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Expression and Regulation of P2 Receptors in Human Circulation

Wang, Lingwei LU (2004)
Abstract (Swedish)
Popular Abstract in Swedish

På senare tid har ett stort antal receptorer för extracellulära nukleotider (P2 receptorer) klonats vilka förmedlar en mängd effekter i hjärtkärlsystemet: inotropa effekter på hjärtat, trombocytaggregation, blodtrycksreglering, frisättning av endoteliala faktorer och stimulering av glatt muskeltillväxt. Redan idag står det klart att P2-receptorer är fysiologiskt och patofysiologiskt av stor betydelse. De första P2-receptorblockerarna och klopidogrel används kliniskt som trombocythämmare vid ischemisk hjärtsjukdom och stroke. Den här studien har bidragit till viktig kunskap om uttryck och regleringar av P2 receptorer på mänskliga glatta muskelceller, endotelceller, trombocyter och röda... (More)
Popular Abstract in Swedish

På senare tid har ett stort antal receptorer för extracellulära nukleotider (P2 receptorer) klonats vilka förmedlar en mängd effekter i hjärtkärlsystemet: inotropa effekter på hjärtat, trombocytaggregation, blodtrycksreglering, frisättning av endoteliala faktorer och stimulering av glatt muskeltillväxt. Redan idag står det klart att P2-receptorer är fysiologiskt och patofysiologiskt av stor betydelse. De första P2-receptorblockerarna och klopidogrel används kliniskt som trombocythämmare vid ischemisk hjärtsjukdom och stroke. Den här studien har bidragit till viktig kunskap om uttryck och regleringar av P2 receptorer på mänskliga glatta muskelceller, endotelceller, trombocyter och röda blodkroppar.



Delarbete I. För att studera mRNA och protein uttryck av P2 receptorer på glatta muskelceller och endotelceller, satte vi upp metoder att kvantifiera mRNA nivåer med hjälp av realtids-PCR och mäta protein nivåer med Western blot. Resultaten visar att P2X1, P2Y2, P2Y12 och P2Y6 är de högst uttryckta P2 receptorerna på glatta muskelceller och kanske viktiga för sammandragande och tillväxtstimulerande effekter av extracellulära nukleotider. P2X4, P2Y11, P2Y1 och P2Y2 är de högst uttryckta P2 receptorerna på endotelceller och kan troligen medverka i utsöndringen av signalsubstanser, såsom NO, PG, EDHF, tPA, vilka gör att kärlväggens glatta muskelceller slappnar av, blodkärlet vidgas och det endogena fibrinolytiska systemet aktiveras.



Delarbete II. Med en ny datorstyrd biomedicinsk modell undersöktes hur P2 receptorer påverkas vid shear stress i glatta muskelceller i blodkärl med intakt endotel. Shear stress reglerade genuttryck på glatta muskelceller mer än på endotelceller i det intakta blodkärlet. Minskat uttryck av de sammandragande P2X1 receptorerna kan vara av betydelse för att reducera blodkärlens tonus och öka blodflödet. Eftersom P2Y2 och P2Y6 receptorer stimulerar tillväxt och migrering av glatta muskelceller, kan ökat uttryck av dessa receptorer kan kanske bildra till blodkärlens ombildning vid shear stress. Det kan förklara varför högt flöde framkallar en ökad kärldiameter och en allmänförstoring av kärlet.



Delarbete III. Vi har satt upp en ny metod för att kvantifiera mRNA nivåer på trombocyter och studera uttrycket av P2 receptorer på mänskliga trombocyter. Resultaten visar att P2Y12, P2X1 och P2Y1 är de viktigaste P2 receptorerna på mänskliga trombocyter. Fortsatta studier och utvecklingen av selektiva antagonister mot P2Y12, P2X1 och P2Y1 receptorer kommer att förhoppningsvis leda till utvecklingen av nya trombocythämmande läkemedel. Dessutom, metoden kan användas att utreda uttryck av mRNA nivåer vilka som helst i mänskliga trombocyter.



