C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states

Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments’ interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506–793) and CaDH2 (residues 683–767) on the structure of actin–tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin–tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle ‘ghost’ fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)–phalloidin or fluorescein-5′-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters Φ_A, Φ_E, Θ_1/2 and N were calculated. Φ_A and Φ_E are angles between the fiber axis and the absorption and emission dipoles, respectively; Θ_1/2 is the angle between the F-actin filament axis and the fiber axis; N is the relative number of randomly oriented fluorophores. Actin–tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the ‘on’ conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca²⁺) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca²⁺) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between ‘off’ (caldesmon and CaDH1) and ‘on’ (S-1 and CaDH2) states in a manner analogous to troponin.     

Binding of divalent copper and calcium ions to poly I

The effect ot Cu²⁺ and Ca²⁺ ions, on the ultraviolet differential (UVD) spectra of single-stranded poly I was studied and the coordination (Δε_b) and conformation (Δε_c) conponents of the spectra calculated The comparison of Δε_b and the UVD spectrum of protonated IMP leads to the conclusion that N(7) ot inosine-5'-monophosphate (IMP) is a coordinating site tor Ca²⁺ and Cu²⁺ ions on the polymer bases. The binding ot Ca²⁺ and Cu²⁺ ions causes differently directed displacements of the four absorption bands of poly I, which are observed in the wavenumber range (50-34) × 10³ cm⁻¹ The calculation of concentration dependencies tor the association constants (K“) ot Ca²⁺ and Cu²⁺ ions binding to poly I bases shows that the binding is cooperative The K“ values for the poly I + Ca²⁺ complex are two orders of magnitude lower than those for the poly 1 + Cu²⁺ complex At low ion concentrations, binding to the poly I phosphates predominates and increases the degree of the polynucleotide helicity. At higher concentrations the spectra are mainly affected by the ion binding to bases, which results in melting of the helical parts of poly I At Ca²⁺ concentrations exceeding 10⁻³ M light-scattering aggregates are formed. The degree of monomer order in them is close to that observed in multistranded helices of poly I     

滨海滩涂地带乌哺鸡竹和淡竹离子含量变化及其与生长及光合作用的关系

在沿海滩涂防护林带低盐区(0.1%)、中盐区(0.2%)和重盐区(0.4%) 3个盐分梯度下,研究了栽植10年的乌哺鸡竹和淡竹Na⁺、K⁺、Ca²⁺、Mg²⁺含量变化及其与生长和光合作用的相关关系.结果表明: 从低盐区到重盐区,乌哺鸡竹的立竹密度和地径分别下降30.4%和28.8%,降幅低于淡竹的44.1%和31.2%;两竹种单株生物量下降,地上器官生物量降幅均显著高于地下器官;乌哺鸡竹和淡竹净光合速率(P_n)和PSⅡ最大光化学效率(F_v/F_m)分别下降57.6%和67.7%、6.1%和7.4%,乌哺鸡竹耐盐能力比淡竹强.随着土壤含盐量的增大,乌哺鸡竹和淡竹各器官Na⁺含量逐渐增加,K⁺、Ca²⁺、Mg²⁺含量逐渐降低.两竹种根Na⁺积累较多,而地上部分K⁺含量较高.盐胁迫环境导致乌哺鸡竹根Ca²⁺含量与淡竹叶片Mg²⁺含量明显下降.两竹种的生物量、P_n、F_v/F_m与Na⁺含量呈显著负相关,与K⁺、Ca²⁺含量呈显著正相关.     

