type="Italic">2+</Emphasis></Superscript>-Induced Cl<Superscript>−</Superscript> Efflux at Rat Distal Colonic Epithelium

With the aid of the halide-sensitive dye 6-methoxy-N-ethylquinolinium iodide (MEQ), changes in intracellular Cl^- concentration were measured to characterize the role of Ca²⁺-dependent Cl^- channels at the rat distal colon. In order to avoid indirect effects of secretagogues mediated by changes in the driving force for Cl^- exit (i.e., mediated by opening of Ca²⁺-dependent K⁺ channels), all experiments were performed under depolarized conditions, i.e., in the presence of high extracellular K⁺ concentrations. The Ca²⁺-dependent secretagogue carbachol induced a stilbene-sensitive Cl^- efflux, which was mimicked by the Ca²⁺ ionophore ionomycin. Surprisingly, the activation of Ca²⁺-dependent Cl^- efflux was resistant against blockers of classical Ca²⁺ signaling pathways such as phospholipase C, protein kinase C and calmodulin. Hence, alternative pathways must be involved in the signaling cascade. One possible signaling molecule seems to be nitric oxide (NO) as the NO donor sodium nitroprusside could induce Cl^- efflux. Vice versa, the NO synthase inhibitor N-ω-monomethyl-arginine (l-NMMA) reduced the carbachol-induced Cl^- efflux. This indicates that NO may be involved in part of the signaling cascade. In order to test the ability of the epithelium to produce NO, the expression of different isoforms of NO synthase was verified by immunohistochemistry. In addition, the cytoskeleton seems to play a role in the activation of Ca²⁺-dependent Cl^- channels. Inhibitors of microtubule association such as nocodazole and colchicine as well as jasplakinolide, a drug that enhances actin polymerization, inhibited the carbachol-induced Cl^- efflux. Consequently, the activation of apical Cl^- channels by muscarinic receptor stimulation differs in signal transduction from the classical phospholipase C/protein kinase C way.     

Methyl-β-cyclodextrin inhibits Ca<Superscript>2+</Superscript>-responses induced by glutoxim and molixan in macrophages

Using Fura-2AM microfluorimetry, we have shown for the first time that methyl-β-cyclodextrin, inducing cholesterol extraction from membranes and raft disruption, significantly inhibits glutoxim- and molixan-induced Ca²⁺-responses in rat peritoneal macrophages. The results suggest that intact rafts are necessary for signaling cascade induced by glutoxim or molixan and leading to intracellular Ca²⁺ concentration increase in macrophages.     

Effect of different methods of Ca<Superscript>2+</Superscript> extraction from PSII oxygen-evolving complex on the Q<Subscript>A</Subscript><Superscript>−</Superscript> oxidation kinetics

Lumenal extrinsic proteins PsbO, PsbP, and PsbQ of photosystem II (PSII) protect the catalytic cluster Mn₄CaO₅ of oxygen-evolving complex (OEC) from the bulk solution and from soluble compounds in the surrounding medium. Extraction of PsbP and PsbQ proteins by NaCl-washing together with chelator EGTA is followed also by the depletion of Ca²⁺ cation from OEC. In this study, the effects of PsbP and PsbQ proteins, as well as Ca²⁺ extraction from OEC on the kinetics of the reduced primary electron acceptor (Q_A ^?) oxidation, have been studied by fluorescence decay kinetics measurements in PSII membrane fragments. We found that in addition to the impairment of OEC, removal of PsbP and PsbQ significantly slows the rate of electron transfer from Q_A ^? to the secondary quinone acceptor Q_B. Electron transfer from Q_A ^? to Q_B in photosystem II membranes with an occupied Q_B site was slowed down by a factor of 8. However, addition of EGTA or CaCl₂ to NaCl-washed PSII did not change the kinetics of fluorescence decay. Moreover, the kinetics of Q_A ^? oxidation by Q_B in Ca-depleted PSII membranes obtained by treatment with citrate buffer at pH 3.0 (such treatment keeps all extrinsic proteins in PSII but extracts Ca²⁺ from OEC) was not changed. The results obtained indicate that the effect of NaCl-washing on the Q_A ^? to Q_B electron transport is due to PsbP and PsbQ extrinsic proteins extraction, but not due to Ca²⁺ depletion.     

