Irregular Ca2 Research Articles

STIM1 participates in the contractile rhythmicity of HL-1 cells by moderating T-type Ca2 + channel activity

STIM1 plays a crucial role in Ca2+ homeostasis, particularly in replenishing the intracellular Ca2+ store following its depletion. In cardiomyocytes, the Ca2+ content of the sarcoplasmic reticulum must be tightly controlled to sustain contractile activity. The presence of STIM1 in cardiomyocytes suggests that it may play a role in regulating the contraction of cardiomyocytes. The aim of the present study was to determine how STIM1 participates in the regulation of cardiac contractility. Atomic force microscopy revealed that knocking down STIM1 disrupts the contractility of cardiomyocyte-derived HL-1 cells. Ca2+ imaging also revealed that knocking down STIM1 causes irregular spontaneous Ca2+ oscillations in HL-1 cells. Action potential recordings further showed that knocking down STIM1 induces early and delayed afterdepolarizations. Knocking down STIM1 increased the peak amplitude and current density of T-type voltage-dependent Ca2+ channels (T-VDCC) and shifted the activation curve toward more negative membrane potentials in HL-1 cells. Biotinylation assays revealed that knocking down STIM1 increased T-VDCC surface expression and co-immunoprecipitation assays suggested that STIM1 directly regulates T-VDCC activity. Thus, STIM1 is a negative regulator of T-VDCC activity and maintains a constant cardiac rhythm by preventing a Ca2+ overload that elicits arrhythmogenic events.

Irregular Ca2+ Oscillations Regulate Transcription via Cumulative Spike Duration and Spike Amplitude

[Ca(2+)](i) oscillations are irregular and heterogeneous. The correlations between NFκB/STAT3-GFP transcription and [Ca(2+)](i) spike amplitude/cumulative spike duration are revealed by simultaneous monitoring in single cells and validated in cell population. [Ca(2+)](i) oscillations regulate transcription through [Ca(2+)](i) spike amplitude and cumulative spike duration. How irregular [Ca(2+)](i) oscillations control transcription is crucial for understanding biological [Ca(2+)](i) signal-regulated events. Agonist-stimulated [Ca(2+)](i) oscillations are universally irregular in their kinetics. How irregular [Ca(2+)](i) oscillations dynamically regulate agonist-stimulated downstream events has not been studied. To overcome the obstacles of irregularity and heterogeneity of [Ca(2+)](i) oscillations, agonist-stimulated [Ca(2+)](i) signaling and NFκB/STAT3-GFP nuclear translocation were simultaneously monitored in each single cell examined. The cause-effect relationship between [Ca(2+)](i) oscillation parameters and transcriptional activities was validated in cell populations through irregular [Ca(2+)](i) oscillations with varied parameters. The time duration of cumulative [Ca(2+)](i) elevations reaching the threshold [Ca(2+)](i) level for a transcriptional factor activation and [Ca(2+)](i) spike amplitude was found to control agonist-stimulated transcription and gene expression.

Abstract 58: Modeling Pathogenesis in Familial Hypertrophic Cardiomyopathy Using Patient-Specific Induced Pluripotent Stem Cells

