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Ionic mechanisms of pacemaker activity in spontaneously contracting atrial HL-1 cells.
Yang Z, Murray KT
(2011) J Cardiovasc Pharmacol 57: 28-36
MeSH Terms: Action Potentials, Biological Clocks, Calcium, Cell Line, Transformed, Diastole, Heart Atria, Humans, Ions, Myocytes, Cardiac, Ryanodine, Sarcolemma, Sarcoplasmic Reticulum, Sinoatrial Node, Thapsigargin
Show Abstract · Added January 20, 2015
Although normally absent, spontaneous pacemaker activity can develop in human atrium to promote tachyarrhythmias. HL-1 cells are immortalized atrial cardiomyocytes that contract spontaneously in culture, providing a model system of atrial cell automaticity. Using electrophysiologic recordings and selective pharmacologic blockers, we investigated the ionic basis of automaticity in atrial HL-1 cells. Both the sarcoplasmic reticulum Ca release channel inhibitor ryanodine and the sarcoplasmic reticulum Ca ATPase inhibitor thapsigargin slowed automaticity, supporting a role for intracellular Ca release in pacemaker activity. Additional experiments were performed to examine the effects of ionic currents activating in the voltage range of diastolic depolarization. Inhibition of the hyperpolarization-activated pacemaker current, If, by ivabradine significantly suppressed diastolic depolarization, with modest slowing of automaticity. Block of inward Na currents also reduced automaticity, whereas inhibition of T- and L-type Ca currents caused milder effects to slow beat rate. The major outward current in HL-1 cells is the rapidly activating delayed rectifier, IKr. Inhibition of IKr using dofetilide caused marked prolongation of action potential duration and thus spontaneous cycle length. These results demonstrate a mutual role for both intracellular Ca release and sarcolemmal ionic currents in controlling automaticity in atrial HL-1 cells. Given that similar internal and membrane-based mechanisms also play a role in sinoatrial nodal cell pacemaker activity, our findings provide evidence for generalized conservation of pacemaker mechanisms among different types of cardiomyocytes.
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14 MeSH Terms
The effects of cellular contraction on aortic valve leaflet flexural stiffness.
Merryman WD, Huang HY, Schoen FJ, Sacks MS
(2006) J Biomech 39: 88-96
MeSH Terms: Animals, Aortic Valve, Fibroblasts, In Vitro Techniques, Myocardial Contraction, Myocytes, Smooth Muscle, Potassium Chloride, Stress, Mechanical, Swine, Thapsigargin
Show Abstract · Added February 12, 2015
The aortic valve (AV) leaflet contains a heterogeneous interstitial cell population composed predominantly of myofibroblasts, which contain both fibroblast and smooth muscle cell characteristics. The focus of the present study was to examine aortic valve interstitial cell (AVIC) contractile behavior within the intact leaflet tissue. Circumferential strips of porcine AV leaflets were mechanically tested under flexure, with the AVIC maintained in the normal, contracted, and contraction-inhibited states. Leaflets were flexed both with (WC) and against (AC) the natural leaflet curvature, both before and after the addition of 90 mM KCl to elicit cellular contraction. In addition, a natural basal tonus was also demonstrated by treating the leaflets with 10 microM thapsigargin to completely inhibit AVIC contraction. Results revealed a 48% increase in leaflet stiffness with AVIC contraction (from 703 to 1040 kPa, respectively) when bent in the AC direction (p=0.004), while the WC direction resulted only in 5% increase (from 491 to 516.5 kPa, respectively--not significant) in leaflet stiffness in the active state. Also, the loss of basal tonus of the AVIC population with thapsigargin treatment resulted in 76% (AC, p=0.001) and 54% (WC, p=0.036) decreases in leaflet stiffness at 5 mM KCl levels, while preventing contraction with the addition of 90 mM KCl as expected. We speculate that the observed layer dependent effects of AVIC contraction are primarily due to varying ECM mechanical properties in the ventricularis and fibrosa layers. Moreover, while we have demonstrated that AVIC contractile ability is a significant contributor to AV leaflet bending stiffness, it most likely serves a role in maintaining AV leaflet tissue homeostasis that has yet to be elucidated.
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10 MeSH Terms
Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is required for activation of NF-kappaB in response to diverse cellular stresses.