The Protective Effect of Mitochondrial ATP-Sensitive K<Superscript>+</Superscript> Channel Opener,Nicorandil, Combined With Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> Exchange Blocker KB-R7943 on Myocardial Ischemia–Reperfusion Injury in Rat

To study the protective effect of mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP channel) opener, nicorandil, combined with Na⁺/Ca²⁺ exchange blocker KB-R7943 on myocardial ischemia–reperfusion injury in isolated rat hearts; the isolated rat heart was perfused by modified Langendorff device, after 15-min balanced perfusion, 45-min ischemia (about left and right coronary perfusion flow reduced to 5% of the original irrigation flow), and 2-h reperfusion were performed. Forty Wistar rats were randomly divided into four groups: control group, nicorandil group, KB-R7943 group, and the combination of nicorandil and KB-R7943 group. After 45-min ischemia and then 2-h reperfusion, the myocardial infarct size was 34.31% in control group, 26.35% in nicorandil group, 28.74% in KB-R7943 group, and 19.23% in combination of nicorandil and KB-R7943 group. SOD activity in coronary perfusion fluid was the highest in the combination of nicorandil and KB-R7943 group, and MDA content was the lowest. In the combination drug group compared with the control group, myocardial ultrastructural injury was significantly reduced. The combination of nicorandil and KB-R7943 significantly reduced myocardial infarct size, significantly reduced myocardial ultrastructural damage, could increase coronary perfusion fluid SOD activity, and reduced MDA levels.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments for the pro-inflammatory cytokine interleukin-36α

Interleukin-36α (IL-36α) is a recently characterised member of the interleukin-1 superfamily. It is involved in the pathogenesis of inflammatory arthritis in one third of psoriasis patients. By binding of IL-36α to its receptor IL-36R via the NF-κB pathway other cytokines involved in inflammatory and apoptotic cascade are activated. The efficacy of complex formation is controlled by N-terminal processing. To obtain a more detailed view on the structure function relationship we performed a heteronuclear multidimensional NMR investigation and here report the ¹H, ¹³C, and ¹⁵N resonance assignments for the backbone and side chain nuclei of the pro-inflammatory cytokine interleukin-36α.     

Determination of the lifetime of D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack> and HD<Superscript>−</Superscript> ions

A method for determining the lifetime of unstable ions is described. The method is based on measuring the decrease in the ion beam current onto a fixed detector with increasing path length of the ion beam from the ion source to the detector. The measurements performed for D^? ₂ and HD^? molecular ions have shown that their lifetimes are 3.5 ± 0.1 and 4.4 ± 0.1 μs, respectively.     

Backbone and Ile-δ1, Leu,Val methyl <Superscript>1</Superscript>H, <Superscript>15</Superscript>N,and <Superscript>13</Superscript>C,chemical shift assignments for <Emphasis Type="Italic">Rhizopus chinensis</Emphasis> lipase

Lipase r27RCL is a 296-residue, 33 kDa monomeric enzyme with high ester hydrolysis activity, which has significant applications in the baking, paper and leather industries. The lipase gene proRCL from Rhizopus microsporus var. chinensis (also Rhizopus chinensis) CCTCC M201021 was cloned as a fusion construct C-terminal to a maltose-binding protein (MBP) tag, and expressed as MBP-proRCL in an Escherichia coli BL21 trxB (DE3) expression system with uniform ²H,¹³C,¹⁵N-enrichment and Ile-δ1, Leu, and Val ¹³CH₃ methyl labeling. The fusion protein was hydrolyzed by Kex2 protease at the recognition site Lys-Arg between residues ?29 and ?28 of the prosequence, producing the enzyme form called r27RCL. Here we report extensive backbone ¹H, ¹⁵N, and ¹³C, as well as Ile-δ1, Leu, and Val side chain methyl, NMR resonance assignments for r27RCL.     