Delarbete IV. I den här studien analyserade vi P2 receptorer hos SLE-patienter. En avtagande nivå av P2Y12 receptorer upptäcktes hos de här patienterna och kan antagas vara en skyddsrespons mot trombotiska komplikationer. Det kan också eventuellt förklara en ökad blödningsrisk hos SLE-patienter.



Delarbete V. Vi har satt upp en ny metod för att kvantifiera mRNA nivåer på röda blodkroppar och utreda P2 receptorernas roll i reglering av extracellulära ATP nivåer. Stimulering av P2Y13 med ADP, som degraderas från ATP, kunde hämma ATP frisättning från erytrocyter. Den negativa återkopplingen kan vara mycket viktig i kontrollen av ATP nivåer i blodplasma och för kontroll av hur blodförsörjningen till olika organ regleras. (Less)
Abstract
P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP and UDP regulating several physiological responses including cardiac function, vascular tone, smooth muscle cell (SMC) proliferation, platelet aggregation, and the release of endothelial factors. The aim of the present study was to investigate the mRNA and protein expression of P2 receptors in human SMCs, endothelial cells (ECs), erythrocytes and platelets, the regulation of P2 receptors under shear stress in human vessels, the changes of platelet P2 receptors in patients with systemic lupus erythematosus (SLE) and the regulation of plasma ATP levels by P2 receptor on erythrocytes. We established methods to quantify P2 receptors in mRNA and protein levels in... (More)
P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP and UDP regulating several physiological responses including cardiac function, vascular tone, smooth muscle cell (SMC) proliferation, platelet aggregation, and the release of endothelial factors. The aim of the present study was to investigate the mRNA and protein expression of P2 receptors in human SMCs, endothelial cells (ECs), erythrocytes and platelets, the regulation of P2 receptors under shear stress in human vessels, the changes of platelet P2 receptors in patients with systemic lupus erythematosus (SLE) and the regulation of plasma ATP levels by P2 receptor on erythrocytes. We established methods to quantify P2 receptors in mRNA and protein levels in human SMCs, ECs, erythrocytes and platelets. Using a novel computerized biomechanical perfusion model, we examined how shear stress regulates P2 receptor expression in human umbilical vein SMCs and ECs. Using microdialysis we established a method to measure ATP demonstrating an important negative feedback system in the control of plasma ATP levels. Our results indicate that P2X1, P2Y2, P2Y12 and P2Y6 are the most expressed P2 receptors in SMCs and are thus probably mediating the contractile and mitogenic actions of extracellular nucleotides. The P2X4, P2Y11, P2Y1 and P2Y2 are the most expressed P2 receptors in ECs and are most likely mediating release of nitric oxide, endothelium dependent hyperpolarizing factor (EDHF) and t-PA induced by extracellular nucleotides. An oligomerisation may occur for the P2Y1 receptor. Shear stress may regulate gene expression in SMCs more than in the endothelium in intact vessels. Decreased expression of the contractile P2X1 receptor in high shear stress could lead to reduced vascular tonus and increased blood flow. Because P2Y2 and P2Y6 receptors stimulate growth and migration of SMCs, the increased expression of these receptors in high shear stress could promote vascular remodeling induced by shear stress. The upregulation of mitogenic P2Y receptors and downregulation of contractile P2X1 receptors in high shear stress is similar to changes seen in the phenotypic shift from a contractile to synthetic SMCs. The first protocol for quantifying mRNA expression in human platelets was developed limiting the P2 receptor drug development targets to P2Y12, P2Y1 and P2X1. Furthermore, the method could be used to study platelet expression for any gene in human materials. mRNA levels in platelets from patients with SLE was compared with controls. A decreased P2Y12 expression at mRNA and protein levels could represent a protective response in SLE against thrombotic complications. Quantifying mRNA expression in human erythrocytes demonstrated selective high expression of P2Y13 receptors. The ATP degradation product ADP activated P2Y13 receptors resulting in inhibition of ATP release. This negative feedback system could be important in the control of plasma ATP levels and tissue circulation. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Ass. Prof Leipziger, Jens, Institute of Physiology, Aarhus University, Denmark