Jiang HY, Wek SA, McGrath BC, Scheuner D, Kaufman RJ, Cavener DR, Wek RC
(2003) Mol Cell Biol 23: 5651-63
MeSH Terms: Animals, Apoptosis, Cell Nucleus, Cells, Cultured, Cycloheximide, Dactinomycin, Dose-Response Relationship, Drug, Endoplasmic Reticulum, Enzyme Activation, Eukaryotic Initiation Factor-2, Heterozygote, Immunoblotting, Luciferases, Mice, Microscopy, Confocal, Microscopy, Fluorescence, NF-kappa B, Phosphorylation, Protein Biosynthesis, Protein Synthesis Inhibitors, Protein Transport, Thapsigargin, Transcription, Genetic, Transcriptional Activation
Show Abstract · Added August 13, 2010
Nuclear factor kappaB (NF-kappaB) serves to coordinate the transcription of genes in response to diverse environmental stresses. In this report we show that phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2) is fundamental to the process by which many stress signals activate NF-kappaB. Phosphorylation of this translation factor is carried out by a family of protein kinases that each respond to distinct stress conditions. During impaired protein folding and assembly in the endoplasmic reticulum (ER), phosphorylation of eIF2alpha by PEK (Perk or EIF2AK3) is essential for induction of NF-kappaB transcriptional activity. The mechanism by which NF-kappaB is activated during ER stress entails the release, but not the degradation, of the inhibitory protein IkappaB. During amino acid deprivation, phosphorylation of eIF2alpha by GCN2 (EIF2AK4) signals the activation of NF-kappaB. Furthermore, inhibition of general translation or transcription by cycloheximide and actinomycin D, respectively, elicits the eIF2alpha phosphorylation required for induction of NF-kappaB. Together, these studies suggest that eIF2alpha kinases monitor and are activated by a range of stress conditions that affect transcription and protein synthesis and assembly, and the resulting eIFalpha phosphorylation is central to activation of the NF-kappaB. The absence of NF-kappaB-mediated transcription and its antiapoptotic function provides an explanation for why eIF2alpha kinase deficiency in diseases such as Wolcott-Rallison syndrome leads to cellular apoptosis and disease.
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24 MeSH Terms
ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs.
Ye J, Rawson RB, Komuro R, Chen X, Davé UP, Prywes R, Brown MS, Goldstein JL
(2000) Mol Cell 6: 1355-64
MeSH Terms: Activating Transcription Factor 6, Amino Acid Motifs, Amino Acid Sequence, Animals, CCAAT-Enhancer-Binding Proteins, Carrier Proteins, Cell Line, DNA-Binding Proteins, Endopeptidases, Endoplasmic Reticulum, Heat-Shock Proteins, Humans, Immunoblotting, Intracellular Membranes, Intracellular Signaling Peptides and Proteins, Membrane Proteins, Molecular Chaperones, Molecular Sequence Data, Mutation, Proprotein Convertases, Protein Binding, Protein Processing, Post-Translational, RNA, Messenger, Sequence Alignment, Serine Endopeptidases, Sterol Regulatory Element Binding Protein 1, Thapsigargin, Transcription Factors, Transcription, Genetic, Tunicamycin
Show Abstract · Added March 5, 2014
ATF6 is a membrane-bound transcription factor that activates genes in the endoplasmic reticulum (ER) stress response. When unfolded proteins accumulate in the ER, ATF6 is cleaved to release its cytoplasmic domain, which enters the nucleus. Here, we show that ATF6 is processed by Site-1 protease (S1P) and Site-2 protease (S2P), the enzymes that process SREBPs in response to cholesterol deprivation. ATF6 processing was blocked completely in cells lacking S2P and partially in cells lacking S1P. ATF6 processing required the RxxL and asparagine/proline motifs, known requirements for S1P and S2P processing, respectively. Cells lacking S2P failed to induce GRP78, an ATF6 target, in response to ER stress. ATF6 processing did not require SCAP, which is essential for SREBP processing. We conclude that S1P and S2P are required for the ER stress response as well as for lipid synthesis.
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30 MeSH Terms
Protein kinase C activator PMA reduces the Ca(2+) response to antigen stimulation of adherent RBL-2H3 mucosal mast cells by inhibiting depletion of intracellular Ca(2+) stores.