Ca-dependence of diastolic properties of cardiac sarcomeres: involvement of titin

The stiffness of the sarcomeres was studied during the diastolic interval of 18 stimulated (0.5 Hz) cardiac trabeculae of rat (pH 7.4; temperature = 25°C). Sarcomere length (SL) and force (F) were measured using, respectively, laser diffraction techniques (resolution: 4 nm) and a silicon strain gauge (resolution: 0.63 μN). Sinusoidal perturbations (frequency = 500 Hz) were imposed to the length of the preparation. The stiffness was evaluated from the corresponding F and SL sinusoids by analysis of both signals together either in the time domain or in the frequency domain. A short burst (duration = 30 ms) of sinusoidal perturbations was repeated at 5 predetermined times during diastole providing 5 measurements of stiffness during the time interval separating two twitches. These measurements revealed that stiffness increases by 30% during diastole, while a simultaneous expansion of the sarcomeres (amplitude = 10-60 nm) was detected. Measurements of the fluorescence of fura-2 under the same conditions revealed a continuous exponential decline of [Ca²⁺]_i from 210 to 90 nM (constant of time 300 ms) during diastole. In order to test the possibility that the increase of sarcomere stiffness and the decline of [Ca²⁺]_i were coupled during diastole of intact trabeculae, we studied the effect of different free Ca²⁺-concentrations ([Ca²⁺]) between 1 and 430 nM on sarcomere stiffness in rat cardiac trabeculae skinned by saponin (n = 17). Stiffness was studied using 500 Hz sinusoidal perturbations of muscle length (ML). We found that, below 70 nM, the stiffness was independent of [Ca²⁺]; between 70 and 200 nM, the stiffness declined with increase of [Ca²⁺]; above 200 nM, the stiffness increased steeply with [Ca²⁺]. The data fitted accurately to the sum of two sigmoids (Hill functions): (1) at [Ca²⁺] < 200 nM the stiffness decreased with [Ca²⁺] (EC₅₀ = 160 ± 13 nM; n = −2.6±0.7) and (2) at [Ca²⁺] > 200 nM, stiffness increased with [Ca²⁺] (EC₅₀ = 3.4±0.3 μM; n = 2.1±0.2) due to attachment of cross-bridges. From these results, it was possible to reproduce accurately the time course of diastolic stiffness observed in intact trabeculae and to predict the effect on stiffness of a spontaneous elevation of the diastolic [Ca²⁺]. Identical stiffness measurements were performed in 4 skinned preparations exposed to a cloned fragment of titin (Ti I-II) which has been shown to exhibit a strong interaction with F-actin in vitro. It was anticipated that Ti I-II would compete with endogenous titin for the same binding site on actin in the I-band. Below 200 nM, Ti I-II (2 μM) eliminated the Ca²⁺-dependence of stiffness. These results are consistent with the hypothesis that the Ca²⁺-sensitivity of the sarcomeres at [Ca²⁺] < 200 nM, i.e. where the myocytes in intact muscle operate during diastole, involves an association between titin molecules and the thin filament.     

'Ca(2+)-induced Ca(2+) entry' or how the L-type Ca(2+) channel remodels its own signalling pathway in cardiac cells

The adjustment of Ca²⁺ entry in cardiac cells is critical to the generation of the force necessary for the myocardium to meet the physiological needs of the body. In this review, we present the concept that Ca²⁺ can promote its own entry through Ca²⁺ channels by different mechanisms. We refer to it under the general term of ‘Ca²⁺-induced Ca²⁺ entry’ (CICE). We review short-term mechanisms (usually termed facilitation) that involve a stimulating effect of Ca²⁺ on the L-type Ca²⁺ current (I_Ca-L) amplitude (positive staircase) or a lessening of Ca²⁺-dependent inactivation of I_Ca-L. This latter effect is related to the amount of Ca²⁺ released by ryanodine receptors (RyR2) of the sarcoplasmic reticulum (SR). Both effects are involved in the control of action potential (AP) duration. We also describe a long-term mechanism based on Ca²⁺-dependent down-regulation of the Kv4.2 gene controlling functional expression of the repolarizing transient outward K⁺ current (I_to) and, thereby, AP duration. This mechanism, which might occur very early during the onset of hypertrophy, enhances Ca²⁺ entry by maintaining Ca²⁺ channel activation during prolonged AP. Both Ca²⁺-dependent facilitation and Ca²⁺-dependent down-regulation of I_to expression favour AP prolongation and, thereby, promote sustained voltage-gated Ca²⁺ entry used to enhance excitation–contraction (EC) coupling (with no change in the density of Ca²⁺ channels per se). These self-maintaining mechanisms of Ca²⁺ entry have significant functions in remodelling Ca²⁺ signalling during the cardiac AP. They might support a prominent role of Ca²⁺ channels in the establishment and progression of abnormal Ca²⁺ signalling during cardiac hypertrophy and congestive heart failure.     

