O ρόλος της ακετυλοχολίνης στη σύσπαση και τον πολλαπλασιασµό των ΛΜΚ (του αναπνευστικού) Απ. Χατζηευθυµίου 2015

Transcription

1 O ρόλος της ακετυλοχολίνης στη σύσπαση και τον πολλαπλασιασµό των ΛΜΚ (του αναπνευστικού) Απ. Χατζηευθυµίου 2015

2 Σύσπαση ΛΜΙ

3 An increase in free intracellular calcium can result from either increased flux of calcium into the cell through calcium channels or by release of calcium from internal stores (e.g., sarcoplasmic reticulum). The free calcium binds to a special calcium binding protein called calmodulin. Calcium-calmodulin activates myosin light chain kinase (MLCK), an enzyme that is capable of phosphorylating myosin light chains (MLC) in the presence of ATP. Myosin light chains are 20-kD regulatory subunits found on the myosin heads. MLC phosphorylation leads to crossbridge formation between the myosin heads and the actin filaments, and hence, smooth muscle contraction.

4 Calmodulin v=lelibchendy As its name suggests, calmodulin is a CALcium MODULated protein. It is abundant in the cytoplasm of all higher cells Calmodulin acts as an intermediary protein that senses calcium levels and relays signals to various calcium-sensitive enzymes, ion channels and other proteins. Calmodulin is a small dumbbell-shaped protein composed of two globular domains connected together by a flexible linker. Each end binds to two calcium ions.

5 Myosin light chain kinase (MLCK) Four different MLCK isoforms exist: MYLK smooth muscle MYLK2 skeletal MYLK3 cardiac MYLK4 novel

6 Myosin light chain kinase (MLCK) Once there is an influx of Ca 2+ into the muscle, either from the sarcoplasmic reticulum or from the extracellular space, contraction of smooth muscle fibres may begin. First, the calcium will bind to calmodulin. This binding will activate MLCK, which will go on to phosphorylate the myosin light chain at serine residue 19. This will enable the myosin crossbridge to bind to the actin filament and allow contraction to begin (through the crossbridge cycle).

7 Myosins Myosins comprise a family of ATP-dependent motor proteins Most myosin molecules are composed of a head, neck, and tail domain. The head domain binds the filamentous actin, and uses ATP hydrolysis to generate force and to "walk" along the filament the neck domain acts as a linker and as a lever arm for transducing force generated by the catalytic motor domain. The neck domain can also serve as a binding site for myosin light chains which are distinct proteins that form part of a macromolecular complex and generally have regulatory functions. The tail domain generally mediates interaction with cargo molecules and/or other myosin subunits. In some cases, the tail domain may play a role in regulating motor activity.

8 Myosins

9 Myosins

10 Myosin II Myosin II is the myosin type responsible for producing muscle contraction in muscle cells. Myosin II contains two heavy chains (about 2000 aa in length) The intermediate neck domain is the region creating the angle between the head and tail. In smooth muscle, a single gene (MYH11) codes for the heavy chains myosin II, but splice variants of this gene result in four distinct isoforms. It also contains 4 light chains, resulting in 2 per head, weighing 20 (MLC 20 ) and 17 (MLC 17 ) kda. These bind the heavy chains in the "neck" region between the head and tail. The MLC 20 is also known as the regulatory light chain and actively participates in muscle contraction The MLC 17 is also known as the essential light chain. Its exact function is unclear, but is believed to contribute to the structural stability of the myosin, head along with MLC 20.

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12 SMOOTH MUSCLE RELAXATION The process of relaxation requires a decreased intracellular Ca 2+ concentration and increased MLC phosphatase activity.

13 Regulation of smooth muscle contraction by caldesmon. At low Ca 2+ concentrations (<10-6 M), caldesmon is a calmodulin binding protein. Like troponin, caldesmon tonically inhibits the ATPase activity of myosin in smooth muscle, reducing the binding of myosin to actin and keeping muscle in the relaxed state. At higher Ca 2+ concentrations, Ca 2+ -calmodulin complex binds to caldesmon, releasing it from actin; thus myosin can interact with actin and the muscle can contract. Phosphorylation by several kinases, including MAP kinase, and dephosphorylation by phosphatases also regulate caldesmon's actin-binding activity.

