Browsing by Subject "Calcium"

Now showing 1 - 20 of 43

Open Access
A mathematical model for persistent post-CSD vasoconstriction.
(PLoS computational biology, 2020-07-15) Xu, Shixin; Chang, Joshua C; Chang, Joshua C; Chow, Carson C; Brennan, KC; Huang, Huaxiong
Cortical spreading depression (CSD) is the propagation of a relatively slow wave in cortical brain tissue that is linked to a number of pathological conditions such as stroke and migraine. Most of the existing literature investigates the dynamics of short term phenomena such as the depolarization and repolarization of membrane potentials or large ion shifts. Here, we focus on the clinically-relevant hour-long state of neurovascular malfunction in the wake of CSDs. This dysfunctional state involves widespread vasoconstriction and a general disruption of neurovascular coupling. We demonstrate, using a mathematical model, that dissolution of calcium that has aggregated within the mitochondria of vascular smooth muscle cells can drive an hour-long disruption. We model the rate of calcium clearance as well as the dynamical implications on overall blood flow. Based on reaction stoichiometry, we quantify a possible impact of calcium phosphate dissolution on the maintenance of F0F1-ATP synthase activity.
Open Access
A modular switch for spatial Ca2+ selectivity in the calmodulin regulation of CaV channels.
(Nature, 2008-02-14) Dick, Ivy E; Tadross, Michael R; Liang, Haoya; Tay, Lai Hock; Yang, Wanjun; Yue, David T
Ca2+/calmodulin-dependent regulation of voltage-gated CaV1-2 Ca2+ channels shows extraordinary modes of spatial Ca2+ decoding and channel modulation, vital for many biological functions. A single calmodulin (CaM) molecule associates constitutively with the channel's carboxy-terminal tail, and Ca2+ binding to the C-terminal and N-terminal lobes of CaM can each induce distinct channel regulations. As expected from close channel proximity, the C-lobe responds to the roughly 100-microM Ca2+ pulses driven by the associated channel, a behaviour defined as 'local Ca2+ selectivity'. Conversely, all previous observations have indicated that the N-lobe somehow senses the far weaker signals from distant Ca2+ sources. This 'global Ca2+ selectivity' satisfies a general signalling requirement, enabling a resident molecule to remotely sense cellular Ca2+ activity, which would otherwise be overshadowed by Ca2+ entry through the host channel. Here we show that the spatial Ca2+ selectivity of N-lobe CaM regulation is not invariably global but can be switched by a novel Ca2+/CaM-binding site within the amino terminus of channels (NSCaTE, for N-terminal spatial Ca2+ transforming element). Native CaV2.2 channels lack this element and show N-lobe regulation with a global selectivity. On the introduction of NSCaTE into these channels, spatial Ca2+ selectivity transforms from a global to local profile. Given this effect, we examined CaV1.2/CaV1.3 channels, which naturally contain NSCaTE, and found that their N-lobe selectivity is indeed local. Disruption of this element produces a global selectivity, confirming the native function of NSCaTE. Thus, differences in spatial selectivity between advanced CaV1 and CaV2 channel isoforms are explained by the presence or absence of NSCaTE. Beyond functional effects, the position of NSCaTE on the channel's amino terminus indicates that CaM can bridge the amino terminus and carboxy terminus of channels. Finally, the modularity of NSCaTE offers practical means for understanding the basis of global Ca2+ selectivity.
Open Access
A mutation in TNNC1-encoded cardiac troponin C, TNNC1-A31S, predisposes to hypertrophic cardiomyopathy and ventricular fibrillation.
(The Journal of biological chemistry, 2012-09) Parvatiyar, MS; Landstrom, AP; Figueiredo-Freitas, C; Potter, JD; Ackerman, MJ; Pinto, JR
Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca(2+) sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca(2+) sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca(2+) affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca(2+) sensitivity of the myofilament, which may alter Ca(2+) handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.
Open Access
A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors.