Kuchtey J, Fewtrell C
(1999) J Cell Physiol 181: 113-23
MeSH Terms: Antigens, Calcium, Cell Adhesion, Cell Line, Enzyme Activation, Fluorescent Dyes, Fura-2, Logistic Models, Mast Cells, Patch-Clamp Techniques, Protein Kinase C, Stimulation, Chemical, Tetradecanoylphorbol Acetate, Thapsigargin
Show Abstract · Added February 19, 2015
Activation of protein kinase C has been shown to reduce the Ca(2+) responses of a variety of cell types. In most cases, the reduction is due to inhibition of Ca(2+) influx, but acceleration of Ca(2+) efflux and inhibition of Ca(2+) store depletion by protein kinase C activation have also been described. For adherent RBL-2H3 mucosal mast cells, results from whole-cell patch clamp experiments suggest that protein kinase C activation reduces Ca(2+) influx, while experiments with intact, fura-2-loaded cells suggest that Ca(2+) influx is not affected. Here we present single-cell data from Ca(2+) imaging experiments with adherent RBL-2H3 cells, showing that antigen-stimulated Ca(2+) responses of phorbol 12-myristate 13-acetate (PMA)-treated cells are more transient than those of control cells. PMA also reduced the response to antigen in the absence of extracellular Ca(2+), indicating that depletion of intracellular Ca(2+) stores is inhibited. If PMA was added after stores had been depleted by thapsigargin, a small decrease in [Ca(2+)](i) was observed, consistent with a slight inhibition of Ca(2+) influx. However, the major effect of PMA on the antigen-stimulated Ca(2+) response is to inhibit depletion of intracellular Ca(2+) stores. We also show that inhibition of protein kinase C did not enhance the Ca(2+) response to antigen, suggesting that inhibition of the Ca(2+) response by activation of protein kinase C does not contribute to the physiological response to antigen.
Copyright 1999 Wiley-Liss, Inc.
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14 MeSH Terms
Heterogeneous responses of cell Ca2+ in human airway epithelium.
Paradiso AM, Brown HA, Ye H, Harden TK, Boucher RC
(1999) Exp Lung Res 25: 277-90
MeSH Terms: Adenosine Triphosphate, Adult, Bradykinin, Calcium, Calcium-Transporting ATPases, Cells, Cultured, Epithelial Cells, Female, Fluorescent Antibody Technique, Indirect, Fura-2, Histamine, Humans, Keratins, Male, Nasal Mucosa, Receptors, Purinergic P2, Thapsigargin
Show Abstract · Added March 21, 2013
The Ca(2+)-mobilizing actions of adenosine 5'-triphosphate (ATP), bradykinin, and histamine were compared in phenotypically distinct human nasal epithelial (HNE) cell types and as a function of time in cell culture. Single-cell measurements of intracellular free Ca2+ (Ca2+i, Fura-2 fluorescence) were recorded in ciliated cells 1-2 days in primary culture, and in nonciliated cells 1-2 days (keratin 14-positive) or 4-5 days (keratin 18-positive) after seeding. No difference in basal Ca2+i was noted between ciliated and nonciliated cell preparations. For ciliated and nonciliated cells studied 1-2 days in culture, ATP, bradykinin, and histamine elicited a cytosolic Ca2+ response in 100% of the cells examined. For nonciliated HNE cells maintained 4-5 days in culture, ATP (10(-4) M) increased cytosolic Ca2+ in all cells tested, but only 85% of the cells responded to bradykinin (10(-5) M) addition, and 65% to histamine (10(-4) M) stimulation. In terms of the absolute change of Ca2+i (delta Ca2+i, peak-basal value), the efficacy was ATP > bradykinin > histamine for the 3 HNE cell preparations. However, the delta Ca2+i in response to agonists was smaller in nonciliated HNE cells studied 1-2 days or 4-5 days in culture as compared to the ciliated cell preparation. Thapsigargin (300 nM), an agent that mobilizes Ca2+i, was equally effective in raising cytosolic Ca2+ in nonciliated (1-2 days and 4-5 days in culture) and ciliated HNE cells. These data show that ciliated cells consistently respond to all agonists, whereas the cytosolic Ca2+ response to ATP, bradykinin, and histamine in nonciliated cells was quantitatively reduced at a comparable time period (1-2 days) and became smaller and less frequent in nonciliated cell preparations maintained 4-5 days in culture. These results demonstrate time-dependent differences in the magnitude and frequency of cytosolic Ca2+ responses to certain agonists, strongly indicating that measurements of Ca2+i in HNE cells must account for the heterogeneity of the cell types and the time cells are maintained in primary culture.
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17 MeSH Terms
Acute regulation of norepinephrine transport: I. protein kinase C-linked muscarinic receptors influence transport capacity and transporter density in SK-N-SH cells.