14 Decrease in the intracellular concentration of Ca 2+ Ca 2+ uptake into the SR is dependent on ATP hydrolysis. This SR Ca,Mg-ATPase, when phosphorylated, binds two Ca 2+ ions, which are then translocated to the luminal side of the SR and released. Mg 2+ is necessary for the activity of the enzyme; it binds to the catalytic site of the ATPase to mediate the reaction. The sarcoplasmic reticular Ca,Mg- ATPase is inhibited by several different pharmacological agents: vanadate, thapsigargin, and cyclopiazonic acid. SR Ca 2+ -binding proteins also contribute to decreased intracellular Ca 2+ levels. Recent studies have identified calsequestrin and calreticulin as SR Ca 2+ - binding proteins in smooth muscle.

15 Decrease in the intracellular concentration of Ca 2+ The plasma membrane also contains Ca,Mg-ATPases, providing an additional mechanism for reducing the concentration of activator Ca 2+ in the cell. This enzyme differs from the SR protein in that it has an autoinhibitory domain that can be bound by calmodulin, causing stimulation of the plasma membrane Ca 2+ pump. Na + /Ca 2+ exchangers are also located on the plasma membrane and aid in decreasing intracellular Ca 2+. This low-affinity antiporter is closely coupled to intracellular Ca 2+ levels and can be inhibited by amiloride and quinidine.

16 Ca 2+ SENSITIZATION MECHANISM AND CONTRACTION OF SMOOTH MUSCLE In addition to the Ca 2+ -dependent activation of MLC kinase, the state of myosin light chain phosphorylation is further regulated by MLC phosphatase, which removes the high-energy phosphate from the light chain of myosin to promote smooth muscle relaxation. MLCP is composed of three subunits: PP1c catalytic subunit (37-kDa), MBS myosin targeting subunit (110- to 130-kDa ) a small subunit of unknown function called M20 (20-kDa). The myosin-binding subunit (MBS), when phosphorylated, inhibits the enzymatic activity of MLC phosphatase, allowing the light chain of myosin to remain phosphorylated, thereby promoting contraction.

17 Ca 2+ SENSITIZATION MECHANISM AND CONTRACTION OF SMOOTH MUSCLE The small G protein RhoA and its downstream target Rho kinase play an important role in the regulation of MLC phosphatase activity. Rho kinase, a serine/threonine kinase, phosphorylates the myosin-binding subunit of MLC phosphatase, inhibiting its activity and thus promoting the phosphorylated state of the myosin light chain.

18 Ca 2+ SENSITIZATION MECHANISM AND CONTRACTION OF SMOOTH MUSCLE Pharmacological inhibitors of Rho kinase, such as fasudil and Y-27632, block its activity by competing with the ATP-binding site on the enzyme. Rho kinase inhibition induces relaxation of isolated segments of smooth muscle contracted to many different agonists. In the intact animal, the pharmacological inhibitors of Rho kinase have been shown to cause relaxation of smooth

19 Χολινεργική διαβίβαση

20 Παρασυµπαθητικό σύστηµα

21 Υποδοχείς ακετυλοχολίνης

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27 M3 signaling pathway

28 M2 signaling pathway

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30 M 3 receptors

31 Λειτουργικός ανταγωνισµός M2-αδρενεργικών υποδοχέων

32 Λειτουργικός ανταγωνισµός M3-αδρενεργικών υποδοχέων

33 Κατανοµή Μ2/Μ3 υποδοχέων στο αναπνευστικό M 2 > M 3 M 1, M 3 M 2, M 3 M 1 M 3

34 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

35 Cell type Presence of muscarinic receptors, ChAT and/or acetylcholine T lymphocyte Muscarinic receptors (M 1 -M 5 ) ChAT Acetylcholine B lymphocyte Muscarinic receptors (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/ monocyte Bronchial epithelium Airway smooth muscle Muscarinic receptors (M 1 /M 2 /M 3 ) ChAT Acetylcholine Muscarinic receptors (M 1 /M 3 ) ChAT Acetylcholine Muscarinic receptors (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

36 Ο ρόλος των επιθηλιακών µουσκαρινικών υποδοχέων

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38 Η επίδραση της ΑΚΧ στον πολλαπλασιασµό

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40 Χολινεργική διαβίβαση στο άσθµα

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43 Neuraminidase IFN-γ NO

44 TNF-a

45 Μεταβολή του φαινότυπου των ΛΜΚ