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.     

Ca2+ sparks in skeletal muscle

The study of Ca²⁺ sparks has led to extensive new information regarding the gating of the Ca²⁺ release channels underlying these events in skeletal, cardiac and smooth muscle cells, as well as the possible roles of these local Ca²⁺ release events in muscle function. Here we review basic procedures for studying Ca²⁺sparks in skeletal muscle, primarily from frog, as well as the basic results concerning the properties of these events, their pattern and frequency of occurrence during fiber depolarization and the mechanisms underlying their termination. Finally, we also consider the contribution of different ryanodine receptor (RyR) isoforms to Ca²⁺ sparks and the number of RyR Ca²⁺ release channels that may contribute to the generation of a Ca²⁺ spark. Over the decade since their discovery, Ca²⁺ sparks have provided a wealth of information concerning the function of Ca²⁺ release channels within their intracellular environment.     

Disposition of water in chloroplast coupling factor 1 (CF1) A neutron scattering analysis

Small-angle neutron scattering experiments were performed in dilute aqueous solutions of chloroplast F₁-ATPase. By contrast variation in ¹H₂O/²H₂O mixtures and when using different concentrations of glycerol in ²H₂O, structural information on the spatial distribution of dry protein and water was obtained. The maximum distance within latent and active CF₁ was 12 nm. the shape of CF₁ was globular. The total volume of CF₁ was 900 nm³, and its dry volume (excluding the volume of one water molecule per two exchangeable hydrogen atoms) was 400 nm³. A volume of 670 nm³ was inaccessible to glycerol at low glycerol concentrations (less than 25%). At higher concentrations (up to 50%) a volume of 460 nm³ was excluded to glycerol. Within the resolution of our experiment (1.6 nm) there was no evidence for particular water-rich regions or of secluded water spaces or any particular places for glycerol exchange. Upon thiol activation of the latent enzyme only small changes in structure were detectable just at the limits of the experimental error. They suggest an enhancement of the surface roughness.     

The effect of Ca on the sulfhydryl reactivity of troponin: Evidence for a Ca-induced conformational charge

Troponin prepared from rabbit skeletal muscle in the presence of dithiothreitol (SH-troponin) was found to have a sulfhydryl content of about 4 moles/1 × 10⁵ g in the presence of a Ca²⁺-chelating agent. The addition of physiological concentrations of Ca²⁺ reduced the reactive sulfhydryl content to 2.0–2.5 moles/1 × 10⁵ g. These sulfhydryl groups are evidently not direct participants in the inhibition of actomyosin superprecipitation, since treatment with N-ethylmaleimide had no effect on the Ca²⁺-sensitizing activity of SH-troponin.

Troponin prepared in the absence of dithiothreitol (S-S-troponin) showed a significant reduction in Ca²⁺-sensitizing activity, relative to SH-troponin. The sulfhydryl groups of S-S-troponin, approx. 2 moles/1 × 10⁵ g were not appreciably affected by Ca²⁺.

It is postulated that the Ca²⁺-sensitive sulfhydryl groups exist at a site which is essential for the regulatory function of troponin and which undergoes a conformational change upon the binding of Ca²⁺.     

Adipokinetic hormone-induced influx of extracellular calcium into insect fat body cells is mediated through depletion of intracellular calcium stores

Adipokinetic hormone I (AKH I) needs extracellular Ca²⁺ for its activating action on glycogen phosphorylase in locust fat body in vitro. TMB-8 reduces this AKH effect significantly, indicating that for a major part, hormone action also requires the mobilization of Ca²⁺ from intracellular stores. Using ⁴⁵Ca²⁺, AKH was shown to stimulate both the influx and the efflux of Ca²⁺. Thapsigargin also enhances the influx of extracellular Ca²⁺ into the fat body cells, indicating that the stimulating effect of AKH on Ca²⁺ influx may be mediated through depletion of intracellular Ca²⁺ stores as well. AKH is known to enhance cAMP levels in locust fat body. We show that elevation of cAMP with forskolin or theophylline leads to activation of glycogen phosphorylase, both in the presence and in the absence of extracellular Ca²⁺. The present data are discussed in an attempt to elucidate further the mechanism underlying transduction of the hormonal signal in locust fat body.     