Background: Hypertrophic cardiomyopathy (HCM) is a prevalent familial cardiac disorder linked to development of heart failure, arrhythmia, and sudden cardiac death. Molecular genetic studies have demonstrated HCM is caused by mutations in genes encoding for the cardiac sarcomere. However, the pathways by which sarcomeric mutations result in myocyte hypertrophy and contractile abnormalities are not well understood. Methods: We aimed to elucidate the molecular mechanisms underlying the development of HCM through the generation of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from dermal fibroblasts of a 10 member family, five of whom carry a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Results: As compared to control iPSC-CMs derived from healthy family members, HCM iPSC-CMs exhibited enlarged cell size, increased atrial natriuretic factor (ANF) expression, nuclear translocation of nuclear factor of activated T-cells (NFAT), and aggravated contractile dysfunction in response to stimulation by β-adrenergic agonists. Interestingly, both video analysis of beating cells and whole cell patch clamping revealed arrhythmia in a significant portion of diseased iPSC-CMs at the single cell level. Ca 2+ imaging demonstrated elevated cytoplasmic Ca 2+ content and irregular transients in HCM iPSC-CMs prior to the onset of cellular hypertrophy, suggesting the HCM phenotype is triggered by dysfunction in Ca 2+ cycling. Treatment of irregular Ca 2+ homeostasis by the Ca 2+ channel blocker verapamil prevented development of cellular hypertrophy and arrhythmia. Conclusions: We hypothesize the cellular abnormalities observed in HCM iPSC-CMs are caused by deficiencies in Ca 2+ regulation. We anticipate our findings will elucidate the mechanisms underlying HCM development and identify novel targets for treatment of the disease.

MioLab: Simulator for cardiac myocyte contractile force of rat based on the dynamics of calcium

BackgroundThe calcium ion (Ca2+) is essential in cardiac electrical activity since it is the direct activator of myofilaments, which cause contraction. For this reason, irregular Ca2+ flow in the cardiac myocyte is one of the main causes of cardiac arrhythmias and contractile dysfunction. In this sense, the formulation of mathematical and computational models of the mammalian ventricular myocyte has played an important role in understanding cardiac physiology. This paper proposes a biophysical model for the rat cardiac myocyte in order to reduce the number of degrees of freedom and provides a mathematical framework that makes computation simpler for our understanding of the complex process of excitation–contraction–relaxation (ECR), uses low-order lumped parameter of the myofilaments and Ca2+handling. The model allows the calculation of contractile force mainly based on the dynamics of Ca2+, provides data on transient Ca2+andenables the analysis of the effects of drugs used in the treatment of cardiac arrhythmias. MethodsThe mathematical model which describes the kinetics of cytosolic Ca2+ and dynamic cross-bridges, was implemented through nine differential equations, auxiliary equations, three 47 biophysical parameters. Each was implemented using C++ programming and its user-friendly interface in the programming language Delphi. The results of each simulation were stored in a text file and its analysis was shown in a graphical interface using executable programs in Matlab®. Additionally, the validation model was performed by comparing both the experimental data of Ca2+ transient and several drug tests. ResultsThe model satisfactorily reproduced the Ca2+ transient, as well as the effects of drugs that cause beta-adrenergic stimulation and the inhibition of the Ca2+ uptake mechanism (SERCA). Changes in the parameters regulating Ca2+ entry through L-type channels produced the oscillation amplitude of the Ca2+ transient known as the syndrome of pulses alternans. Changes in parameters related to the Na+–Ca2+ exchange has already stabilized the transient Ca2+ and produced a decrease in amplitude of the contractile force. ConclusionThe simulator proved to be a tool to study and understand the mechanisms that involve the kinetics of Ca2+ and the dynamics of cross-bridges in the unit heart muscle, as well as a tool to analyze the possible factors that may cause arrhythmias and study the effects of drugs that are used in the treatment of cardiovascular diseases in general.

Impact of hypoxia, simulated ischemia and reperfusion in HL-1 cells on the expression of FKBP12/FKBP12.6 and intracellular calcium dynamics