The function of Ca channel subtypes in exocytotic secretion: new perspectives from synaptic and non-synaptic release

By mediating the Ca²⁺ influx that triggers exocytotic fusion, Ca²⁺ channels play a central role in a wide range of secretory processes. Ca²⁺ channels consist of a complex of protein subunits, including an ₁ subunit that constitutes the voltage-dependent Ca²⁺-selective membrane pore, and a group of auxiliary subunits, including β, γ, and ₂–δ subunits, which modulate channel properties such as inactivation and channel targeting. Subtypes of Ca²⁺ channels are constituted by different combinations of ₁ subunits (of which 10 have been identified) and auxiliary subunits, particularly β (of which 4 have been identified). Activity-secretion coupling is determined not only by the biophysical properties of the channels involved, but also by the relationship between channels and the exocytotic apparatus, which may differ between fast and slow types of secretion. Colocalization of Ca²⁺ channels at sites of fast release may depend on biochemical interactions between channels and exocytotic proteins. The aim of this article is to review recent work on Ca²⁺ channel structure and function in exocytotic secretion. We discuss Ca²⁺ channel involvement in selected types of secretion, including central neurotransmission, endocrine and neuroendocrine secretion, and transmission at graded potential synapses. Several different Ca²⁺ channel subtypes are involved in these types of secretion, and their function is likely to involve a variety of relationships with the exocytotic apparatus. Elucidating the relationship between Ca²⁺ channel structure and function is central to our understanding of the fundamental process of exocytotic secretion.     

Characterisation and selectivity of divalent metal ions binding by citrus and sugar-beet pectins

A scale of selectivity for the binding of calcium and some heavy metal ions by citrus and sugar-beet pectins was set up by pH-measurements. The same order of selectivity was found for the two pectins, decreasing as follows: Cu²⁺ Pb²⁺ Zn²⁺ > Cd²⁺ Ni²⁺ ≥ Ca²⁺. Binding isotherms for Ca²⁺, Cu²⁺, Ni²⁺, Pb²⁺ and Zn²⁺ ions have shown a greater binding level when the ionic strength decreased and when the pectin concentration increased in the presence of 0.1 M NaNO₃. By comparing binding isotherms, the same order of selectivity was found as by pH-studies. Scatchard plots and Hill index evaluation showed for all ions and all pectins anticooperative interactions in water. In the presence of 0.1 M NaNO₃, citrus pectins displayed cooperative interactions for all metal ions. In contrast, for sugar-beet pectins, cooperative interactions only occured with Cu²⁺ and Pb²⁺. With Ca²⁺, Ni²⁺ and Zn²⁺ sugar-beet pectins displayed Scatchard plots which could not be distinguished from an anticooperative binding. This difference of behaviour could be related to the presence of acetyl groups decreasing the affinity of Me²⁺ for sugar-beet pectins.     

The Ca 2+ leak paradox and rogue ryanodine receptors: SR Ca 2+ efflux theory and practice

