Overview of Signaling PDF

07/11/2023 Overview of Signaling Transmitters Transmitters Hormones   ...

07/11/2023 Overview of Signaling Transmitters Transmitters Hormones    Second Growth Messangers Ion Tyrosine Factors Channels Kinase Protein Kinases Hormones: Steroids mRNA Thyroid Synthesis Protein Synthesis Monomeric G protein OFF Rho GTP Pi GDP GEF GAP GTP GDP Rho ON Heterotirmeric G protein OFF ON 1  07/11/2023 Monomeric G protein The fraction of any small GTPase prevalent at a given timepoint in the GTP- bound active state is primarily the result of the relative activity of its GEF(s) and GAP(s). GEF, Guanine Nucleotide Exchange Factor GAP, GTP-ase activating protein GDI, , Guanine Nucleotide Dissociation Inhibitor REM GEM Ras Rho Rab Rap Arf Ran Rheb Rit Miro RGK GARCIA-RANEA JA AND VALENCIA A.FEBS Lett 434: 219–225, 1998. 2  07/11/2023 Cell Functions under Regulation of Rho GTPases: 1. Actin cytoskeletal rearrangement, Ras cell proliferation morphogenesis, cell motility Rho cytoskeletal dynamics 2. Secretion, endocytosis, phagocytosis, Rab membrane trafficking vesicle shuttling Rap cellular adhesion 3. Cell-cell adhesion and cell-extracellular Arf vesicular transport matrix adhesion Ran nuclear transport 4. NADPH oxidase activity (Rac) Rheb mTOR pathway 5. Gene transcription RGK 6. Cell cycle progression Rit 7. Mitogenesis and transformation Miro mitochondrial transport 8. Apoptosis Wennerberg K, 2005. J. Cell. Sci. 118: 843–6 Role of a RabGAP in the stimulation of glucose transport by insulin. Activation of Rab organizes the fusion of the GLUT4 carrier-containing vesicles with the plasma membrane. 3  07/11/2023 Modulation of myosin phosphorylation in smooth muscle and non-muscle cells Statins MLCK myosin light-chain kinase MLCP myosin light-chain phosphatase PLC-β β-Isoforms of phospholipase C RhoGEF Rho-specific guanine nucleotide exchange factor; IP3 inositol-1,4,5-trisphosphate DAG, diacylglycerol PKC protein kinase C CaM calmodulin ROK Rho-kinase cGK cGMPdependent kinase PKA cAMP-dependent kinase (protein kinase A) Wettschureck J Mol Med (2002) 80:629–638 J. H. Brown et. Al. Circ Res. 2006;98:730-742.) 4  07/11/2023 OLEH POCHYNYUK Exp Biol Med 232:1258–1265, 2007 REM RGK REM RAD GEM/Kir GEM Ras Rho Rab Rap Arf Ran Rheb Rit Miro RGK (Rem, Rad and Gem/Kir) GARCIA-RANEA JA AND VALENCIA A.FEBS Lett 434: 219–225, 1998. 5  07/11/2023 Calcium channel interacting proteins C Jarvis et al. Current Opinion in Cell Biology 2007, 19:474–482 Effects of Rem on calcium channels expressed in HEK293 cells Rem+ No Rem Rem Phosphorylation of Ca2+ channel Finlin et al. PNAS 2003, 100:14469–14474 6  07/11/2023 300 Effects of REM-/- on Capacitance (pF) 200 * cell dimension 100 Wild-type Rem-/- 0 4000 mean cell surface area 3500 (um2) 3000 * 2500 2000 Wild type Rem-/- central z-plane optical sections (di-8-ANEPPS) mean cross section area * (um2) WGA-stained histological sections Evaluation of heart size wild-type Rem-/- 7  07/11/2023 Effects of REM-/- on L-type calcium current Wild type REM-/- 0.5 nA 0.5 nA 100 ms 100 ms +10 mV -40 mV 500 ms mV -40 -20 0 20 40 60 300 Capacitance (pF) 200 -10 100 Wild-type Rem-/- 0 Wild type -20 REM-/- pA/pF Effects of RAD-/- on L-type calcium current Vtest (mV) -40 -20 0 20 40 60 0 WT Rad-/- Vtest -35 mV -10 current density (pA/pF) -20 mV - wt -/- -20 Rad 0 mV 5 pA/pF 100 ms 1.0 Normalized conductance 0.8 0.6 0.4 0.2 0.0 -60 -40 -20 0 20 40 Vtest (mV) 8  07/11/2023 Effects of Rad-/- on intracellular calcium transients WT F340/F380 Rad-/- F340/F380 F340/F380 +KN93 KN93= CAMK inhibitor Effects of Rad-/- cardiac action potential Wt Rad-/- 40 40 Vm (mV) Vm (mV) 0 0 -40 -40 -80 -80 10 ms 10 ms Rad-/- 0.1 Hz The absence of Rad expression mimics tonic β- adrenergic stimulation of electrical and contractile properties without gross cardiac hypertrophy, and without arrhythmia at physiologically-relevant frequencies. 