AimsTo establish a cardiac cell culture model for simulated ischemia and reperfusion and in this model investigate the impact of simulated ischemia and reperfusion on expression of the calcium handling proteins FKBP12 and FKBP12.6, and intracellular calcium dynamics. MethodsHL-1 cell cultures were exposed to normoxia (as control), hypoxia, simulated ischemia (HEDA) or HEDA+reactive oxygen species (ROS) for up to 24h and after HEDA, with or without ROS, followed or not by simulated reperfusion (REPH) for 6h. Viability was analyzed with a trypan blue exclusion method. Cell lysates were analyzed with real-time PCR and Western blot (WB) for FKBP12 and FKBP12.6. Intracellular Ca2+measurements were performed using dual-wavelength ratio imaging in fura-2 loaded cells. ResultsA time-dependent drop in viability was shown after HEDA (P<0.001). Viability was not further influenced by addition of ROS or REPH. The general patterns of FKBP12 and FKBP12.6 mRNA expression showed upregulation after hypoxia, downregulation after ischemia and normalization after reperfusion, which was partially attenuated if ROS was added during HEDA. The protein contents were unaffected after hypoxia, tended to increase after ischemia and, for FKBP12.6, a further increase after reperfusion was shown. Hypoxia or HEDA, with or without REPH, resulted in a decreased amplitude of the Ca2+ peak in response to caffeine. In addition, cells subjected to HEDA for 3h or HEDA for 3h followed by 6h of REPH displayed irregular Ca2+ oscillations with a decreased frequency. ConclusionA threshold for cell survival with respect to duration of ischemia was established in our cell line model. Furthermore, we could demonstrate disturbances of calcium handling in the sarcoplasmic reticulum as well as alterations in the expressions of the calcium handling proteins FKBP12 and FKBP12.6, why this model may be suitable for further studies on ischemia and reperfusion with respect to calcium handling of the sarcoplasmic reticulum.

Neuronal Mechanisms of Voluntary and Involuntary Movement in the Parafascicular (CM-Pf) Complex of the Thalamus in Patients with Spasmodic Torticollis

The responses of 144 neurons in the parafascicular CM-Pf complex of the human thalamus recorded by microelectrodes techniques in 18 stereotaxic neurosurgical operations were studied in patients with spasmodic torticollis. Two previously classified types of neurons with occasional irregular (type A) and volleytype Ca2+-dependent activity (type B) were shown to have high reactivity during performance of verbally controlled voluntary movements. Concordant occurrence of activatory-inhibitory responses of neurons of the two (A and B) types at the stage of presentation of verbal orders were found, with synergistic activatory responses from these neurons at the stage of performing the movement and subsequently. Execution of a voluntary movement could be accompanied by rapid increases in intercellular interactions between closelying neurons in the form of local synchronization with stabilization of rhythmic oscillatory (3–5 Hz) neuron activity. Differences in the responses of neurons were seen during voluntary movements without and with the involvement of the affected axial neck muscles, as well as during involuntary dystonic movements. These data provide evidence of the involvement of the nonspecific CM-Pf complex of the thalamus in the pathological mechanisms of spasmodic torticollis, which may have applied value in relation to the surgical treatment of this disease.

Carbon monoxide exposure enhances arrhythmia after cardiac stress: involvement of oxidative stress

Arrhythmias following cardiac stress are a key predictor of death in healthy population. Carbon monoxide (CO) is a ubiquitous pollutant promoting oxidative stress and associated with hospitalization for cardiovascular disease and cardiac mortality. We investigated the effect of chronic CO exposure on the occurrence of arrhythmic events after a cardiac stress test and the possible involvement of related oxidative stress. Wistar rats exposed chronically (4weeks) to sustained urban CO pollution presented more arrhythmic events than controls during recovery after cardiac challenge with isoprenaline in vivo. Sudden death occurred in 22% of CO-exposed rats versus 0% for controls. Malondialdehyde (MDA), an end-product of lipid peroxidation, was increased in left ventricular tissue of CO-exposed rats. Cardiomyocytes isolated from CO-exposed rats showed higher reactive oxygen species (ROS) production (measured with MitoSox Red dye), higher diastolic Ca(2+) resulting from SR calcium leak and an higher occurrence of irregular Ca(2+) transients (measured with Indo-1) in comparison to control cells after a high pacing sequence. Acute treatment with a ROS scavenger (N-acetylcysteine, 20mmol/L, 1h) prevented this sequence of alterations and decreased the number of arrhythmic cells following high pacing. Chronic CO exposure promotes oxidative stress that alters Ca(2+) homeostasis (through RYR2 and SERCA defects) and thereby mediates the triggering of ventricular arrhythmia after cardiac stress that can lead to sudden death.