Effect of H2O on energy-dependent oxidoreduction of cytochrome b

The rate of oxidation of the long-wavelength cytochrome b (b₅₆₆) on addition of oligomycin to ATP-supplemented anaerobic rat-liver mitochondria was strongly inhibited when ²H₂O was substituted for medium water. This effect was dependent on added substrate, was reversed by uncoupling agents, and was absent in sub-mitochondrial particles. At the same phosphate potential, b₅₆₆ reduction was favoured in ²H₂O in comparison with H₂O medium. The redox state of b₅₆₆ may be controlled by the phosphate potential via an intramembrane acid-base equilibrium which is shifted by ²H₂O.     

An investigation of water splitting in the Kok scheme of photosynthetic oxygen evolution

The involvement of OH bond breaking in the 4 dark reactions of the Kok scheme of photosynthetic oxygen evolution was investigated using Chlorella and spinach chloroplasts. When the photosynthetic material was suspended in a ²H₂O based medium, the reaction rates in all 4 cases were only slightly reduced as compared to the rates observed in an H₂O based medium. This was evidence that these rate processes were probably not limited by the breaking of an OH bond. Observations were also made on the yields of O₂ from dark adapted Chlorella subjected to a sequence of brief saturating light flashes. The oscillating flash yield sequence observed with algae suspended in ²H₂O showed greater damping of the oscillations than when the algae were suspended in H₂O. A computer fit of the Kok model to these results revealed a slightly higher proportion of misses, (i.e. absorbed quanta that do not drive photochemistry) in the ²H₂O case.     

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.     

Muscarinic receptor-mediated intracellular Ca mobilization in embryonic chick heart cells

Activation of muscarinic receptors of heart cells elevates the intracellular Ca²⁺ concentration. The increase is considered to be due to influx of extracellular Ca²⁺. We show that intracellular Ca²⁺ mobilization is involved. Cell suspensions prepared from hearts of 6-day-old chick embryos were loaded with the fluorescent Ca²⁺ chelator chlortetracycline. Muscarinic stimulation induces a dose-dependent fluorescence decrease (ED₅₀=2.6 × 10⁻⁶ M) indicating intracellular Ca²⁺ mobilization.     

Dihydropyridine-sensitive Ca channel in aneurally cultured human muscles Relationship between high-affinity binding site and inhibition of calcium uptake

Dihydropyridine-sensitive Ca²⁺ channels and the relationship between binding of dihydropyridine derivatives and depolarization-induced Ca²⁺ uptake have been studied in aneurally cultured human muscle. Analysis of the equilibrium binding of the 1,4-dihydropyridine derivative (+)-PN200-110 revealed a single high-affinity binding site with a K_d of 0.15±0.05 nM and a B_max of 87±12 fmol/mg protein. Inhibition of (+)-[³H]PN200-110 binding by nitrendipine revealed a K_i of 0.8 nM for the nitrendipine-receptor complex. Depolarization of cultured human muscle achieved by elevating the K⁺ concentration increased the uptake ⁴⁵Ca²⁺ which was inhibited by nitrendipine with an IC₅₀ of 1.1 nM. This study demonstrates that aneurally cultured human muscle has dihydropyridine-sensitive voltage-dependent Ca²⁺ channels which are functional when the fibers are depolarized.     

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.     

Acid phosphatases bind to the main high density lipoprotein apolipoprotein A-I

Light-dependent Ca²⁺ efflux via the Ca²⁺/H⁺ antiport in the photosynthetic purple sulfur bacterium Chromatium vinosum was inhibited by three phenothiazines: chlorpromazine; trifluoperazine and phenothiazine. The inhibitors had no effect on Ca²⁺ uptake by C. vinosum in the dark nor any effect on the light-dependent efflux of either Na⁺ or Tl⁺ catalyzed, respectively, by the C. vinosum Na⁺/H⁺ or K⁺/H⁺ antiports. Ruthenium red and LaCl₃, neither of which inhibited light-dependent Ca²⁺ efflux in C. vinosum, markedly inhibited Ca²⁺ uptake in the dark by C. vinosum cells. Ruthenium red had no effect on the uptake of either Na⁺or the K⁺ analog T1⁺ by C. vinosum cells in the dark. These results have been interpreted in terms of two separate Ca²⁺ transport systems in C. vinosum: (i) a phenothiazine-sensitive and ruthenium red, La³⁺-insensitive Ca²⁺/H⁺ antiport responsible for Ca²⁺ efflux in the light; and (ii) a ruthenium red and La³⁺-sensitive but phenothiazine-insensitive Ca²⁺ uptake system.     