(PLoS One, 2016) Koh, James; Hogue, Joyce A; Sosa, Julie A
The functional impact of intratumoral heterogeneity has been difficult to assess in the absence of a means to interrogate dynamic, live-cell biochemical events in the native tissue context of a human tumor. Conventional histological methods can reveal morphology and static biomarker expression patterns but do not provide a means to probe and evaluate tumor functional behavior and live-cell responsiveness to experimentally controlled stimuli. Here, we describe an approach that couples vibratome-mediated viable tissue sectioning with live-cell confocal microscopy imaging to visualize human parathyroid adenoma tumor cell responsiveness to extracellular calcium challenge. Tumor sections prepared as 300 micron-thick tissue slices retain viability throughout a >24 hour observation period and retain the native architecture of the parental tumor. Live-cell observation of biochemical signaling in response to extracellular calcium challenge in the intact tissue slices reveals discrete, heterogeneous kinetic waveform categories of calcium agonist reactivity within each tumor. Plotting the proportion of maximally responsive tumor cells as a function of calcium concentration yields a sigmoid dose-response curve with a calculated calcium EC50 value significantly elevated above published reference values for wild-type calcium-sensing receptor (CASR) sensitivity. Subsequent fixation and immunofluorescence analysis of the functionally evaluated tissue specimens allows alignment and mapping of the physical characteristics of individual cells within the tumor to specific calcium response behaviors. Evaluation of the relative abundance of intracellular PTH in tissue slices challenged with variable calcium concentrations demonstrates that production of the hormone can be dynamically manipulated ex vivo. The capability of visualizing live human tumor tissue behavior in response to experimentally controlled conditions opens a wide range of possibilities for personalized ex vivo therapeutic testing. This highly adaptable system provides a unique platform for live-cell ex vivo provocative testing of human tumor responsiveness to a range of physiological agonists or candidate therapeutic compounds.
Open Access
A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes.
(J Gen Physiol, 1988-02) Marchetti, C; Premont, RT; Brown, AM
Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.
Open Access
Alterations in cardiac adrenergic signaling and calcium cycling differentially affect the progression of cardiomyopathy.
(J Clin Invest, 2001-04) Freeman, K; Lerman, I; Kranias, EG; Bohlmeyer, T; Bristow, MR; Lefkowitz, RJ; Iaccarino, G; Koch, WJ; Leinwand, LA
The medical treatment of chronic heart failure has undergone a dramatic transition in the past decade. Short-term approaches for altering hemodynamics have given way to long-term, reparative strategies, including beta-adrenergic receptor (betaAR) blockade. This was once viewed as counterintuitive, because acute administration causes myocardial depression. Cardiac myocytes from failing hearts show changes in betaAR signaling and excitation-contraction coupling that can impair cardiac contractility, but the role of these abnormalities in the progression of heart failure is controversial. We therefore tested the impact of different manipulations that increase contractility on the progression of cardiac dysfunction in a mouse model of hypertrophic cardiomyopathy. High-level overexpression of the beta(2)AR caused rapidly progressive cardiac failure in this model. In contrast, phospholamban ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy, in hypertrophic cardiomyopathy mice. Cardiac expression of a peptide inhibitor of the betaAR kinase 1 not only prevented systolic dysfunction and exercise intolerance but also decreased cardiac remodeling and hypertrophic gene expression. These three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of betaAR signaling or excitation-contraction coupling can provide therapeutic benefit.
Open Access
Analysis of the mouse transcriptome for genes involved in the function of the nervous system.
(Genome Res, 2003-06) Gustincich, Stefano; Batalov, Serge; Beisel, Kirk W; Bono, Hidemasa; Carninci, Piero; Fletcher, Colin F; Grimmond, Sean; Hirokawa, Nobutaka; Jarvis, Erich D; Jegla, Tim; Kawasawa, Yuka; LeMieux, Julianna; Miki, Harukata; Raviola, Elio; Teasdale, Rohan D; Tominaga, Naoko; Yagi, Ken; Zimmer, Andreas; Hayashizaki, Yoshihide; Okazaki, Yasushi; RIKEN GER Group; GSL Members
We analyzed the mouse Representative Transcript and Protein Set for molecules involved in brain function. We found full-length cDNAs of many known brain genes and discovered new members of known brain gene families, including Family 3 G-protein coupled receptors, voltage-gated channels, and connexins. We also identified previously unknown candidates for secreted neuroactive molecules. The existence of a large number of unique brain ESTs suggests an additional molecular complexity that remains to be explored.A list of genes containing CAG stretches in the coding region represents a first step in the potential identification of candidates for hereditary neurological disorders.