CD14<Superscript>++</Superscript>CD16<Superscript>−</Superscript> and CD14<Superscript>+</Superscript>CD16<Superscript>+</Superscript> human monocyte adhesion to endothelial cells

Based on the difference in the CD14 and CD16 expression, two subsets of monocytes were identified in human and other mammalian blood. These subsets have different patterns of adhesion molecules and chemokine receptors that suggests the different mode of their interaction with endothelium and tissue traffic. Here, we investigated the ability of CD14⁺CD16⁺ and CD14⁺⁺CD16⁻ monocytes to adhere to endothelial cell monolayer in presence or absence of pro- and anti-inflammatory cytokines. We demonstrated that CD14⁺CD16⁺ monocytes had a higher level of adhesion to intact monolayer of endothelial cells than CD14⁺⁺CD16⁻ monocytes. Adhesion of CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ monocytes significantly increased in the presence of TNFα or its combination with other cytokines. IFNγ and IL-4 alone did not affect the adhesion of monocytes. These results show that CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ monocytes can be recruited to the inflamed endothelium, but CD14⁺CD16⁺ monocytes adhere to endothelial cells without inflammations twice as strongly as CD14⁺⁺CD16⁻ monocytes.     

The effect of MII α-conotoxin and its N-terminal derivatives on Ca<Superscript>2+</Superscript>- and Na<Superscript>+</Superscript>-signals induced by nicotine in SH-SY5Y neuroblastoma cells

Nicotinic acetylcholine receptors (nAChRs) are involved in the regulation of intracellular Ca²⁺-dependent processes both in normal and pathological states. α-Conotoxins from the venom of Conus marine mollusks are a valuable tool for the investigation of the pharmacological action and functional role of nAChRs. Analogues of α-conotoxin MII labeled by Bolton-Hunter reagent (BH-MII) or fluorescein isothiocyanate (FITC-MII) on the N-terminal glycine residue have been synthesized in the present work. Fluorescence microscopy studies of SH-SY5Y neuroblastoma cells loaded with Ca²⁺ indicator Fura-2, or by both Ca²⁺ indicator Fluo-4 and Na⁺ indicator SBFI, were used to test the effect of MII modification on its ability to block Ca²⁺ and Na⁺ signals induced by nicotine. Measurements in SH-SY5Y cells showed that kinetics of the increase and recovery of the concentration of free Ca²⁺ ([Ca²⁺] _i ) upon nicotine application and washout was different from that for free Na⁺ ([Na⁺] _i ), this being evidence of differences in the mechanism of Ca²⁺ and Na⁺ homeostasis regulation. MII suppressed the nicotine-induced increase of [Ca²⁺] _i and [Na⁺] _i in a concentration-dependent manner. An additional tyrosine residue added to the N-terminus of one of the MII derivatives caused a significant decrease in the inhibitory action of MII; this decrease was even more pronounced when a large FITC label was introduced into MII. The BH-MII derivative had an inhibitory effect similar to that of unmodified α-conotoxin. MII and its iodinated derivatives are promising tools for radioligand assays.     

Structures and stability of N<Subscript>13</Subscript><Superscript>+</Superscript> and N<Subscript>13</Subscript><Superscript>−</Superscript> clusters

The structures and stabilities of eleven N₁₃ ⁺ and N₁₃ ^– isomers have been investigated with second-order Møller–Plesset (MP2) and density functional theory (DFT) methods. Five N₁₃ ⁺ isomers and six N₁₃ ^– isomers are all reasonable local minima on their potential energy hypersurfaces. The most stable N₁₃ ⁺ cation is structure C-2 with C_2v symmetry, which contains a pentazole ring and two N₄ open chains. It is different from those of the N₇ ⁺ and N₉ ⁺ clusters, but similar to the N₁₁ ⁺ cluster. Meanwhile, the most stable N₁₃ ^– structure A-2 is composed of a pentazole ring and a six-membered ring connected by two nitrogen atoms. It is not only different from those of the N₇ ^– and N₉ ^– clusters, but also from the N₁₁ ^– cluster. The decomposition pathways of structures C-2 and A-2 were investigated at the B3LYP/(aug)-cc-pVDZ level. From the barrier heights of the structures C-2 and A-2 decomposition processes, it is suggested that C-2 is difficult to observe experimentally and A-2 may be observed as a short-lived species. Figure Optimized geometrical parameters of N₁₃ ⁺ isomer C-2