THE CRYSTAL STRUCTURE OF VLADIMIRITE, WITH A REVISED CHEMICAL FORMULA, Ca4(AsO4)2(AsO3OH){middle dot}4H2O

Two samples of vladimirite, one from Bou Azzer, Morocco, and the other from a new occurrence in Copiapo, Chile (designated as R100075 and R080001, respectively), were examined with an electron microprobe, single-crystal X-ray diffraction, and Raman spectroscopy. Our results show that vladimirite is monoclinic with space group P 2 1 / c and unit-cell parameters a 5.8279(2), b 10.1802(4), c 22.8944(10) A, β 96.943(2)°, and V 1348.35(9) A 3 for R100075 and a 5.8220(1), b 10.1750(2), c 22.8816(6) A, β 96.902(1)°, and V 1345.66(5) A 3 for R080001. The structure determinations, with R 1 = 0.022 and 0.023 for R100075 and R080001, respectively, yielded an ideal chemical formula Ca 4 (AsO 4 ) 2 (AsO 3 OH)·4H 2 O (Z = 4) for this mineral, in contrast to Ca 5 (AsO 4 ) 2 (AsO 3 OH) 2 ·5H 2 O (Z = 3) documented in the literature. The chemical compositions for R100075 and R080001 are Ca 4.03 (AsO 4 ) 2 (As 0.99 O 3 OH)·4H 2 O and Ca 3.97 (AsO 4 ) 2 (As 1.01 O 3 OH)·4H 2 O, with trace Zn and Mn, respectively. The structure is characterized by undulating layers formed by the four nonequivalent, rather irregular Ca polyhedra [Ca1O 6 (H 2 O), Ca2O 6 (H 2 O), Ca3O 4 (H 2 O) 3 , and Ca4O 5 (H 2 O) 3 ] linked through the sharing of edges and vertices. These undulating layers are parallel to (010) and are interconnected by AsO 4 and AsO 3 OH tetrahedra, as well as hydrogen bonds, along the b axis. Vladimirite is remarkable inasmuch as one of the hydrogen-bonded O–H···O distances (O12H–H···O3) is only 2.465(2) A, which is the shortest donor–acceptor distance of all known Ca-bearing arsenate minerals, similar to the short donor–acceptor distances observed in several synthetic compounds containing AsO 3 OH groups.

A polymer chip‐based technology for the investigation of small resistance arteries

Growing consensus links hypertension, a primary cardiovascular risk factor, to elevated peripheral resistance. Advanced research on peripheral resistance arteries (RA) is therefore essential to improve our knowledge regarding hypertension and existing treatment strategies. Current experimental approaches (i.e., pressure myography) are time intensive, not high throughput ready and require highly skilled personnel. To overcome these limitations, we have designed and fabricated a microfluidic device that allows RAs to be reversibly loaded, fixed, and perfused on a polymer chip (AoC). The current study aims to validate the AoC in relation to the classical cannulation approach.Dose response curves for phenylephrine (PE) and acetylcholine (ACh) were virtually identical for mesenteric RAs tested on the AoC and those studied on a conventional pressure myograph. Arteries kept in culture on the AoC for 24h prior to functional testing showed fully maintained responses to PE and ACh. On‐chip vessel loading with Fura‐2 revealed irregular Ca2+ oscillations that were synchronized following PE treatment.This study presents a fast, easy to manage, low cost, scalable and high throughput‐ready technology that will drastically facilitate microvascular research. Based on its innovative features, the AoC has great potential for application in drug development and on‐site clinical use (personalized medicine).