Open Access
Assessing the nonlinear association of environmental factors with antibiotic resistance genes (ARGs) in the Yangtze River Mouth, China.
(Scientific reports, 2023-11) Miao, Jiazheng; Ling, Yikai; Chen, Xiaoyuan; Wu, Siyuan; Liu, Xinyue; Xu, Shixin; Umar, Sajid; Anderson, Benjamin D
The emergence of antibacterial resistance (ABR) is an urgent and complex public health challenge worldwide. Antibiotic resistant genes (ARGs) are considered as a new pollutant by the WHO because of their wide distribution and emerging prevalence. The role of environmental factors in developing ARGs in bacterial populations is still poorly understood. Therefore, the relationship between environmental factors and bacteria should be explored to combat ABR and propose more tailored solutions in a specific region. Here, we collected and analyzed surface water samples from Yangtze Delta, China during 2021, and assessed the nonlinear association of environmental factors with ARGs through a sigmoid model. A high abundance of ARGs was detected. Amoxicillin, phosphorus (P), chromium (Cr), manganese (Mn), calcium (Ca), and strontium (Sr) were found to be strongly associated with ARGs and identified as potential key contributors to ARG detection. Our findings suggest that the suppression of ARGs may be achieved by decreasing the concentration of phosphorus in surface water. Additionally, Group 2A light metals (e.g., magnesium and calcium) may be candidates for the development of eco-friendly reagents for controlling antibiotic resistance in the future.
Open Access
Barnacle cement: a polymerization model based on evolutionary concepts.
(2009-11) Dickinson, Gary H.
The tenacity by which barnacles adhere has sparked a long history of scientific investigation into their adhesive mechanisms. To adhere, barnacles utilize proteinaceous cement that rapidly polymerizes and forms adhesive bonds underwater, and is insoluble once polymerized. Although progress has been made towards understanding the chemical properties of cement proteins, the biochemical mechanisms of cement polymerization remain largely unknown. In this dissertation, I used evolutionary concepts to elucidate barnacle cement polymerization. Well-studied biological phenomena (blood coagulation in vertebrates and invertebrates) were used as models to generate hypotheses on proteins/biochemical mechanisms involved in cement polymerization. These model systems are under similar selective pressures to cement polymerization (life or death situations) and show similar chemical characteristics (soluble protein that quickly/efficiently coagulates). I describe a novel method for collection of unpolymerized cement. Multiple, independent techniques (AFM, FTIR, chemical staining for peroxidase and tandem mass spectroscopy) support the validity of the collection technique. Identification of a large number of proteins besides ‘barnacle cement proteins’ with mass spectrometry, andobservations of hemocytes in unpolymerized cement inspired the hypothesis that barnacle cement is hemolymph. A striking biochemical resemblance was shown between barnacle cement polymerization and vertebrate blood coagulation. Clotted fibrin and polymerized cement were shown to be structurally similar (mesh of fibrous protein) but biochemically distinct. Heparin, trypsin inhibitor and Ca2+ chelators impeded cement polymerization, suggesting trypsin and Ca2+ involvement in polymerization. The presence/activity of a cement trypsin-like serine protease was verified and shown homologous to bovine pancreatic trypsin. Protease activity may activate cement structural precursors, allowing loose assembly with other structural proteins and surface rearrangement. Tandem mass spectrometry and Western blotting revealed a homologous protein to human coagulation factor XIII (fibrin stabilizing factor: transglutaminase that covalently cross-links fibrin monomers). Transglutaminase activity was verified and may covalently cross-link assembled cement monomers. Similar to other protein coagulation systems, heritable defects occur during cement polymerization. High plasma protein concentration combined with sub-optimal enzyme, and/or cofactor concentrations and sub-optimal physical/muscular parameters (associated with hemolymph release) results in improperly cured cement in certain individuals when polymerization occurs in contact with low surface energy silicone and its associated leached molecules.
Open Access
Beyond the cardiac myofilament: hypertrophic cardiomyopathy- associated mutations in genes that encode calcium-handling proteins.
