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Signal transduction and calcium sensitivity of smooth muscle contraction

Bonnevier, Johan LU (2005)
Abstract (Swedish)
Popular Abstract in Swedish

Glatt muskulatur ingår i flera fysiologiskt viktiga organsystem och är involverade i flera sjukdomsprocesser. Glatt muskelkontraktion regleras av variationer i intracellulärt calcium; höjda nivåer aktiverar kinaset MLCK som fosforylerar myosin och initierar kontraktion. Myosin fosfatas (MLCP) avfosforylerar myosin vilket leder till relaxation. Flera intracellulära signalvägar påverkar denna fosforyleringsprocess genom att justera kontraktionens kalciumkänslighet, framförallt genom påverkan på MLCP-aktiviteten. Ett genomgående tema i denna avhandling har varit att studera några av dessa signalvägar och försöka ta reda på dess cellulära mekanismer och fysiologiska roll. I arbete I visar vi med... (More)
Popular Abstract in Swedish

Glatt muskulatur ingår i flera fysiologiskt viktiga organsystem och är involverade i flera sjukdomsprocesser. Glatt muskelkontraktion regleras av variationer i intracellulärt calcium; höjda nivåer aktiverar kinaset MLCK som fosforylerar myosin och initierar kontraktion. Myosin fosfatas (MLCP) avfosforylerar myosin vilket leder till relaxation. Flera intracellulära signalvägar påverkar denna fosforyleringsprocess genom att justera kontraktionens kalciumkänslighet, framförallt genom påverkan på MLCP-aktiviteten. Ett genomgående tema i denna avhandling har varit att studera några av dessa signalvägar och försöka ta reda på dess cellulära mekanismer och fysiologiska roll. I arbete I visar vi med hjälp av kalciummätningar med Fura-2 att den cell permeabla cGMP-analogen 8-Br-cGMP relaxerar glatt tarmmuskulatur genom att de intracellulära kalciumnivåerna sänks, men även genom att sänka systemets kalciumkänslighet. Genom att använda möss som saknar genen för cGMP-aktiverat kinas (cGK) i glatt muskel, kunde vi visa att cGMP relaxationen beror på aktivering av cGK. Vi visade genom att använda ?-toxin från staphylococcus aureus att cGMP nedreglerar kalciumkänsligheten och inducerar relaxation i koncentrationer i nanomolar nivå. Korsaktivering av cAMP aktiverat kinas (PKA) krävde tusenfaldigt högre koncentrationer. I arbete II påvisade vi en ny relaxationsmekanism för cGK som relaxerar glatt muskel genom att sänka dess kalciumkänslighet och sänka fosforyleringsgraden av det MLCPreglerande proteinet CPI-17. I arbete III undersökte vi subenhetexpressionen av MLCP i hypertrof glatt tarmmuskelvävnad. En ökning i ration mellan CPI-17 och MLCPs katalytiska subenhet kunde associeras med ökade svar vid aktivering av protein kinas C (PKC), Rho kinas (ROCK) cGK signalvägarna. Hypertrof vävnad hade ökad agonistkänslighet, vilket tyder på att de förändrade intracellulära signalvägarna leder till ökad retbarhet i tarmen. I arbete IV undersökte vi vilken roll förändrad kalciumkänslighet har i upprätthållandet av den toniska fasen i agonistmedierade kärlkontraktioner. Genom att ersätta kalcium med strontium fann vi att en ökning av kalciumkänsligheten inte är tillräcklig för att upprätthålla denna toniska fas som istället upprätthålls med hjälp av en kalciumspecifik mekanism som depolariserar cellmembranet. Signalvägarna som påverkar kalciumkänslighet är beroende av att signalmolekylerna kan kopplas samman och signalöverföring ske. Strukturer som föreslagits vara dockningsplattformar för flera av signalvägarna som påverkar kalciumkänslighet är caveolae. I arbete V hade vi tillgång till möss som helt saknar det caveolaestabiliserande proteinet caveolin-1. Vi kunde visa att dessa möss helt saknar caveolae. Receptorsvar efter stimulering med endothelin-1 var attenuerade i avsaknad av caveolin-1 och caveolae. Ökningen i kalciumkänslighet efter aktivering av Rho kinas respektive protein kinas C signalvägarna var emellertid helt oförändrad, vilket tyder på att caveolae främst är viktiga för att reglera de intracellulära kalciumnivåerna i den glatta muskulaturen. (Less)
Abstract
Smooth muscles are components of several physiologically important organs and often involved in disease. Smooth muscle contraction is regulated by variations in intracellular [Ca2+]; increased levels activate the myosin light chain kinase (MLCK) which phosphorylates myosin and initiates contraction. Myosin phosphatase (MLCP) dephosphorylates myosin and promotes relaxation. Several cellular signaling pathways modulate the phosphorylation process by altering the Ca2+ sensitivity, mainly by influencing the MLCP activity. A general objective of this thesis was to study the cellular mechanisms and the physiological role of some of these pathways. In (I) we show, using Fura-2 measurements of intracellular [Ca2+], that the cell permeable cGMP... (More)
Smooth muscles are components of several physiologically important organs and often involved in disease. Smooth muscle contraction is regulated by variations in intracellular [Ca2+]; increased levels activate the myosin light chain kinase (MLCK) which phosphorylates myosin and initiates contraction. Myosin phosphatase (MLCP) dephosphorylates myosin and promotes relaxation. Several cellular signaling pathways modulate the phosphorylation process by altering the Ca2+ sensitivity, mainly by influencing the MLCP activity. A general objective of this thesis was to study the cellular mechanisms and the physiological role of some of these pathways. In (I) we show, using Fura-2 measurements of intracellular [Ca2+], that the cell permeable cGMP analogue 8-Br-cGMP induced relaxation in intestinal smooth muscle by lowering intracellular [Ca2+] and decreasing Ca2+ sensitivity. Using transgenic mice lacking cGMP activated kinase in smooth muscle, we show that the cGMP relaxant effect was due to activation of cGK. Using tissue permeabilized with staphylococcus aureus ?