Knockdown of TRIC-B from tric-a-/-mice Alters Intracellular Ca2+ Signaling in Skeletal and Cardiac Muscles

Trimeric intracellular cation (TRIC) channel subtypes are present in the endo/sarcoplasmic reticulum (SR) and nuclear membranes of muscle cells and other tissues. Knockout mice lacking both TRIC-A and TRIC-B channels suffer lethal embryonic cardiac failure due to dysfunctional intracellular Ca2+ signaling in the mutant cardiomyocytes (Yazawa et al., Nature 448, 78-82). The lethality associated with double knockout of tric-a and tric-b prevents physiological assessment of TRIC channels in adult tissues. Here we took advantage of the viable tric-a-/- mice and employed RNAi-mediated knockdown of tric-b, in order to examine the physiological function of TRIC channels in adult muscle cells. We used electroporation-mediated delivery of shRNA against tric-b into the flexor digitorum brevis (FDB) muscles of living tric-a-/- mice. Individual FDB fibers with knockdown of TRIC-B were used to examine the Ca2+ sparks properties in response to osmotic stress, and voltage-induced Ca2+ release under voltage clamp. Compared with the tric-a-/- muscle treated with control shRNA, acute knockdown of TRIC-B leads to significant reduction of the amplitude of Ca2+ sparks accompanied with prolongation of the duration of Ca2+ sparks. In neonatal cardiomyocytes isolated from the tric-a-/- mice, knockdown of TRIC-B led to significant perturbation of Ca2+ signaling from the SR, evidenced by irregular intracellular Ca2+ signaling and reduced frequency of spontaneous Ca2+ oscillations. These results indicate that disruption of TRIC function can alter intracellular Ca2+ signaling in skeletal and cardiac muscles and this may underlie an increased susceptibility of these tissues to various physiological stresses.

Extracellular ATP activates an Arabidopsis plasma membrane Ca2+-permeable conductance

Extracellular ATP has been found to elevate cytosolic free Ca2+ in Arabidopsis thaliana and trigger gene transcription, suggesting that it acts as a plant cell regulator. Recent findings place extracellular ATP upstream of Arabidopsis thaliana NADPH oxidase activity and plasma membrane Ca2+-permeable channels in the root epidermis. Here we show that increasing extracellular ATP concentration evokes a larger but more irregular Ca2+ influx conductance in root epidermal protoplasts. This may help modulate changes in cytosolic free Ca2+ as a second messenger and help explain the dose-dependent effects of extracellular ATP on cell function. The receptors for ATP and the downstream plasma membrane Ca2+ channels remain unknown at the protein or gene level. No equivalents of animal ATP receptors have been identified in higher plant genomes. We propose here that annexins could perceive extracellular ATP and participate in Ca2+ influx.

Correlating short-term Ca2+ responses with long-term protein expression after activation of single T cells

In order to elucidate the dynamics of cellular processes that are induced in context with intercellular communication, defined events along the signal transduction cascade and subsequent activation steps have to be analyzed on the level of individual cells and correlated with each other. Here we present an approach that allows the initiation of cell-cell or cell-particle interactions and the analysis of cellular reactions within various regimes while the identity of each individual cell is preserved. It utilizes dielectrophoresis (DEP) and microfluidics in a lab-on-chip system. With high spatial and temporal precision we contacted single T cells with functionalized microbeads and monitored their immediate cytosolic Ca(2+) response. After this, the cells were released from the chip system and cultivated further. Expression of the activation marker molecule CD69 was analyzed the next day and correlated with the previously recorded Ca(2+) signal for each individual cell. We found a significant difference in the patterns of Ca(2+) traces between activated and non-activated cells, which shows that Ca(2+) signals in T cells can provide early information about a later reaction of the cell. Although T cells are non-excitable cells, we also observed irregular Ca(2+) transients upon exposure to the DEP field only. These Ca(2+) signals depended on exposure time, electric field strength and field frequency. By minimizing their occurrence rate, we could identify experimental conditions that caused the least interference with the physiology of the cell.