(Current molecular medicine, 2012-06) Landstrom, AP; Ackerman, MJ
Traditionally regarded as a genetic disease of the cardiac sarcomere, hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease and a significant cause of sudden cardiac death. While the most common etiologies of this phenotypically diverse disease lie in a handful of genes encoding critical contractile myofilament proteins, approximately 50% of patients diagnosed with HCM worldwide do not host sarcomeric gene mutations. Recently, mutations in genes encoding calcium-sensitive and calcium-handling proteins have been implicated in the pathogenesis of HCM. Among these are mutations in TNNC1- encoded cardiac troponin C, PLN-encoded phospholamban, and JPH2-encoded junctophilin 2 which have each been associated with HCM in multiple studies. In addition, mutations in RYR2-encoded ryanodine receptor 2, CASQ2-encoded calsequestrin 2, CALR3-encoded calreticulin 3, and SRI-encoded sorcin have been associated with HCM, although more studies are required to validate initial findings. While a relatively uncommon cause of HCM, mutations in genes that encode calcium-handling proteins represent an emerging genetic subset of HCM. Furthermore, these naturally occurring disease-associated mutations have provided useful molecular tools for uncovering novel mechanisms of disease pathogenesis, increasing our understanding of basic cardiac physiology, and dissecting important structure-function relationships within these proteins.
Open Access
Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs.
(Elife, 2015-01-09) Madden, Lauran; Juhas, Mark; Kraus, William E; Truskey, George A; Bursac, Nenad
Existing in vitro models of human skeletal muscle cannot recapitulate the organization and function of native muscle, limiting their use in physiological and pharmacological studies. Here, we demonstrate engineering of electrically and chemically responsive, contractile human muscle tissues ('myobundles') using primary myogenic cells. These biomimetic constructs exhibit aligned architecture, multinucleated and striated myofibers, and a Pax7(+) cell pool. They contract spontaneously and respond to electrical stimuli with twitch and tetanic contractions. Positive correlation between contractile force and GCaMP6-reported calcium responses enables non-invasive tracking of myobundle function and drug response. During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength. In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes. Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.
Open Access
Ca2+ channel nanodomains boost local Ca2+ amplitude.
(Proc Natl Acad Sci U S A, 2013-09-24) Tadross, Michael R; Tsien, Richard W; Yue, David T
Local Ca(2+) signals through voltage-gated Ca(2+) channels (CaVs) drive synaptic transmission, neural plasticity, and cardiac contraction. Despite the importance of these events, the fundamental relationship between flux through a single CaV channel and the Ca(2+) signaling concentration within nanometers of its pore has resisted empirical determination, owing to limitations in the spatial resolution and specificity of fluorescence-based Ca(2+) measurements. Here, we exploited Ca(2+)-dependent inactivation of CaV channels as a nanometer-range Ca(2+) indicator specific to active channels. We observed an unexpected and dramatic boost in nanodomain Ca(2+) amplitude, ten-fold higher than predicted on theoretical grounds. Our results uncover a striking feature of CaV nanodomains, as diffusion-restricted environments that amplify small Ca(2+) fluxes into enormous local Ca(2+) concentrations. This Ca(2+) tuning by the physical composition of the nanodomain may represent an energy-efficient means of local amplification that maximizes information signaling capacity, while minimizing global Ca(2+) load.
Open Access
Ca2+-Mediated Thermal Sensing in Plants
(2017) Xue, Yan
Temperature is an omnipresent environmental factor that shapes the growth, development and survival of plants. However, global warming has been an inevitable process and caused unusual temperature patterns across the world. As a consequence, forestry as well as agricultural plants are reportedly facing challenges from their environment. Several temperature responses in plant have been described, including short-term responses (such as acclimation) that increase tolerance towards sudden temperature stresses; as well as long-term responses (for example vernalization and flowering) that adjust growth and development to cope with seasonal temperature changes. However, the molecular mechanisms of how plants perceive temperature changes remain poorly understood. It has been observed for decades that one earliest response of plants towards low temperature is a transient increase of the cytosolic free Ca2+ concentration ([Ca2+]i). Considering the highly conserved role of [Ca2+]i increases in mediating thermal perception in animals, it has been speculated that [Ca2+]i increases may also play a role in thermal perception in plants. Nevertheless, despite intensive efforts, the molecular components responsible for cold-induced [Ca2+]i increases remain elusive. In this study, we carried out Ca2+-imaging-based forward genetic screen in Arabidopsis thaliana, isolated mutants defective in cold-induced [Ca2+]i increases (coca) and identified corresponding genes responsible for the coca phenotype through physical mapping. One of the mutants, named coca1, is highly specific to low temperature perception versus other stimuli, including osmotic, ionic and oxidative stimuli. coca1 displays compromised cold-induced [Ca2+] increases in both cotyledons and roots, as well as reduced growth fitness under ambient cool temperature. COCA1 encodes the dynamin-related protein 1A (DRP1A) and is localized on the plasma membrane. Our pharmacological studies showed that DRP1A acts upstream of plasma membrane rigidification and may mediate temperature perception by modification of membrane curvature which in turn opens Ca2+ channels. Alternatively, DRP1A may regulate endocytosis and channel activity through endocytosis signaling. Identification of coca1 as the first Arabidopsis mutant defective in cold-induced [Ca2+]i increases and DRP1A as a key player in thermal perception will greatly extend our understanding of plant adaptation to temperature changes, open up new avenues for studying Ca2+ signaling towards other stimuli and provide potential molecular genetic targets for engineering cold-resistant crops.