Ca²⁺ efflux from the sarcoplasmic reticulum (SR) is routed primarily through SR Ca²⁺ release channels (ryanodine receptors, RyRs). When clusters of RyRs are activated by trigger Ca²⁺ influx through L-type Ca²⁺ channels (dihydropyridine receptors, DHPR), Ca²⁺ sparks are observed. Close spatial coupling between DHPRs and RyR clusters and the relative insensitivity of RyRs to be triggered by Ca²⁺ together ensure the stability of this positive-feedback system of Ca²⁺ amplification. Despite evidence from single channel RyR gating experiments that phosphorylation of RyRs by protein kinase A (PKA) or calcium-calmodulin dependent protein kinase II (CAMK II) causes an increase in the sensitivity of the RyR to be triggered by [Ca²⁺]_i there is little clear evidence to date showing an increase in Ca²⁺ spark rate. Indeed, there is some evidence that the SR Ca²⁺ content may be decreased in hyperadrenergic disease states. The question is whether or not these observations are compatible with each other and with the development of arrhythmogenic extrasystoles that can occur under these conditions. Furthermore, the appearance of an increase in the SR Ca²⁺ “leak” under these conditions is perplexing. These and related complexities are analyzed and discussed in this report. Using simple mathematical modeling discussed in the context of recent experimental findings, a possible resolution to this paradox is proposed. The resolution depends upon two features of SR function that have not been confirmed directly but are broadly consistent with several lines of indirect evidence: (1) the existence of unclustered or “rogue” RyRs that may respond differently to local [Ca²⁺]_i in diastole and during the [Ca²⁺]_i transient; and (2) a decrease in cooperative or coupled gating between clustered RyRs in response to physiologic phosphorylation or hyper-phosphorylation of RyRs in disease states such as heart failure. Taken together, these two features may provide a framework that allows for an improved understanding of cardiac Ca²⁺ signaling.     

An effect of trypsin on the actin-myosin interaction

Three different lines of evidence were obtained to show that trypsin modifies the actin-myosin interaction: (I) At trypsin to actomyosin or myosin ratios between 1 to 300 and 1 to 500, 30 min of trypsin treatment causes an 8-fold increase in the Ca²⁺-modified ITPase activity of actomyosin but has no effect on the Ca²⁺-modified ITPase activity of myosin alone. At these same trypsin to actomyosin ratios, the Mg²⁺ + Ca²⁺-modified ATPase activity increases by 10–30% during the first 1–2 min of trypsin digestion, and then decreases rapidly to less than 20% of its original activity after 60 min of digestion. Trypsin has no effect on the Mg²⁺ + Ca²⁺-modified ATPase activity of pure myosin. (2) The rate of turbidity response of reconstituted actomyosin suspensions is first increased and then decreased by trypsin treatment. At trypsin to actomyosin ratios of 1 to 3000, rate of turbidity response is maximal after 5 min of trypsin digestion and then decreases; after 60 min, the turbidity response is much slower than the response of the control actomyosin. (3) Supercontracted sarcomeres, shortened to less than 50% of their initial length, are lengthened to 70% of their initial length by 4 min of trypsin treatment. Myosin B from such lengthened sarcomeres has less than 35% of its myosin converted to light meromyosin and heavy meromyosin.

These results show that trypsin modifies the actin-myosin interaction in at least two ways: (1) a very rapid initial modification that increases the Mg²⁺ + Ca²⁺-modified ATPase activity and the rate of turbidity increase, and (2) a slower modification that decreases the Mg²⁺ + Ca²⁺-modified ATPase activity and rate of turbidity response, and that lengthens contracted sarcomeres. Tryptic modification is not due to cleavage of myosin to light and heavy meromyosin. Since tryptic modification occurs more rapidly than conversion of myosin to light and heavy meromyosin, all heavy meromyosin preparations will be modified.     