Identification and physiological activity of survival factor released from cardiomyocytes during ischaemia and reperfusion

We carried out a screening of survival factors released from cells exposed to simulated ischaemia and reperfusion (sI/R) using the embryonic rat heart-derived cell line, H9c2 cells, and examined the physiological role of the identified factor. The culture medium supernatant of H9c2 cells exposed to sI/R was separated by column chromatography and the fractions examined for survival activity. The protein with survival activity was identified by mass spectrometry, and its physiological role was examined in the models of ischaemia. Cell survival activity was detected in at least three fractions of the cell supernatant collected during sI/R and subjected to a series of column chromatographic steps. Among the proteins measured by mass spectrometry and western blotting, a p36 protein identified as a glycolytic enzyme, lactate dehydrogenase muscle subunit (M-LDH), showed strong survival activity. H(2)O(2)-induced intracellular calcium overload in H9c2 cells and irregular Ca(2+) transients in adult rat cardiomyocytes were both found to be inhibited by pretreatment with M-LDH. M-LDH also lowered the frequency and amplitude of early afterdepolarizations induced by H(2)O(2) in adult rat cardiomyocytes and suppressed the ischaemia-reperfusion-induced reduction of cardiac output from mouse working heart preparations. M-LDH was found to increase the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), which plays a role in H9c2 cell survival. M-LDH released from cardiomyocytes after hypoxia and reoxygenation has a role in protecting the heart from oxidative stress-induced injury through an intracellular signal transduction pathway involving ERK1/2.

Intracellular Ca2+ dynamics of hippocampal interneurons following nicotinic acetylcholine receptor activation

Ca2+ permeability of central nicotinic acetylcholine receptors (nAChRs), especially the α7 subunits, are exceptionally high and this important feature provide a special functional importance for these receptors at the system level. Although studies at the cellular level extensively characterized the molecular properties of Ca2+ influx following nAChR activation, much less is known about the time-related Ca2+ dynamics during nicotine administration in integration units of neurons. Such studies are of particular relevance to understanding in situ nonsynaptic actions of nicotine. Puff ejection of drugs produce a rapid drug delivery and elimination from the cell surface allowing the activation of extrasynaptic receptors within desensitization time-frame. In this report we provide evidence that rapid nicotine application is able to produce irregular Ca2+ transients in the dendrites of stratum radiatum interneurons in the hippocampal CA1 region. Potential components and mechanisms of nAChR-mediated Ca2+ influx are discussed in details to demonstrate the unique feature of activation of nAChRs involved in nonsynaptic function in interneurons as compared to other types of nicotinic activity.

Irregular spiking in free calcium concentration in single, human platelets. Regulation by modulation of the inositol trisphosphate receptors.

Fluorescence ratio imaging indicates that immobilized, aspirin-treated platelets, loaded with Fura-2, respond to inositol 1,4,5-trisphosphate- (InsP3)-generating agonists such as thrombin by high-frequency, irregular rises in cytosolic [Ca2+]i with spikes that vary in peak level and peak-to-peak interval. This differs from the regular [Ca2+]i oscillations observed in other, larger cells. We found that the thiol-reactive compounds thimerosal (10 microm) and U73122 (10 microm) evoked similar irregular Ca2+ responses in platelets, but in this case in the absence of InsP3 generation. Thrombin-induced spiking was acutely abolished by inhibiting phospholipase C or elevating intracellular cAMP levels, while spiking with sulfhydryl reagents was only partially blocked by cAMP elevation. Confocal laser scanning microscopy using fluo-3-loaded platelets indicated that, with all agonists or conditions, the irregular spikes were almost instantaneously raised in various regions within a single platelet. When using saponin-permeabilized platelets, we found that InsP3-induced Ca2+ release from stores was stimulated by modest Ca2+ concentrations, pointing to a mechanism of InsP3-dependent Ca2+-induced Ca2+ release (CICR). This process was completely inhibitable by heparin. The Ca2+ release by InsP3, but not the CICR sensor, was negatively regulated by cAMP elevation. Thimerosal treatment did not release Ca2+ from intracellular stores, but markedly potentiated the stimulatory effect of InsP3. In contrast, U73122 caused a heparin/cAMP-insensitive Ca2+ leak from stores that differed from those used by InsP3. Taken together, these results demonstrate that InsP3 receptor channels play a crucial role in the irregular, spiking Ca2+ signal of intact platelets, even when induced by agents such as thimerosal or U73122 which do not stimulate InsP3 formation. The irregular Ca2+ release events appear to be subjected to extensive regulation by: (a) InsP3 level, (b) the potentiating effect of elevated Ca2+ on InsP3 action via CICR, (c) InsP3 channel sensitization by sulfhydryl (thimerosal) modification, (d) InsP3 channel-independent Ca2+ leak with U73122, and (e) down-regulation via cAMP elevation. The observation that individual Ca2+ peaks were generated in various parts of a platelet at similar intervals and amplitudes points to effective cooperation of the various stores in the Ca2+-release process.