Open Access
Calcium-based plasticity model explains sensitivity of synaptic changes to spike pattern, rate, and dendritic location.
(Proceedings of the National Academy of Sciences of the United States of America, 2012-03) Graupner, Michael; Brunel, Nicolas
Multiple stimulation protocols have been found to be effective in changing synaptic efficacy by inducing long-term potentiation or depression. In many of those protocols, increases in postsynaptic calcium concentration have been shown to play a crucial role. However, it is still unclear whether and how the dynamics of the postsynaptic calcium alone determine the outcome of synaptic plasticity. Here, we propose a calcium-based model of a synapse in which potentiation and depression are activated above calcium thresholds. We show that this model gives rise to a large diversity of spike timing-dependent plasticity curves, most of which have been observed experimentally in different systems. It accounts quantitatively for plasticity outcomes evoked by protocols involving patterns with variable spike timing and firing rate in hippocampus and neocortex. Furthermore, it allows us to predict that differences in plasticity outcomes in different studies are due to differences in parameters defining the calcium dynamics. The model provides a mechanistic understanding of how various stimulation protocols provoke specific synaptic changes through the dynamics of calcium concentration and thresholds implementing in simplified fashion protein signaling cascades, leading to long-term potentiation and long-term depression. The combination of biophysical realism and analytical tractability makes it the ideal candidate to study plasticity at the synapse, neuron, and network levels.
Open Access
Controlled and cardiac-restricted overexpression of the arginine vasopressin V1A receptor causes reversible left ventricular dysfunction through Gαq-mediated cell signaling.
(Circulation, 2011-08) Li, Xue; Chan, Tung O; Myers, Valerie; Chowdhury, Ibrul; Zhang, Xue-Qian; Song, Jianliang; Zhang, Jin; Andrel, Jocelyn; Funakoshi, Hajime; Robbins, Jeffrey; Koch, Walter J; Hyslop, Terry; Cheung, Joseph Y; Feldman, Arthur M
Background
[Arg8]-vasopressin (AVP) activates 3 G-protein-coupled receptors: V1A, V2, and V1B. The AVP-V1A receptor is the primary AVP receptor in the heart; however, its role in cardiac homeostasis is controversial. To better understand AVP-mediated signaling in the heart, we created a transgenic mouse with controlled overexpression of the V1A receptor.
Methods and results
The V1A receptor transgene was placed under the control of the tetracycline-regulated, cardiac-specific α-myosin heavy chain promoter (V1A-TG). V1A-TG mice had a normal cardiac function phenotype at 10 weeks of age; however, by 24 weeks of age, tetracycline-transactivating factor/V1A-TG mouse hearts had reduced cardiac function, cardiac hypertrophy, and dilatation of the ventricular cavity. Contractile dysfunction was also observed in isolated adult cardiac myocytes. When V1A receptor transgene was induced to be expressed in adult mice (V1A-TG(Ind)), left ventricular dysfunction and dilatation were also seen, albeit at a later time point. Because the V1A receptor mediates cell signaling through Gα(q) protein, we blocked Gα(q) signaling by crossing tetracycline-transactivating factor/V1A mice with transgenic mice that expressed a small inhibitory peptide against Gα(q). Gα(q) blockade abrogated the development of the heart failure phenotype in tetracycline-transactivating factor/V1A-TG mice. The heart failure phenotype could be reversed by administration of doxycycline.