Arrhythmogenic Ca(2+) release from cardiac myofilaments

We investigated the initiation of Ca²⁺waves underlying triggered propagated contractions (TPCs) occurring in rat cardiac trabeculae under conditions that simulate the functional non-uniformity caused by mechanical or ischemic local damage of the myocardium. A mechanical discontinuity along the trabeculae was created by exposing the preparation to a small constant flow jet of solution with a composition that reduces excitation–contraction coupling in myocytes within that segment. Force was measured and sarcomere length as well as [Ca²⁺]_i were measured regionally. When the jet-contained Caffeine, BDM or Low-[Ca²⁺], muscle-twitch force decreased and the sarcomeres in the exposed segment were stretched by shortening of the normal regions outside the jet. During relaxation the sarcomeres in the exposed segment shortened rapidly. Short trains of stimulation at 2.5 Hz reproducibly caused Ca²⁺-waves to rise from the borders exposed to the jet. Ca²⁺-waves started during force relaxation of the last stimulated twitch and propagated into segments both inside and outside of the jet. Arrhythmias, in the form of non-driven rhythmic activity, were triggered when the amplitude of the Ca²⁺-wave increased by raising [Ca²⁺]_o. The arrhythmias disappeared when the muscle uniformity was restored by turning the jet off. We have used the four state model of the cardiac cross bridge (Xb) with feedback of force development to Ca²⁺ binding by Troponin-C (TnC) and observed that the force–Ca²⁺ relationship as well as the force–sarcomere length relationship and the time course of the force and Ca²⁺ transients in cardiac muscle can be reproduced faithfully by a single effect of force on deformation of the TnC·Ca complex and thereby on the dissociation rate of Ca²⁺. Importantly, this feedback predicts that rapid decline of force in the activated sarcomere causes release of Ca²⁺ from TnC.Ca²⁺,which is sufficient to initiate arrhythmogenic Ca²⁺ release from the sarcoplasmic reticulum. These results show that non-uniform contraction can cause Ca²⁺-waves underlying TPCs, and suggest that Ca²⁺ dissociated from myofilaments plays an important role in the initiation of arrhythmogenic Ca²⁺-waves.     

Interactions between dihydropyridine receptors and ryanodine receptors in striated muscle

Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca²⁺ channels in the surface/transverse tubular membrane and ryanodine receptor Ca²⁺ release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation–contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein–protein interaction remain unknown. The trigger for Ca²⁺ release through ryanodine receptors in cardiac muscle is a Ca²⁺ influx through the L-type Ca²⁺ channel. The Ca²⁺ entering through the surface membrane Ca²⁺ channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated.     

Reciprocal coupling between troponin C and myosin crossbridge attachment

The attachment of cycling myosin crossbridges to actin and the resultant muscle contraction are regulated in skeletal muscle by the binding of Ca2+ to the amino-terminal, regulatory sites of the troponin C (TnC) subunit of the thin filament protein troponin. Conversely, the attachment of crossbridges to actin has been shown to alter the affinity of TnC for Ca2+. In this study, fluorescently labeled TnC incorporated into reconstituted thin filaments was used to investigate the relationship between crossbridge attachment to actin and structural changes in the amino-terminal region of TnC. Fluorescence intensity changes were measured under the following conditions: saturating [Ca2+] in the absence of crossbridges, rigor crossbridge attachment in the presence and absence of Ca2+, and cycling crossbridge attachment. The percent of heavy meromyosin crossbridges associated with the thin filaments under these conditions was also determined. The results show that, in addition to the binding of Ca2+ to TnC, the attachment of both rigor and cycling crossbridges to actin alters the structure of TnC near the regulatory, Ca2+-specific sites of the molecule. A differential coupling between weakly versus strongly bound crossbridge states and TnC structure was detected, suggesting a possible differential regulation of these states by conformational changes in TnC. These findings illustrate a reciprocal coupling, via thin filament protein interactions, between structural changes in TnC and the attachment of myosin crossbridges to actin, such that each can influence the other, and indicate that TnC is not simply an on-off switch but may exist in a number of different conformations.     

镁转运体MGT7参与拟南芥对高钙环境的适应

高Ca²⁺环境对许多植物的生长不利, 因此研究植物对高Ca²⁺环境的适应机制非常重要。研究发现, 拟南芥(Ara- bidopsis thaliana)镁转运体MGT7功能缺失突变体mgt7-1和mgt7-2具有高Ca²⁺敏感表型: 在高Ca²⁺培养基上, 相对于野生型Col-0, 突变体叶鲜重显著下降, 但根长无显著差异。高Ca²⁺对MGT7启动子活性和包括MGT7在内的镁转运体基因表达无显著调节作用。Col-0与mgt7突变体之间, 在外加Ca²⁺诱导细胞质Ca²⁺瞬时升高和Ca²⁺含量方面无显著差异; 但是, 在正常和高Ca²⁺培养基上, mgt7突变体的Mg含量均显著低于Col-0。高Ca²⁺显著抑制Col-0和mgt7突变体内Mg的积累。因此我们假设, mgt7突变体的高Ca²⁺敏感表型是由于其体内Mg含量下降导致的。进一步的研究证实, 只有增加培养基中Mg²⁺的含量, 而不是N、P、K和S, 才可以使突变体的高Ca²⁺敏感表型得到恢复。     

