O ρόλος της ακετυλοχολίνης στη σύσπαση και τον πολλαπλασιασµό των ΛΜΚ του αναπνευστικού

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1 O ρόλος της ακετυλοχολίνης στη σύσπαση και τον πολλαπλασιασµό των ΛΜΚ του αναπνευστικού Απ. Χατζηευθυµίου Αν. Καθηγήτρια Ιατρικής Φυσιολογίας Βιοφυσική µεµβρανών 2014

2 Σύζευξη διέγερσης-συστολής στις ΛΜΙ

3 Μυοσίνη Myosins are a large superfamily of motor proteinsthat move along actinfilaments, while hydrolyzing ATP. About 20 classesof myosin have been distinguished on the basis of the sequence ofamino acidsin their ATP-hydrolyzing motor domains. The differentclassesof myosin also differ in structure of theirtail domains.

4 Myosin II Myosin IIwas first studied for its role in musclecontraction, but it functions also in non-muscle cells. Myosin II includes two heavy chains. The globular motor domainof eachheavy chaincatalyzes ATP hydrolysis and interacts with actin. Each heavy chain continues into a tail domainin which heptadrepeat sequencespromote dimerizationby interacting to form a rod-like a-helical coiled coil. Two light chains, designated essentialand regulatory, wrap around the neck region of each myosin II heavy chain. In addition toregulatoryroles, light chains may help to stiffen the neck regions.

5 Myosins Myosin Ihas only one heavy chainwith a single globular motor domain. Its relatively short tail lacks the heptad repeats that would be involved in dimerization viaformation of acoiled coil. The Myosin VItail domain includes a short segment of heptad repeats. Myosin VI is found to be either monomeric or dimeric under different conditions. Myosin Vhas two heavy chains like myosin II. But myosin V has a longer neck regionthat has6binding sitesforcalmodulin light chains.

6 MyosinIIheads interact with actin filaments in areaction cyclethat may be summarized as follows: ATP bindingcauses a conformationalchangethat causes myosin to let go of actin. The active site closes, andatp is hydrolyzed, as a conformational change (cocking of the head) results in myosin weakly binding actin, at a different place on the filament. P i releaseresults in conformational change that leads to stronger myosin binding, and the power stroke. ADP dissociation leaves the myosin head tightly bound to actin. In the absence of ATP, this state results in muscle rigidity called rigor.

7 Καλµοδουλίνη As its name suggests, calmodulin is a CALcium MODULated protein. It isabundant in the cytoplasm of all higher cells Calmodulinacts as an intermediary protein that senses calcium levelsand relays signals to various calcium-sensitive enzymes, ionchannels and other proteins. Calmodulin is a small dumbbellshaped proteincomposed of two globular domains connected together by a flexible linker. Calcium binding exposes nonpolarsurfaces of calmodulin, which then bind to non-polar regions on the target proteins.

8 Myosin light-chain kinase (MYLK or MLCK) A serine/threonine-specific protein kinase thatphosphorylatesthe regulatory light chain of myosin II Four different MLCK isoforms exist: MYLK smooth muscle MYLK2 skeletal MYLK3 cardiac MYLK4 novel First, the calcium will bind tocalmodulin. This binding will activate MLCK, which will go on to phosphorylate themyosin light chainatserine residue 19. This will enable the myosincrossbridgeto bind to theactin filamentand allow contraction to begin (through the crossbridge cycle).

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10 Major structural domains of IP 3 R Five-domain structural model of mouse IP3 receptor 1. N-terminal coupling/suppressor domain, IP3-binding core domain, internal coupling domain, transmembrane/ channel-forming domain, gatekeeper domain. The signal for IP3 binding is transferred through both the N-terminal and internal coupling domains to the gatekeeper domain, which triggers a conformational change in the activation gate formed within the transmembrane/ channel-forming domain.

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12 Τερµατισµός συστολής The MLC phosphatase(mlcρ) removes phosphate from the phosphorylatedmlc to induce smooth muscle relaxation. The MLC phosphatase is a holoenzyme and consistsof three subunits: a 37-kDa catalytic subunit, a 20-kDavariable subunit, and a 110- to 130-kDa myosin-binding subunit. The myosin-binding subunit, when phosphorylated,inhibits the enzymatic activity of MLC phosphatase,allowing the light chain of myosin to remain phosphorylated,thereby promoting contraction.

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14 Ευαισθητοποίηση στο ασβέστιο RoA kinase The RhoA/Rho-kinaseplays an important role in the regulation of MLC phosphatase activity. The Rho-kinase, a serine/threoninekinase, phosphorylatesthe myosin binding subunit of MLC phosphatase, resulting in an inhibition of its activity and thus promoting the phosphorylatedstate of the MLC In addition to the RhoA/Rho-kinase system, an involvement of protein kinasec (PKC) and its downstream, a 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI- 17), in agonist- induced Ca 2+ sensitization has also been suggested