Conclusions
Our results demonstrate a role for V1A-mediated signaling in the development of heart failure and support a role for V1A blockade in the treatment of patients with elevated levels of vasopressin.
Open Access
Coupling of beta2-adrenoceptor to Gi proteins and its physiological relevance in murine cardiac myocytes.
(Circ Res, 1999-01-08) Xiao, RP; Avdonin, P; Zhou, YY; Cheng, H; Akhter, SA; Eschenhagen, T; Lefkowitz, RJ; Koch, WJ; Lakatta, EG
-Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (betaAR) signal transduction. Although several of these models have altered betaAR subtypes, the specific functional sequelae of betaAR stimulation in murine heart, particularly those of beta2-adrenergic receptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta2AR stimulation on contraction, [Ca2+]i transient, and L-type Ca2+ currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergic receptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-32P]GTP azidoanilide into alpha subunits of Gi2 and Gi3 after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Gi proteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gs and Gi signaling, resulting in null inotropic response, unless the Gi signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of beta2AR-coupled Gi proteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling.
Open Access
Cyclosporine A inhibits Ca2+-dependent stimulation of the Na+/H+ antiport in human T cells.
(J Cell Biol, 1986-08) Rosoff, PM; Terres, G
The cyclic undecapeptide cyclosporine A (CsA) is a potent immunosuppressive agent that inhibits the initial activation of T lymphocytes. This agent appears to be most effective in blocking the action of mitogens such as concanavalin A and the calcium ionophore A23187, which cause an influx of Ca2+, but not those that may act by alternate mechanisms. These observations suggest that CsA may block a Ca2+-dependent step in T cell activation. We have shown that stimulation of the T3-T cell receptor complex-associated Ca2+ transporter activates the Na+/H+ antiport (Rosoff, P. M., and L. C. Cantley, 1985, J. Biol. Chem., 260: 14053-14059). The tumor-promoting phorbol esters, which are co-mitogenic for T cells, activate the exchanger by a separate pathway which is mediated by protein kinase C. Both the rise in intracellular Ca2+ and intracellular pH may be necessary for the successful triggering of cellular activation. In this report we show that CsA blocks the T3-T cell receptor-stimulated, Ca2+ influx-dependent activation of Na+/H+ exchange, but not the phorbol ester-mediated pathway in a transformed human T cell line. CsA inhibited mitogen-stimulation of interleukin-2 production in a separate cell line. CsA also inhibited vasopressin stimulation of the antiporter in normal rat kidney fibroblasts, but had no effect on serum or 12-O-tetradecanoyl phorbol 13-acetate stimulation. CsA did not affect serum or vasopressin or serum stimulation of normal rat kidney cell proliferation. CsA also had no effect on lipopolysaccharide or phorbol ester stimulation of Na+/H+ exchange activity or induction of differentiation in 70Z/3 pre-B lymphocytes in which these events are initiated by the protein kinase C pathway. These data suggest that mechanisms of activation of Na+/H+ exchange that involve an elevation in cytosolic Ca2+ are blocked by CsA but that C kinase-mediated regulation is unaffected. The importance of the Na+/H+ antiport in the regulation of growth and differentiation of T cells is discussed.
Open Access
Direct In Vivo Manipulation and Imaging of Calcium Transients in Neutrophils Identify a Critical Role for Leading-Edge Calcium Flux.
(Cell Rep, 2015-12-15) Tobin, DM
Calcium signaling has long been associated with key events of immunity, including chemotaxis, phagocytosis, and activation. However, imaging and manipulation of calcium flux in motile immune cells in live animals remain challenging. Using light-sheet microscopy for in vivo calcium imaging in zebrafish, we observe characteristic patterns of calcium flux triggered by distinct events, including phagocytosis of pathogenic bacteria and migration of neutrophils toward inflammatory stimuli. In contrast to findings from ex vivo studies, we observe enriched calcium influx at the leading edge of migrating neutrophils. To directly manipulate calcium dynamics in vivo, we have developed transgenic lines with cell-specific expression of the mammalian TRPV1 channel, enabling ligand-gated, reversible, and spatiotemporal control of calcium influx. We find that controlled calcium influx can function to help define the neutrophil's leading edge. Cell-specific TRPV1 expression may have broad utility for precise control of calcium dynamics in other immune cell types and organisms.
