ATP activates a Reactive Oxygen Species-dependent oxidative stress response and secretion of pro-inflammatory cytokines in macrophages

Secretion of the proinflammatory cytokines, interleukin (IL)-1β and IL-18, usually requires two signals. The first, due to microbial products such as lipopolysaccharide, initiates transcription of the cytokine genes and accumulation of the precursor proteins. Cleavage and secretion of the cytokines is mediated by caspase-1, in association with an inflammasome containing Nalp3, which can be activated by binding of extracellular ATP to purinergic receptors. We show that treatment of macrophages with ATP results in production of reactive oxygen species (ROS), which stimulate the phosphatidylinositol 3-kinase (PI3K) pathway and subsequent Akt and ERK1/2 activation. ROS exerts its effect through glutathionylation of PTEN (phosphatase and tensin homologue deleted from chromosome 10), whose inactivation would shift the equilibrium in favor of PI3K. ATP-dependent ROS production and PI3K activation also stimulate transcription of genes required for an oxidative stress response. In parallel, ATP-mediated ROS-dependent PI3K is required for activation of caspase-1 and secretion of IL-1β and IL-18. Thus, an increase in ROS levels in ATP-treated macrophages results in activation of a single pathway that promotes both adaptation to subsequent exposure to oxidants or inflammation, and processing and secretion of proinflammatory cytokines

Activation and inactivation of neuronal nitric oxide synthase: characterization of Ca2+-dependent [125I]Calmodulin binding

Constitutive isoforms of nitric oxide synthase (NOS) are activated by transient binding of Ca(2+)/Calmodulin. Here, we characterize the binding of Calmodulin to purified neuronal NOS (nNOS). [125I]Calmodulin bound to a single class of non-interacting and high affinity sites on nNOS. [125I]Calmodulin binding achieved rapid saturation, was linear with nNOS concentration, and exhibited a strict dependence on [Ca(2+)]. Neither affinity nor extent of [125I]Calmodulin binding was affected by L-arginine, NADPH or Tetrahydrobiopterin. Native Calmodulin and engineered Calmodulin homologs [i.e., duplicated N-terminal (CaMNN)] potently displaced [125I]Calmodulin. CaMNN supported nNOS catalysis, but required approximately five-fold more Ca(2+) for comparable activity with native Calmodulin. Taken with results from kinetic analyses of [125I]Calmodulin association and dissociation, our findings suggest four sequential steps in activation of nNOS by Calmodulin: (1) Ca(2+) binds to Calmodulin's C-lobe, (2) the C-lobe of Calmodulin binds NOS, (3) Ca(2+) binds to the N-lobe of Calmodulin, and (4) the N-lobe binds to nNOS. Activation of nNOS only occurs after completion of step (4), with the displacement of nNOS's autoinhibitory insert. Upon intracellular Ca(2+) sequestration, deactivation of nNOS would proceed in reverse order

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state

Remodelling of the contractile apparatus within smooth muscle cells is an essential process that allows effective contractile activity over a wide range of cell lengths. The thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. The structure of this folded molecule has been controversial. Using negative stain electron microscopy of individual folded molecules from turkey gizzard we show they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Smooth muscle heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin whose features are identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2-D crystals on lipid. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2-D crystal. The tail of the intact myosin is bent sharply and consistently at two positions close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. The first segment runs between the heads from the head-tail junction. Unexpectedly, the other segments associate only with the blocked head rather than both heads, such that the second bend lies at a specific position near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young’s modulus of about 0.5 GPa. The folded tail of the intact molecule is less flexible indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule

A theory of Plasma Membrane Calcium Pump stimulation and activity

The ATP-driven Plasma Membrane Calcium pump or Ca(2+)-ATPase (PMCA) is characterized by a high affinity to calcium and a low transport rate compared to other transmembrane calcium transport proteins. It plays a crucial role for calcium extrusion from cells. Calmodulin is an intracellular calcium buffering protein which is capable in its Ca(2+) liganded form of stimulating the PMCA by increasing both the affinity to calcium and the maximum calcium transport rate. We introduce a new model of this stimulation process and derive analytical expressions for experimental observables in order to determine the model parameters on the basis of specific experiments. We furthermore develop a model for the pumping activity. The pumping description resolves the seeming contradiction of the Ca(2+):ATP stoichiometry of 1:1 during a translocation step and the observation that the pump binds two calcium ions at the intracellular site. The combination of the calcium pumping and the stimulation model correctly describes PMCA function. We find that the processes of calmodulin-calcium complex attachment to the pump and of stimulation have to be separated. Other PMCA properties are discussed in the framework of the model. The presented model can serve as a tool for calcium dynamics simulations and provides the possibility to characterize different pump isoforms by different type-specific parameter sets.Comment: 24 pages, 6 figure

Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics.

Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fu displaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators

Fast-Response Calmodulin-Based Fluorescent Indicators Reveal Rapid Intracellular Calcium Dynamics

Faithful reporting of temporal patterns of intracellular Ca 2 + dynamics requires the working range of indicators to match the signals. Current genetically encoded calmodulin-based fluorescent indicators are likely to distort fast Ca 2 + signals by apparent saturation and integration due to their limiting fluorescence rise and decay kinetics. A series of probes was engineered with a range of Ca 2 + affinities and accelerated kinetics by weakening the Ca 2 + -calmodulin-peptide interactions. At 37 °C, the GCaMP3-derived probe termed GCaMP3 fast is 40-fold faster than GCaMP3 with Ca 2 + decay and rise times, t 1/2 , of 3.3 ms and 0.9 ms, respectively, making it the fastest to-date. GCaMP3 fast revealed discreet transients with significantly faster Ca 2 + dynamics in neonatal cardiac myocytes than GCaMP6f. With 5-fold increased two-photon fluorescence cross-section for Ca 2 + at 940 nm, GCaMP3 fast is suitable for deep tissue studies. The green fluorescent protein serves as a reporter providing important novel insights into the kinetic mechanism of target recognition by calmodulin. Our strategy to match the probe to the signal by tuning the affinity and hence the Ca 2 + kinetics of the indicator is applicable to the emerging new generations of calmodulin-based probe

High Speed Two-Photon Imaging of Calcium Dynamics in Dendritic Spines: Consequences for Spine Calcium Kinetics and Buffer Capacity

Rapid calcium concentration changes in postsynaptic structures are crucial for synaptic plasticity. Thus far, the determinants of postsynaptic calcium dynamics have been studied predominantly based on the decay kinetics of calcium transients. Calcium rise times in spines in response to single action potentials (AP) are almost never measured due to technical limitations, but they could be crucial for synaptic plasticity. With high-speed, precisely-targeted, two-photon point imaging we measured both calcium rise and decay kinetics in spines and secondary dendrites in neocortical pyramidal neurons. We found that both rise and decay kinetics of changes in calcium-indicator fluorescence are about twice as fast in spines. During AP trains, spine calcium changes follow each AP, but not in dendrites. Apart from the higher surface-to-volume ratio (SVR), we observed that neocortical dendritic spines have a markedly smaller endogenous buffer capacity with respect to their parental dendrites. Calcium influx time course and calcium extrusion rate were both in the same range for spines and dendrites when fitted with a dynamic multi-compartment model that included calcium binding kinetics and diffusion. In a subsequent analysis we used this model to investigate which parameters are critical determinants in spine calcium dynamics. The model confirmed the experimental findings: a higher SVR is not sufficient by itself to explain the faster rise time kinetics in spines, but only when paired with a lower buffer capacity in spines. Simulations at zero calcium-dye conditions show that calmodulin is more efficiently activated in spines, which indicates that spine morphology and buffering conditions in neocortical spines favor synaptic plasticity

Rate-dependent Ca2+ signalling underlying the force-frequency response in rat ventricular myocytes: A coupled electromechanical modeling study