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Western blotting, Cardiovascular system, Kardiovaskulära systemet, vascular remodeling, systemic lupus erythematosus, smooth muscle cell, shear stress, real-time PCR, platelets, P2 receptor, gene expression, erythrocyte, ATP release, endothelium
pages
151 pages
publisher
Lingwei Wang, Dep. of Cardiology, Lund University Hospital, Lund , Sweden,
defense location
Segerfalksalen, Wallenberg Neuroscience Center, Lund, Sweden.
defense date
2004-05-18 10:15
ISBN
91-628-5959-5
language
English
LU publication?
yes
id
37c6b6b8-5c80-49f4-a0bc-55a8d95274d3 (old id 466884)
date added to LUP
2007-09-26 16:35:25
date last changed
2016-09-19 08:45:11
@phdthesis{37c6b6b8-5c80-49f4-a0bc-55a8d95274d3,
  abstract     = {P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP and UDP regulating several physiological responses including cardiac function, vascular tone, smooth muscle cell (SMC) proliferation, platelet aggregation, and the release of endothelial factors. The aim of the present study was to investigate the mRNA and protein expression of P2 receptors in human SMCs, endothelial cells (ECs), erythrocytes and platelets, the regulation of P2 receptors under shear stress in human vessels, the changes of platelet P2 receptors in patients with systemic lupus erythematosus (SLE) and the regulation of plasma ATP levels by P2 receptor on erythrocytes. We established methods to quantify P2 receptors in mRNA and protein levels in human SMCs, ECs, erythrocytes and platelets. Using a novel computerized biomechanical perfusion model, we examined how shear stress regulates P2 receptor expression in human umbilical vein SMCs and ECs. Using microdialysis we established a method to measure ATP demonstrating an important negative feedback system in the control of plasma ATP levels. Our results indicate that P2X1, P2Y2, P2Y12 and P2Y6 are the most expressed P2 receptors in SMCs and are thus probably mediating the contractile and mitogenic actions of extracellular nucleotides. The P2X4, P2Y11, P2Y1 and P2Y2 are the most expressed P2 receptors in ECs and are most likely mediating release of nitric oxide, endothelium dependent hyperpolarizing factor (EDHF) and t-PA induced by extracellular nucleotides. An oligomerisation may occur for the P2Y1 receptor. Shear stress may regulate gene expression in SMCs more than in the endothelium in intact vessels. Decreased expression of the contractile P2X1 receptor in high shear stress could lead to reduced vascular tonus and increased blood flow. Because P2Y2 and P2Y6 receptors stimulate growth and migration of SMCs, the increased expression of these receptors in high shear stress could promote vascular remodeling induced by shear stress. The upregulation of mitogenic P2Y receptors and downregulation of contractile P2X1 receptors in high shear stress is similar to changes seen in the phenotypic shift from a contractile to synthetic SMCs. The first protocol for quantifying mRNA expression in human platelets was developed limiting the P2 receptor drug development targets to P2Y12, P2Y1 and P2X1. Furthermore, the method could be used to study platelet expression for any gene in human materials. mRNA levels in platelets from patients with SLE was compared with controls. A decreased P2Y12 expression at mRNA and protein levels could represent a protective response in SLE against thrombotic complications. Quantifying mRNA expression in human erythrocytes demonstrated selective high expression of P2Y13 receptors. The ATP degradation product ADP activated P2Y13 receptors resulting in inhibition of ATP release. This negative feedback system could be important in the control of plasma ATP levels and tissue circulation.},
  author       = {Wang, Lingwei},
  isbn         = {91-628-5959-5},
  keyword      = {Western blotting,Cardiovascular system,Kardiovaskulära systemet,vascular remodeling,systemic lupus erythematosus,smooth muscle cell,shear stress,real-time PCR,platelets,P2 receptor,gene expression,erythrocyte,ATP release,endothelium},
  language     = {eng},
  pages        = {151},
  publisher    = {Lingwei Wang, Dep. of Cardiology, Lund University Hospital, Lund , Sweden,},
  school       = {Lund University},
  title        = {Expression and Regulation of P2 Receptors in Human Circulation},
  year         = {2004},
}