Two bombesin analogues discriminate between neuromedin B- and bombesin-induced calcium flux in a lung cancer cell line

We examined the profile of two bombesin (BN) antagonists, (CH₃)₂CHCO-His-Trp-Ala-Val- -Ala-His-Leu-NHCH₃] (ICI 216140) and [ -Phe⁶,des-Met¹⁴]BN(6–14)ethylamide (DPDM-BN EA), against neuromedin B-induced Ca²⁺ mobilization in the small cell lung cancer (SCLC) line NCI-H345. Neuromedin B (NMB), a BN-like peptide sharing sequence homology with ranatensin, elicited a concentration-dependent Ca²⁺ release (in part) from intracellular stores. Sequential addition of NMB attenuated Ca²⁺ mobilization. Desensitization occurred between BN and NMB; depletion of intracellular Ca²⁺ is a likely mechanism because thapsigargin stimulated Ca²⁺ release after a maximally desensitizing dose of NMB. ICI 216140 and DPDM-BN EA competitively inhibited BN-induced Ca²⁺ transients. In contrast, these compounds antagonized NMB-stimulated Ca²⁺ transients in a noncompetitive manner. The pharmacological profiles obtained support receptor heterogeneity for BN-like peptides on this SCLC line, underscoring the need for thorough examination of dose-response relationships when investigating effects of BN analogues on intact cells.     

Calcium ion control of platelet thrombosthenin ATPase activity

This study demonstrates that Ca²⁺ regulates thrombosthenin ATPase activity, likening the control of platelet contraction to that of cardiac and skeletal muscle. Thrombosthenin, the platelet contractile protein, was isolated by repeated low ionic strength and isoelectric precipitation. Thrombosthenin superprecipitation and ATPase activity were measured in 10⁻⁴ M CaCl₂ (high ionized Ca²⁺) and 0.25 mM ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) (low ionized Ca²⁺). In both high and low Ca²⁺, superprecipitation, measured as an increase in turbidity, ocurred shortly after addition of ATP. ATP hydrolysis by thrombosthenin, which proceeded linearly for several hours, was greater in high Ca²⁺ (approx. 2.3 nmoles·mg⁻¹·min⁻¹) than in low Ca²⁺ (approx. 1.8 nmoles·mg⁻¹·min⁻¹). This difference, when analyzed by the Student's t-test for paired samples was highly significant (P < 0.001). Thrombosthenin ATPase activity was not significantly altered by azide, an inhibitor of mitochondrial ATPase, nor by ouabain, an inhibitor of (Na⁺ + K⁺)-activated ATPase. The dependence of thrombosthenin activation on ionized Ca²⁺, measured with the use of CaEGTA buffers, was studied. The Ca²⁺-dependent portion of thrombosthenin ATPase was half maximal at 4.5·10⁻⁷ M Ca²⁺. This corresponds to an apparent binding constant of 2.2·10⁶ M⁻¹, a value that is comparable to that of skeletal and cardiac muscle. These data suggest that a Ca²⁺ control mechanism similar to that of the troponin-tropomyosin complex of muscle exists in the platelet.     