Tight Junction Dynamics in the Frog Urinary Bladder

In a previous study in frog skin (Castro et al., J. Memb. Biol. 134:15-29, 1993), it was shown that TJs experimentally disrupted by a selective deposition of BaSO₄ could be re-sealed upon addition of Ca²⁺to the apical solution; in the absence of apical Ca²⁺, the normal Ca²⁺ activity of the Na₂SO₄-Ringer's bathing the basolateral side was not able to induce TJ resealing. We now show that apical Ca²⁺also activates the TJ sealing mechanism in frog urinary bladders. Three known procedures were utilized to increase TJ permeability, all in the absence of apical Ca²⁺: (i) exposure to high positive transepithelial clamping potentials; (ii) exposure of the apical surface to hypertonic solutions; and (iii) selective deposition of BaSO₄ in the TJs. The resealing of the TJs was promoted by raising the concentration of Ca²⁺ in the apical solution. This effect of Ca²⁺ is not impaired by the presence of Ca²⁺ channel blockers (nifedipine, verapamil, Mn²⁺ or Cd²⁺) in the apical solution, indicating that junction resealing does not depend on Ca²⁺ entering the cells through the apical membrane. TJ resealing that occurs in response to raised apical Ca²⁺ most likely results from a direct effect of Ca²⁺, entering the disrupted TJs from the apical solution and reaching the zonula adhaerens Ca²⁺ receptors (E-cadherins). Protein kinase C (PKC) must play a significant role in the control of TJ assembly in this tight epithelia since the PKC inhibitor (H7) and the activator (diC8) markedly affect TJ recovery after disruption by apical hypertonicity. H7 treated tissues show marked recuperation of conductance even in the absence of apical Ca²⁺. In contrast, diC8 prevents tissue recuperation which normally occurs after addition of Ca²⁺ to the apical solution.     

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.     

Tumour-promoting phorbol esters inhibit agonist-induced phosphatidate formation and Ca flux in human platelets

Tumour-promoting phorbol esters (phorbol-12-myristate-13-acetate, PMA; phorbol-12,13-dibutyrate, PDBu) but not 4β-phorbol, activate protein kinase C. Using human platelets pre-labelled with quin2 or ³²PO₄ we examined the effects of these compounds on human platelet cytosolic free Ca²⁺ ([Ca²⁺]_j) and on [³²]phosphatidic acid ([³²P]PtdOH). PMA and PDBu, but not 4β-phorbol inhibited thrombin-, PAF- and vasopressin-induced elevation of [Ca²⁺], and [²⁺P]PtdOH formation. It is suggested that protein kinase C may act to terminate the transduction processes that link receptor occupancy to cellular activation.     

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca²⁺ in brain microsomes after Ca²⁺ loading by the Ca²⁺-ATPase or by the Na⁺/Ca²⁺ exchanger. The results show that in microsomes loaded with Ca²⁺ by the Ca²⁺-ATPase, Ins(1,4,5)P₃ (5 μM) release 21±2% of the total Ca²⁺ accumulated, and that in the microsomes loaded with Ca²⁺ by the Na²⁺/Ca²⁺ exchanger, Ins(1,4,5)P₃ released 28±3% of the total Ca²⁺ accumulated. These results suggest that receptors of Ins(1,4,5)P₃ may be co-localized with the Na²⁺/Ca²⁺ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P₃ receptors in the plasma membrane where the Na²⁺/Ca²⁺ exchanger is normally present, or both. We also found that Ins(1,4,5)P₃ inhibited the Ca²⁺-ATPase by 33.7%, but that it had no significant effect on the Na²⁺/Ca²⁺ exchanger.     