Open Access
Disruption of STIM1-mediated Ca²⁺ sensing and energy metabolism in adult skeletal muscle compromises exercise tolerance, proteostasis, and lean mass.
(Molecular metabolism, 2022-03) Wilson, Rebecca J; Lyons, Scott P; Koves, Timothy R; Bryson, Victoria G; Zhang, Hengtao; Li, TianYu; Crown, Scott B; Ding, Jin-Dong; Grimsrud, Paul A; Rosenberg, Paul B; Muoio, Deborah M
Objective
Stromal interaction molecule 1 (STIM1) is a single-pass transmembrane endoplasmic/sarcoplasmic reticulum (E/SR) protein recognized for its role in a store operated Ca²⁺ entry (SOCE), an ancient and ubiquitous signaling pathway. Whereas STIM1 is known to be indispensable during development, its biological and metabolic functions in mature muscles remain unclear.
Methods
Conditional and tamoxifen inducible muscle STIM1 knock-out mouse models were coupled with multi-omics tools and comprehensive physiology to understand the role of STIM1 in regulating SOCE, mitochondrial quality and bioenergetics, and whole-body energy homeostasis.
Results
This study shows that STIM1 is abundant in adult skeletal muscle, upregulated by exercise, and is present at SR-mitochondria interfaces. Inducible tissue-specific deletion of STIM1 (iSTIM1 KO) in adult muscle led to diminished lean mass, reduced exercise capacity, and perturbed fuel selection in the settings of energetic stress, without affecting whole-body glucose tolerance. Proteomics and phospho-proteomics analyses of iSTIM1 KO muscles revealed molecular signatures of low-grade E/SR stress and broad activation of processes and signaling networks involved in proteostasis.
Conclusion
These results show that STIM1 regulates cellular and mitochondrial Ca²⁺ dynamics, energy metabolism and proteostasis in adult skeletal muscles. Furthermore, these findings provide insight into the pathophysiology of muscle diseases linked to disturbances in STIM1-dependent Ca²⁺ handling.
Open Access
Earlier onset and greater severity of disordered mineral metabolism in diabetic patients with chronic kidney disease.
(Diabetes care, 2012-05) Wahl, Patricia; Xie, Huiliang; Scialla, Julia; Anderson, Cheryl AM; Bellovich, Keith; Brecklin, Carolyn; Chen, Jing; Feldman, Harold; Gutierrez, Orlando M; Lash, Jim; Leonard, Mary B; Negrea, Lavinia; Rosas, Sylvia E; Anderson, Amanda Hyre; Townsend, Raymond R; Wolf, Myles; Isakova, Tamara; Chronic Renal Insufficiency Cohort Study Group
Disordered mineral metabolism is a common complication of chronic kidney disease (CKD) and a novel risk factor for CKD progression, cardiovascular disease, and mortality. Although diabetes is the leading cause of CKD and is associated with worse clinical outcomes than other etiologies, few studies have evaluated mineral metabolism in CKD according to diabetes status.Using the Chronic Renal Insufficiency Cohort Study, we tested the hypothesis that diabetes is independently associated with lower serum calcium and higher serum phosphate, parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23).Compared with participants without diabetes (n = 1,936), those with diabetes (n = 1,820) were more likely to have lower estimated glomerular filtration rate (eGFR), lower serum albumin, and higher urinary protein excretion (all P < 0.001). Unadjusted serum phosphate, PTH, and FGF23 levels were higher and calcium was lower among those with compared with those without diabetes (all P < 0.001). After multivariate adjustment, diabetes remained a significant predictor of serum phosphate, PTH, and FGF23 but not calcium. The eGFR cut point at which 50% of participants met criteria for secondary hyperparathyroidism or elevated FGF23 was higher in participants with diabetes compared with those without (PTH: eGFR 30-39 vs. 20-29, P < 0.001; FGF23: eGFR 50-59 vs. 40-49, P < 0.001).Disordered mineral metabolism begins earlier in the course of CKD and is more severe among CKD patients with compared with those without diabetes. Future studies should explore mechanisms for these differences and whether they contribute to excess risks of adverse clinical outcomes among diabetic patients with CKD.

Browsing by Subject "Calcium"

Background

Methods and results

Conclusions

Objective

Methods

Results

Conclusion