15 Για να προκληθεί χάλασητων λείων µυϊκών ινών απαιτείται η αποµάκρυνση των ιόντων ασβεστίου. Η αποµάκρυνση αυτή επιτελείται µε τις αντλίες ασβεστίου, µέσω των οποίων αντλούνται ιόντα ασβεστίου έξω από το λείο µυϊκό κύτταρο προς τον εξωκυττάριοχώρο ή προς το σαρκοπλασµατικό δίκτυο

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17 Yποδοχείς ακετυλοχολίνης

18 Μουσκαρινικοί υποδοχείς

19 Cell type Presence of muscarinic receptors, ChAT and/or acetylcholine T lymphocyte Muscarinicreceptors(M 1 -M 5 *) ChAT Acetylcholine B lymphocyte Muscarinicreceptors(M 1 -M 5 *) ChAT Acetylcholine Mast cell Muscarinic receptors (M 1 ) ChAT Acetylcholine Neutrophil Muscarinic receptors (M 1 /M 2 /M 3 ) ChAT Eosinophil Muscarinic receptors (M 1 ) ChAT Macrophage/m onocyte Bronchial epithelium Airway smooth muscle Muscarinic receptors (M 1 /M 2 /M 3 ) ChAT Acetylcholine Muscarinicreceptors(M 1 /M 3 ) ChAT Acetylcholine Muscarinicreceptors(M 2 /M 3 ) ChAT Functional effects of acetylcholine Increased cytotoxicity Cytokine production Proliferation Proliferation Inhibition of histamine release Chemotaxis LTB 4 production# unknown LTB 4 production# Release of monocyte, eosinophil and neutrophil chemotactic factors Pro-inflammatory gene expression * Further characterization is necessary: putative presence of muscarinic M1 M5 receptors shows high variability and is based on their presence in mononuclear leukocytes [83]. # Presence of muscarinic receptors on macrophages and neutrophils suggests their involvement in LTB4 production by sputum cells [93].

20 M 2 > M 3 M 1, M 3 M 2, M 3 M 1 M 3

21 ΕΠΙΘΗΛΙΑΚΑ ΚΥΤΤΑΡΑ Έκκριση βλέννας Έκκριση ύδατος- ηλεκτρολυτών Απελευθέρωση προσταγλανδινών Έκκριση σεροτονίνης Απελευθέρωση φλεγµονωδών παραγόντων Απελευθέρωση χηµειοτακτικών παραγόντων (για ουδετερόφλικα και µακροφάγα) Απελευθέρωση CSF (granulocyte macrophage-colony stimulation factor) Πολλαπλασιασµός επιθηλιακών κυττάρων NO προσταγλανδίνες

22 Οι Μ1, Μ3 & Μ5 υποδοχείς συνδέονται µε G πρωτεΐνες της Gq οικογένειας Οι Μ2 & Μ4 υποδοχείς συνδέονται µε G πρωτεΐνες της Gi/o οικογένειας

23 Trimeric G-proteins 7-TM receptors associate with G-proteins. G-proteins bind to the intracellular IL2 and IL3 parts of the receptors. Trimeric G-proteins are a complex of α-, β- and γ subunits. In their inactive form, G- protein α subunit binds GDP; upon ligand binding, this GDP is exchanged to GTP resulting in the active form of the Gα, which dissociates from the complex and associates to effector proteins. Finally, GTP is hydrolyzed by the Gα and the inactivated Gα re-associates with the Gβγ-7- TM receptor complex.

24 Based on their function, Gα subunits have different types: (1) Gαs: stimulation of adenylyl-cyclase leading to increase of camp (2) Gαi: inhibition of adenylyl-cyclase leading to decrease of camp (3) Gαq: activation of PLC (4) G12: activation of RhoGEF

25 IP 3 receptor and PLC signaling cascade system Μ 3 υποδοχέας Phospholipase C cleaves phophatidyl-inositol-4,5-bisphosphate (PIP2) found in theplasma membraneinto the soluble IP3 and the membrane resident diacylglycerol (DAG). IP3 initiates a rise in intracellular Ca 2+, DAG activates Protein kinase C (PKC)

26 Η δράση της ΑΚΧ µέσω Μ 3 υποδοχέων

27 Η δράση της ΑΚΧ µέσω Μ 2 υποδοχέων

28 Προσυναπτικοί Μ 2 υποδοχείς

29 Λειτουργικός ανταγωνισµός µε τους β- αδρενεργικούς υποδοχείς (2)

30 Ευαισθητοποίηση στο ασβέστιο

31 Model for regulation of MLC phosphorylation by Rho, Rho-kinase, and myosin phosphatase Rho-kinaseis able to regulate the phosphorylation of MLC by the direct phosphorylation of MLCand by the inactivation of myosin phosphatasethrough the phosphorylationof MBS. MLC, myosin light chain; cat, catalytic subunit; MBS, myosin-binding subunit. Rho-kinaseand myosin phosphatase thus coordinately regulate the phosphorylationstate of MLC, which is thought to induce smooth muscle contraction and stress fibber formation in non-muscle cells.

32 Η επίδραση της ΑΚΧ στον πολλαπλασιασµό των ΛΜΙ

33 Η δράση της ΑΚΧ µέσω Μ 2 υποδοχέων

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35 TNF-a

36 Neuraminidase IFN-γ NO

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