Rate-dependent effects on the Ca2+ sub-system in a rat ventricular myocyte are investigated. Here, we employ a deterministic mathematical model describing various Ca2+ signalling pathways under voltage clamp (VC) conditions, to better understand the important role of calmodulin (CaM) in modulating the key control variables Ca2+/calmodulin-dependent protein kinase-II (CaMKII), calcineurin (CaN), and cyclic adenosine monophosphate (cAMP) as they affect various intracellular targets. In particular, we study the frequency dependence of the peak force generated by the myofilaments, the force-frequency response (FFR). Our cell model incorporates frequency-dependent CaM-mediated spatially heterogenous interaction of CaMKII and CaN with their principal targets (dihydropyridine (DHPR) and ryanodine (RyR) receptors and the SERCA pump). It also accounts for the rate-dependent effects of phospholamban (PLB) on the SERCA pump; the rate-dependent role of cAMP in up-regulation of the L-type Ca2+ channel (ICa;L); and the enhancement in SERCA pump activity via phosphorylation of PLB.Our model reproduces positive peak FFR observed in rat ventricular myocytes during voltage-clamp studies both in the presence/absence of cAMP mediated -adrenergic stimulation. This study provides quantitative insight into the rate-dependence of Ca2+-induced Ca2+-release (CICR) by investigating the frequency-dependence of the trigger current (ICa;L) and RyR-release. It also highlights the relative role of the sodium-calcium exchanger (NCX) and the SERCA pump at higher frequencies, as well as the rate-dependence of sarcoplasmic reticulum (SR) Ca2+ content. A rigorous Ca2+ balance imposed on our investigation of these Ca2+ signalling pathways clarifies their individual roles. Here, we present a coupled electromechanical study emphasizing the rate-dependence of isometric force developed and also investigate the temperature-dependence of FFR. Our model provides mechanistic biophysically based explanations for the rate-dependence of CICR, generating useful and testable hypotheses. Although rat ventricular myocytes exhibit a positive peak FFR in the presence/absence of beta-adrenergic stimulation, they show a characteristic increase in the positive slope in FFR due to the presence of Norepinephrine or Isoproterenol. Our study identifies cAMP-mediated stimulation, and rate-dependent CaMKII-mediated up-regulation of ICa;L as the key mechanisms underlying the aforementioned positive FFR

The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle

Resting skeletal muscle is a major contributor to adaptive thermogenesis, i.e., the thermogenesis that changes in response to exposure to cold or to overfeeding. The identification of the “furnace” that is responsible for increased heat generation in resting muscle has been the subject of a number of investigations. A new state of myosin, the super relaxed state (SRX), with a very slow ATP turnover rate has recently been observed in skeletal muscle (Stewart et al. in Proc Natl Acad Sci USA 107:430–435, 2010). Inhibition of the myosin ATPase activity in the SRX was suggested to be caused by binding of the myosin head to the core of the thick filament in a structural motif identified earlier by electron microscopy. To be compatible with the basal metabolic rate observed in vivo for resting muscle, most myosin heads would have to be in the SRX. Modulation of the population of this state, relative to the normal relaxed state, was proposed to be a major contributor to adaptive thermogenesis in resting muscle. Transfer of only 20% of myosin heads from the SRX into the normal relaxed state would cause muscle thermogenesis to double. Phosphorylation of the myosin regulatory light chain was shown to transfer myosin heads from the SRX into the relaxed state, which would increase thermogenesis. In particular, thermogenesis by myosin has been proposed to play a role in the dissipation of calories during overfeeding. Up-regulation of muscle thermogenesis by pharmaceuticals that target the SRX would provide new approaches to the treatment of obesity or high blood sugar levels

Subdivisions of the Auditory Midbrain (N. Mesencephalicus Lateralis, pars dorsalis) in Zebra Finches Using Calcium-Binding Protein Immunocytochemistry

The midbrain nucleus mesencephalicus lateralis pars dorsalis (MLd) is thought to be the avian homologue of the central nucleus of the mammalian inferior colliculus. As such, it is a major relay in the ascending auditory pathway of all birds and in songbirds mediates the auditory feedback necessary for the learning and maintenance of song. To clarify the organization of MLd, we applied three calcium binding protein antibodies to tissue sections from the brains of adult male and female zebra finches. The staining patterns resulting from the application of parvalbumin, calbindin and calretinin antibodies differed from each other and in different parts of the nucleus. Parvalbumin-like immunoreactivity was distributed throughout the whole nucleus, as defined by the totality of the terminations of brainstem auditory afferents; in other words parvalbumin-like immunoreactivity defines the boundaries of MLd. Staining patterns of parvalbumin, calbindin and calretinin defined two regions of MLd: inner (MLd.I) and outer (MLd.O). MLd.O largely surrounds MLd.I and is distinct from the surrounding intercollicular nucleus. Unlike the case in some non-songbirds, however, the two MLd regions do not correspond to the terminal zones of the projections of the brainstem auditory nuclei angularis and laminaris, which have been found to overlap substantially throughout the nucleus in zebra finches