Effect of polymeric cosolutes on calcium pectinate gelation. Part 3. Gum arabic and overview

Addition of gum arabic (average M_r≈450 kDa; 0.5–2.0 wt%) to solutions of low methoxy pectin (DE 31; 2.0 wt%; pH≈2.9–3.0) with stoichiometric Ca²⁺ caused massive increases in G′ and G″ in the pre-gel state at 90 °C (attributed to segregative interactions promoting formation of calcium-mediated ‘egg-box’ junctions between pectin chains) but had little effect on the gels formed on cooling to 5 °C. This is in marked contrast to the behaviour of other polymeric cosolutes studied in the investigations reported in the two preceding papers, which caused large reductions in gel moduli (attributed to excessive association of calcium pectinate into large aggregated bundles); the difference is tentatively ascribed to strengthening of the calcium pectinate network by divalent counterions to the uronate residues in gum arabic. When the complication of cation exchange was eliminated by extensive dialysis of gum arabic against 100 mM Na⁺ and use of the final dialysate in preparation of mixtures with calcium pectinate, massive increases in G′ and G″ at high temperature were again observed, but with accompanying reductions in moduli at low temperature, which, at gum arabic concentrations above 1.0 wt%, arose from collapse of the developing calcium pectinate network during cooling. The tentative conclusion from this work, and from the two preceding papers, is that enthalpically unfavourable (segregative) interactions between low methoxy pectin and polymeric cosolutes can be relieved in two ways: (i) Ca²⁺-mediated self-association of pectin into compact ordered assemblies which occupy less of the total volume, and (ii) conformational rearrangement of the cosolute molecules to minimise segmental interactions with pectin; conformational rearrangement is inhibited by chain stiffness and by branching; thus polymeric cosolute molecules of limited flexibility are more effective in promoting self-association of pectin than more flexible molecules of comparable size, and branched molecules are more effective than linear chains of comparable stiffness.     

Effect of lignans isolated from Hernandia nymphaeifolia on estrogenic compounds-induced calcium mobilization in human neutrophils

The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17β-estradiol, tamoxifen and clomiphene)-induced Ca²⁺ mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca²⁺–containing medium, the lignans (50–100 μM) inhibited 10 μM 17β-estradiol- and 5 μM tamoxifen-induced increases in intracellular free Ca²⁺ levels ([Ca²⁺]_i) without changing 25 μM clomiphene-induced [Ca²⁺]_i increase. 17β-estradiol and tamoxifen increased [Ca²⁺]_i by causing Ca²⁺ influx and Ca²⁺ release because their responses were partly reduced by removing extracellular Ca²⁺. In contrast, clomiphene solely induced Ca²⁺ release. The effect of the lignans on these two Ca²⁺ movement pathways underlying 17β-estradiol- and tamoxifen-induced [Ca²⁺]_i increases was explored. All the lignans (50–100 μM) inhibited 10 μM 17β-estradiol-and 5 μM tamoxifen-induced Ca²⁺ release, and 17β-estradiol-induced Ca²⁺ influx. However, only 100 μM epi-aschantin was able to reduce tamoxifen-induced Ca²⁺ influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca²⁺ signaling in human neutrophils in a multiple manner.     

Contrasting effects of PACAP and carbachol on [Ca]i and inositol phosphates in human neuroblastoma NB-OK-1 cells

The effects of PACAPs on [Ca²⁺]_i were compared to those of carbachol in human neuroblastoma NB-OK-1 cells. PACAP(1–27) and PACAP(1–38) increased [Ca²⁺]_i in a biphasic manner: a transient rise and a secondary plateau. The transient phase reflected the mobilization of [Ca²⁺]_i pool(s) via the inositol phosphate pathway. The modest sustained plateau required extracellular Ca²⁺. Carbachol also increased [Ca²⁺]_i in a biphasic manner, but it mobilized intracellular Ca²⁺ pool(s) with a higher efficacy than PACAPs, then greatly increased Ca²⁺ entry, this being accompanied by a more marked and prolonged elevation of IP₃ and IP₄ than with PACAPs. It is likely that cAMP-mediated phosphorylations due to PACAPs facilitated desensitization at the PACAP receptor-phospholipase C level, so that there was less Ca²⁺ handling through PACAP receptors than with muscarinic M₁ receptors.