Apparsundaram S, Galli A, DeFelice LJ, Hartzell HC, Blakely RD
(1998) J Pharmacol Exp Ther 287: 733-43
MeSH Terms: Biological Transport, Carrier Proteins, Cell Line, Cell Membrane, Egtazic Acid, Enzyme Activation, Methacholine Chloride, Norepinephrine, Norepinephrine Plasma Membrane Transport Proteins, Protein Kinase C, Receptors, Muscarinic, Symporters, Thapsigargin
Show Abstract · Added December 10, 2013
Using SK-N-SH cells, we observe that muscarinic acetylcholine receptor activation by methacholine (MCh) rapidly and selectively diminishes l-NE transport capacity (Vmax) with little or no change in norepinephrine (NE) Km and without apparent effects on membrane potential monitored directly under current clamp. Over the same time frame, MCh exposure reduces the density of [3H]nisoxetine binding sites (Bmax) in intact cells but not in total membrane fractions, consistent with a loss of transport capacity mediated by sequestration of transporters rather than changes in intrinsic transport activity or protein degradation. Similar changes in NE transport and [3H]nisoxetine binding capacity are observed after phorbol ester (beta-PMA) treatment. Inhibition of PKC by antagonists and downregulation of PKC by chronic treatment with phorbol esters abolishes beta-PMA-mediated effects but produce only a partial blockade of MCh-induced effects. Neither muscarinic acetylcholine receptor nor PKC activation require extracellular Ca++ to diminish NET activity. In contrast, treatment of cells with the Ca++/ATPase antagonist, thapsigargin in Ca++-free medium, eliminates the staurosporine-insensitive component of MCh regulation. These findings were further corroborated by the ability of [1, 2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester application in Ca++-free medium to abolish NET regulation by MCh. Although they may contribute to basal NET expression, we could not implicate CaMKII-, PKA- or nitric oxide-linked pathways in MCh regulation. Together, these findings 1) provide evidence in support of G-protein coupled receptor-mediated regulation of catecholamine transport, 2) reveal intracellular Ca++-sensitive, PKC-dependent and -independent pathways that serve to regulate NET expression and 3) indicate that the diminished capacity for NE transport evident after mAChR and PKC activation involves a redistribution of NET protein.
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13 MeSH Terms
Metabotropic glutamate receptor (mGluR)-mediated potentiation of cyclic AMP responses does not require phosphoinositide hydrolysis: mediation by a group II-like mGluR.
Winder DG, Conn PJ
(1995) J Neurochem 64: 592-9
MeSH Terms: Animals, Brain, Calcium, Calcium-Transporting ATPases, Cyclic AMP, Cycloleucine, Glycine, Hydrolysis, In Vitro Techniques, Intracellular Membranes, Neurotoxins, Phosphatidylinositols, Protein Kinase C, Rats, Receptors, Metabotropic Glutamate, Resorcinols, Terpenes, Thapsigargin
Show Abstract · Added May 19, 2014
Metabotropic glutamate receptors (mGluRs) in the CNS are coupled to a variety of second messenger systems, the best characterized of which is activation of phosphoinositide hydrolysis. Recently, we found that activation of mGluRs in rat brain slices by the selective mGluR agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) potentiates cyclic AMP (cAMP) responses elicited by activation of other receptors coupled to Gs. It has been suggested that mGluR-mediated potentiation of cAMP responses is secondary to activation of phosphoinositide hydrolysis. However, preliminary evidence suggests that this is not the case. Therefore, we designed a series of experiments to test more fully the hypothesis that mGluR-mediated potentiation of cAMP responses is secondary to phosphoinositide hydrolysis. Inhibitors of both protein kinase C and intracellular calcium mobilization failed to antagonize 1S,3R-ACPD-stimulated potentiation of cAMP responses. Further, coapplication of phorbol esters and 1S,3R-ACPD induced a cAMP response that was greater than additive. Finally, (RS)-3,5-dihydroxyphenylglycine, a selective agonist of mGluRs coupled to phosphoinositide hydrolysis, failed to potentiate cAMP responses, whereas (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine, an mGluR agonist that does not activate mGluRs coupled to phosphoinositide hydrolysis, elicited a robust potentiation of cAMP responses. In total, these data strongly suggest that mGluR-mediated potentiation of cAMP responses is not secondary to activation of phosphoinositide hydrolysis and is likely mediated by a group II mGluR.
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18 MeSH Terms