Ragged spiking of free calcium in ADP-stimulated human platelets: regulation of puff-like calcium signals in vitro and ex vivo.

1. Human platelets respond to agonists of G protein (G(q))-coupled receptors by generating an irregular pattern of spiking changes in cytosolic Ca2+ ([Ca2+]i). We have investigated the ADP-induced Ca2+ responses of single, Fluo-3-loaded platelets in the presence or absence of autologous plasma or whole blood under flow conditions. 2. In plasma-free platelets, incubated in buffer medium, baseline separated [Ca2+]i peaks always consisted of a rapid rising phase (median time 0.8 s) which was abruptly followed by a slower, mono-exponential decay phase. The decay constant differed from platelet to platelet, ranging from 0.23 +/- 0.02 to 0.63 +/- 0.03 s(-1) (mean +/- S.E.M., n = 3-5), and was used to identify individual Ca2+ release events and to determine the Ca2+ fluxes of the events. 3. Confocal, high-frequency measurements of adherent, spread platelets (diameter 3-5 microm) indicated that different optical regions had simultaneous patterns of both low- and high-amplitude Ca2+ release events. 4. With or without plasma or flowing blood, the ADP-induced Ca2+ signals in platelets had the characteristics of irregular Ca2+ puffs as well as more regular Ca2+ oscillations. Individual [Ca2+]i peaks varied in amplitude and peak-to-peak interval, as observed for separated Ca2+ puffs within larger cells. On the other hand, the peaks appeared to group into periods of ragged, shorter-interval Ca2+ release events with little integration, which were alternated with longer-interval events. 5. We conclude that the spiking Ca2+ signal generated in these small cells has the characteristics of a 'poor' oscillator with an irregular frequency being reactivated from period to period. This platelet signal appears to be similar in an environment of non-physiological buffer medium and in flowing, whole blood.

Triad proteins and intracellular Ca2+ transients during development of human skeletal muscle cells in aneural and innervated cultures.