50 ms 9  07/11/2023 cAMP compartmentation hypothesis 1. cAMP produced by a given receptor does not have access to all the PKAs 2. Activated PKAs do not phosphorylate all the possible substrates This implies: 1. A specific location of cAMP signaling molecules inside the cell 2. Mechanisms that limit the diffusion of cAMP Steinberg & Brunton, Ann Rev Pharmacol Toxicol 41:751-73 (2001) Spatial compartmentalization of signal transduction 10  07/11/2023 cAMP synthesis ATP cAMP 9 isoforms of adenylyl cyclase (AC): AC1-9 AC in heart: AC9 might be present at mRNA level AC5 and AC6 proteins are present Sompartmentalizalt AC4 and AC7 proteins are present AC5, AC6 ― sequence and functional similarities ― activators: GS, forskolin ― inhibitors: GI, G, Ca2+, PKA phosphorylation AC5 purinergic receptors PKC AC6  adrenergic receptors AC4, AC7 ― sequence and functional similarities to AC2 ― activators: G if activated GS, is present ― inhibitors: PKA phosphorylation The big family of cyclic nucleotide PDEs 11  07/11/2023 PDE subtypes hydrolyzing cAMP in cardiac myocytes AC cAMP targets PDE breakdown + MIMX, nimodipine PDE1 Ca2+- Calmodulin cAMP, cGMP EHNA PDE2 + ` cAMP, cGMP cGMP Milrinone, cilostamide PDE3 _ cAMP Ro-201724, rolipram PDE4 cAMP 5 ’AMP cGMP synthesys GTP cGMP pGC is located in the plasma membrane GC acivators: Atrial Natriuretic Pepetide Brain Natriuretic Pepetide C-type Natriuretic Pepetide Receptor types: NPR-A intrinsic GC activity high affinity for ANP and BNP NPR-B intrinsic GC activity, more specific for CNP NPR-C no enzymatic activity, acts via Gi sGC is located intracellulary activator: NO 12  07/11/2023 The enzymes involved in cAMP and cGMP synthesis and degradation and the complex interplay between the 2 signaling pathways in cardiac myocytes cAMP effectors: PKA CaL, PLB, RyR2 TNI, phosphatase inhibitor PKA activation of cAMP response element binding protein (CREB) (transcription factor) HCN channel, exchange protein cGMP effectors: PKG (calcium channel inhibition) PDE (opposes positive effects of cAMP on cardiac cells) Fischmeister Circ Res. 2006;99:816-828.) cAMP response to β-adrenergic stimulation C460W/E583M Coleus forskohlii  cAMP Forskolin (100 µM) ICNG Patch- 20 0 mV clamp 16 Isoprenaline ICNG @ -50mV (nA) (100 nM) -50 mV 200 ms 12 8 4 I/V 0 0 2 4 6 8 10 12 14 16 18 20 Time (min) External Internal 120 [NaCl] 120 [CsCl] 20 [CsCl] 20 [NaCl] 0 Ca / 0 Mg pCa 8.5 Rochais et al. (2004) J. Biol. Chem. 279, 52095-52105. 13  07/11/2023 PDE3 and PDE4 control cAMP microdomains generated on -adrenergic stimulation Ext. ICNG   AC   5/6 Gs Int. ATP cAMP PDE 3 PDE 4 Hormonal specificity C460W/E583M 1 2  *** cAMP ICNG (13) 30 ICNG density (pA/pF) 20 10 (13) (9) (9) 0 basal ISO + basal ISO + ICI 118551 CGP 20712A ICI-118,551 is a selective β2 receptor antagonist. CGP 20712 is a specific β1 receptor antagonist, Rochais et al. (2006) Circ. Res. 98, 1081-1088. 14  07/11/2023 cAMP compartmentation β1-AR β2-AR Ext. Ext. AC AC Gs Gs PDE 3 PDE 4 Rochais et al. J. Biol. Chem. 279, 52095-52105 (2004) Circ. Res. 98, 1081-1088 (2006) Macromolecular complexes involving cAMP PDEs R AC Gs ATP cAMP PKA C C RII RII PDE AKAP P 5’-AMP effector cardiac contraction Fischmeister et al. (2006) Circ Res 99, 816-828. 