-toxin, we show that cGMP induced Ca2+ desensitization and relaxation in a nano-molar concentration interval. Cross-activation of cAMP dependent kinase by cGMP was minimal and occurred at ~1000-fold higher cGMP concentrations. In (II) we show that cGK relaxed by reversing Ca2+ sensitization via a novel relaxant pathway involving dephosphorylation of a regulatory protein, CPI-17. In (III) the subunit composition of the MLCP was examined in hypertrophic intestinal tissue. An increase in the ratio of CPI-17/relative to the catalytic subunit of MLCP was associated with increased responsiveness to protein kinase C, Rho kinase and cGK. Hypertrophic tissue had increased responsiveness to contractile agonist, suggesting that altered cellular signaling influences the reactivity of the intestine. In (IV) the role of Ca2+ sensitization mechanisms during agonist induced tonic contractions in vascular tissue was examined. Using replacement of Ca2+ with Sr2+, we show that Ca2+ sensitization was not sufficient to maintain tonic force. Ca2+ dependent steps causing membrane depolarization are needed. Ca2+ sensitization is influenced by cellular targeting of the signaling molecules. Structures of proposed importance are the membrane associated caveolae. In (V) caveolin-1 deficient mice were used. We show that smooth muscle from these animals completely lack caveolae. The receptor mediated responses to endothelin-1 were attenuated in intestinal tissue from these mice. However, Ca2+ sensitization via small G-proteins and protein kinase C was unaffected, suggesting that caveolae primarily affect steps involved in Ca2+ elevation. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • professor Aalkjaer, Christian, Aarhus universitet, Fysiologisk institut.
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Fysiologi, Physiology, hypertrophy, CPI-17, MYPT1, myosin phosphatase, Rho kinase, PKC, smooth muscle, cGK
pages
112 pages
publisher
Department of Experimental Medical Science, Lund Univeristy
defense location
Segerfalksalen, Wallenberg Neurocentrum, BMC A, Lund.
defense date
2005-05-04 09:15
ISSN
1652-8220
ISBN
91-85439-29-0
language
English
LU publication?
yes
id
71a673ac-a3f6-42fa-825c-60f7625462de (old id 544722)
date added to LUP
2007-09-10 11:28:17
date last changed
2016-09-19 08:44:52
@phdthesis{71a673ac-a3f6-42fa-825c-60f7625462de,
  abstract     = {Smooth muscles are components of several physiologically important organs and often involved in disease. Smooth muscle contraction is regulated by variations in intracellular [Ca2+]; increased levels activate the myosin light chain kinase (MLCK) which phosphorylates myosin and initiates contraction. Myosin phosphatase (MLCP) dephosphorylates myosin and promotes relaxation. Several cellular signaling pathways modulate the phosphorylation process by altering the Ca2+ sensitivity, mainly by influencing the MLCP activity. A general objective of this thesis was to study the cellular mechanisms and the physiological role of some of these pathways. In (I) we show, using Fura-2 measurements of intracellular [Ca2+], that the cell permeable cGMP analogue 8-Br-cGMP induced relaxation in intestinal smooth muscle by lowering intracellular [Ca2+] and decreasing Ca2+ sensitivity. Using transgenic mice lacking cGMP activated kinase in smooth muscle, we show that the cGMP relaxant effect was due to activation of cGK. Using tissue permeabilized with staphylococcus aureus ?-toxin, we show that cGMP induced Ca2+ desensitization and relaxation in a nano-molar concentration interval. Cross-activation of cAMP dependent kinase by cGMP was minimal and occurred at ~1000-fold higher cGMP concentrations. In (II) we show that cGK relaxed by reversing Ca2+ sensitization via a novel relaxant pathway involving dephosphorylation of a regulatory protein, CPI-17. In (III) the subunit composition of the MLCP was examined in hypertrophic intestinal tissue. An increase in the ratio of CPI-17/relative to the catalytic subunit of MLCP was associated with increased responsiveness to protein kinase C, Rho kinase and cGK. Hypertrophic tissue had increased responsiveness to contractile agonist, suggesting that altered cellular signaling influences the reactivity of the intestine. In (IV) the role of Ca2+ sensitization mechanisms during agonist induced tonic contractions in vascular tissue was examined. Using replacement of Ca2+ with Sr2+, we show that Ca2+ sensitization was not sufficient to maintain tonic force. Ca2+ dependent steps causing membrane depolarization are needed. Ca2+ sensitization is influenced by cellular targeting of the signaling molecules. Structures of proposed importance are the membrane associated caveolae. In (V) caveolin-1 deficient mice were used. We show that smooth muscle from these animals completely lack caveolae. The receptor mediated responses to endothelin-1 were attenuated in intestinal tissue from these mice. However, Ca2+ sensitization via small G-proteins and protein kinase C was unaffected, suggesting that caveolae primarily affect steps involved in Ca2+ elevation.},
  author       = {Bonnevier, Johan},
  isbn         = {91-85439-29-0},
  issn         = {1652-8220},
  keyword      = {Fysiologi,Physiology,hypertrophy,CPI-17,MYPT1,myosin phosphatase,Rho kinase,PKC,smooth muscle,cGK},
  language     = {eng},
  pages        = {112},
  publisher    = {Department of Experimental Medical Science, Lund Univeristy},
  school       = {Lund University},
  title        = {Signal transduction and calcium sensitivity of smooth muscle contraction},
  year         = {2005},
}