Dihydropyridine receptors (DHPRs), ryanodine receptors (RyRs), and triadin are major components of triads of mature skeletal muscle and play crucial roles in Ca2+ release in excitation-contraction (E-C) coupling. We investigated the expression and localization of these proteins as well as intracellular Ca2+ transients during development of human muscle cells cultured aneurally and innervated with rat spinal cord. mRNAs encoding skeletal muscle isoforms of the DHPR alpha1 subunit (alpha1S-DHPR), the RyR, and triadin were scarce in myoblasts and increased remarkably after myotube formation. Immunocytochemically, alpha1S-DHPR was expressed after myoblast fusion and localized mainly within the cytoplasmic area of aneural myotubes whereas the cardiac isoform (alpha1C-DHPR) was abundant along the plasma membrane. RyRs and triadin were both detected after myotube formation and colocalized in the cytoplasm of aneural myotubes and innervated muscle fibers. Along the plasma membrane of aneural myotubes, colocalization of alpha1C-DHPR with the RyR was more frequently observed than that of alpha1S-DHPR. In innervated muscle fibers, alpha1S-DHPR and RyR were colocalized first along the plasma membrane and later in the cytoplasmic area and formed regular double rows of cross-striation. The alpha1C-DHPR diminished after innervation. In Ca2+ imaging, spontaneous irregular slow Ca2+ oscillations were observed in aneurally cultured myotubes whereas nerve-driven regular fast oscillations were observed in innervated muscle fibers. Both caffeine and depolarization induced Ca2+ transients in aneurally cultured myotubes and innervated muscle fibers. In aneurally cultured myotubes, depolarization-induced Ca2+ transients were highly dependent on extracellular Ca2+, suggesting immaturity of the Ca2+ release system. This dependence remarkably decreased after innervation. Our present results show that these proteins are expressed differently in aneurally cultured myotubes than in adult skeletal muscle, that Ca2+ release in aneurally cultured myotubes is different from in adult skeletal muscle, and that innervation induces formation of a mature skeletal muscle-like excitation-contraction coupling system in cultured human muscle cells.

Ca2+ Entry Induced by Cyclic ADP-ribose in Intact T-Lymphocytes

Cyclic ADP-ribose (cADPr) is a potent Ca2+-mobilizing natural compound (Lee, H. C., Walseth, T. F., Bratt, G. T., Hayes, R. N., and Clapper, D. L. (1989) J. Biol. Chem. 264, 1608-1615) which has been shown to release Ca2+ from an intracellular store of permeabilized T-lymphocytes (Guse, A. H., Silva, C. P., Emmrich, F., Ashamu, G., Potter, B. V. L., and Mayr, G. W. (1995) J. Immunol. 155, 3353-3359). Microinjection of cADPr into intact single T lymphocytes dose dependently induced repetitive but irregular Ca2+ spikes which were almost completely dependent on the presence of extracellular Ca2+. The Ca2+ spikes induced by cADPr could be blocked either by co-injection of cADPr with the specific antagonist 8-NH2-cADPr, by omission of Ca2+ from the medium, or by superfusion of the cells with Zn2+ or SK-F 96365. Ratiometric digital Ca2+ imaging revealed that single Ca2+ spikes were initiated at several sites ("hot spots") close to the plasma membrane. These hot spots then rapidly formed a circular zone of high Ca2+ concentration below the plasma membrane which subsequently propagated like a closing optical diaphragm into the center of the cell. Taken together these data indicate a role for cADPr in Ca2+ entry in T-lymphocytes.

Pulsatile intracellular calcium release does not depend on fluctuations in inositol trisphosphate concentration

Many hormones, neurotransmitters and growth factors evoke in their target cells oscillations in the free internal Ca2+ concentration [( Ca2+]i). In electrically non-excitable cells these fluctuations are due to periodic release of Ca2+ from intracellular reservoirs, stimulated by the internal messenger inositol trisphosphate (InsP3). Most models at present invoke fluctuating levels of InsP3 as a key component in generating the oscillations in [Ca2+]i. InsP3 injected into intact cells evokes irregular and transient oscillatory Ca2+-dependent current responses, but the intracellular InsP3 concentration is not constant in such experiments. Here we monitor changes in [Ca2+]i by measuring Ca2+-activated Cl- current in single internally perfused mouse pancreatic acinar cells and show that acetylcholine (ACh), acting through muscarinic receptors, evokes regular and repetitive current pulses which are mimicked by InsP3 applied through a patch pipette. To exclude the possibility that InsP3 is periodically phosphorylated or degraded, we replaced it by the non-metabolizable InsP3 analogue inositol trisphosphorothioate (InsPS3), which also evokes regular pulses of Ca2+-activated Cl- current. These effects are independent of external Ca2+, but abolished by high intracellular concentrations of a Ca2+-chelator. We conclude that repetitive pulses of intracellular Ca2+ release occur even when the concentration of InsP3 is constant.