15  07/11/2023 out pGC  1-AR in PI3Kγ PDE3 B cGMP AKAP79 PDE5 Gs PDE2 cGMP AC sGC cAMP AKAP15 RyR2 PDE4 SR Ca2+ PDE3 Ca2+ CST2PP2APP1 LTCC Serca2 PLB mAKAP PDE2 PDE3 PDE4D3 Gs  2-AR arrestin Ca2+ A PDE4D5 AKAP79 Gi myomegali n PDE4D3 cAMP PDE3 T-tubule myofilaments PKA A CREB Golgi PDE2 PDE4A1 ICER PDE3A mAKAP myomegali nucleus Epac1 n ERK5 PDE4D3 PDE4D 3 Fischmeister et al. (2006) Circ Res 99, 816-828. NITRIC OXIDE 16  07/11/2023 What is Nitric Oxide? First described in 1979 as the endothelial derived relaxing factor Used by the body as a signaling molecule. Serves different functions depending on body system. i.e. neurotransmitter, vasodilator, bactericide. Environmental Pollutant First gas known to act as a biological messenger Acute vascular effect: vasodilation Chronic vascular effect: the prevention of the formation of atherosclerotic plaques NO is synthesized upon the cleavage of L-arginine into L-citrulline by NO synthases (NOS) tetrahydrobiopterin (BH4), an essential cofactor of eNOS NO signaling in the myocardium constitutive NOS isoforms NOS1 (nNOS localized with RyR) NOS3 (eNOS localized in caveolae) low output enzymes and produce NO in phase with myocyte contraction due to Ca2+-calmodulin regulation [Ca2+]i, dependent isoforms although NO is a highly diffusible signaling molecule, signaling via NOS1 and NOS3 is compartmentalized, and NOS1 and NOS3 differentially modulate cardiac function cGMP dependent and independent effects Inducible NOS isoform NOS2 (iNOS) it is present during many pathophysiological conditions of the myocardium (e.g. ischemia–reperfusion injury, septicemia, aging, heart failure, etc., induced by cytikines, TNα). high output enzyme (1000-fold more NO than eNOS) harmful effects (s-nitrosylation, peroxynitrite production, formation of highly reactive ONOO-) independent of [Ca2+]i, 17  07/11/2023 Nitric oxide downstream signalling NO regulates cardiovascular function through two distinct pathways: an indirect pathway by the activation of sGC and its downstream stimulation of PKG PKG reduces vascular tone, acts on VSMC proliferation, acts on platelet aggregation induces cardiac positive lusitropic effects a direct pathway by the S-nitrosylation of proteins S-nitrosylation alter protein activity (RYR, TNI, LTCC, Hbg...) prevents cysteine thiol groups from irreversible oxidation (e.g. mediated by peroxynitrite; ONOO–). NOS–NO signalling in cardiovascular tissues 18  07/11/2023 NOS–NO pathway in vascular beds in health and disease Bimodal activity of nitric oxide on vasomotor tone vasodilator effect induced by increased endothelial release of NO due to reduction of intracellular Ca2+ concentration vasoconstriction induced by very low concentrations of NO likely to be due to an increase of intracellular Ca2+ concentration Bimodal action of nitric oxide on myocardial contractility low concentrations of NO increase myocardial contractility (0.05 μM), high concentrations exert a negative inotropic effect (10 μM) ADPRC = adenosine diphosphate ribosyl cyclase; cADPR = cyclic adenosine β-NAD=β-nicotinamide adenine dinucleotide 19  07/11/2023 Regulation of cardiac myocyte function by specific nitric oxide synthases in the healthy myocardium in pathological states NO activates the sGC–cGMP–PKG pathway Cell shortening in a - decreased activation of the sGC–cGMP–PKG pathway - TnI phosphorylation - iNOS → NO → reacts with failing human superoxide (O2) → peroxynitrite (ONOO−), - reduces mitochondrial O2 consumption cardiacofmyocyte - nNOS-mediated S-nitrosylation RYR2 and defective modulation - reduces LTCC activity of Ca -handling proteins favour cytosolic Ca2+ overload 2+ - attenuates β1-adrenergic responsiveness - translocation of nNOS to the caveolae to limit Ca2+ influx (LTCC) - PLB phosphorylation S-nitrosylation reduces myofilament Ca2+ sensitivity eNOS and nNOS cooperate to attenuate inotropic responsiveness (such as to catecholamines) and to promote cardiac myocyte relaxation 20