Hypoosmotic shock activates Ca channels in isolated nerve terminals

Influence of hypotonic swelling on Ca²⁺ (⁴⁵Ca²⁺) uptake in rat brain synaptosomes was studied. A decrease in medium osmolality from 310 to 260-180 mOsm led to a progressive stimulation of ⁴⁵Ca²⁺ accumulation. The effect was blocked by verapamil (IC₅₀ = 5 μM), CoCl₅₀ = 58 μM) and retained at a fixed concentration of external sodium indicating the involvement of Ca²⁺ channels rather than Na⁺/Ca²⁺ exchange in swelling-induced Ca²⁺ influx. The populations of calcium channels observed in hypoosmotic and depolarizing conditions are different in three aspects: (i) kinetics of ⁴⁵Ca²⁺ entry; (ii) insensitivity to dihydropyridines and ω-conotoxin GVIA; (iii) insensitivity to preliminary depolarization by high potassium. The effects of swelling and depolarization on Ca²⁺ uptake were additive. No change in membrane potential monitored with diS-C₃-(5) was recorded during synaptosome hypotonic swelling. The results suggest the existence in synaptosomal plasma membrane of volume-dependent calcium-permeable channels with properties distinct from those of the voltage-dependent calcium channels. Activation of these channels may constitute an early event in volume regulation of nerve terminals in anisoosmotic conditions.     

Vasopressin elevates cytosolic calcium in small cell lung cancer cells

The ability of vasopressin to elevate cytosolic Ca²⁺ in small cell lung cancer (SCLC) cells was investigated. Ten nanomolar vasopressin elevated the cytosolic Ca²⁺ in 6 of 8 SCLC cell lines that were loaded with Fura-2 AM. Using SCLC cell line NCI-H345, the effect of vasopressin was dose dependent, being maximal at 100 nM, where the cytosolic Ca²⁺ was elevated from 150 to 210 nM. Because addition of 1 mM EGTA had no effect on the vasopressin response, vasopressin released Ca²⁺ from intracellular pools. Also, oxytocin weakly elevated the cytosolic Ca²⁺. The response to vasopressin was strongly blocked by [(β-mercapto-β,β-cyclopentamethylene propionic acid)¹,O-MeTyr²,Arg⁸]vasopressin and weakly blocked by [(β-mercapto-β,β-cyclopentamethylene propionic acid)¹,O-MeTyr²,Orn⁸]vasotocin. These data suggest that V₁ vasopressin receptors are present on SCLC cells.     

Fluoxetine-induced inhibition of synaptosomal [H] 5-HT release: Possible CA-channel inhibition

Fluoxetine, a selective 5-HT uptake inhibitor, inhibited 15 mM K⁺-induced [³H] 5-HT release from rat spinal cord and cortical synaptosomes at concentrations > 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K⁺ used to depolarize the synaptosomes and the concentration of external Ca²⁺. Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [³H] 5-HT release induced by the Ca²⁺-ionophore A 23187 or Ca²⁺-independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K⁺-induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca²⁺ channels and Ca²⁺ entry. Whereas fluoxetine and paroxetine inhibited binding of [³H] nitrendipine to the dihydropyridine-sensitive L-type Ca²⁺ channel, the less selective uptake inhibitors did not alter binding. The dihydropyridine antagonist nimodipine partially blocked fluoxetine-induced inhibition of release. Moreover enhanced K⁺-stimulated release due to the dihydropyridine agonist Bay K 8644 was reversed by fluoxetine. Fluoxetine also inhibited the K⁺-induced increase in intracellular free Ca²⁺ in fura-2 loaded synaptosomes. These data are consistent with the suggestion that fluoxetine inhibits K⁺-induced [³H] 5-HT release by antagonizing voltage-dependent Ca²⁺ entry into nerve terminals.     

The infrared absorption of amino acid side chains

Amino acid side chains play fundamental roles in stabilising protein structures and in catalysing enzymatic reactions. These fields are increasingly investigated by infrared spectroscopy at the molecular level. To help the interpretation of the spectra, a review of the infrared absorption of amino acid side chains in H₂O and ²H₂O is given. The spectral region of 2600–900 cm⁻¹ is covered.