Seminario molecular..! cms & lml

Uric acid attenuates nitric oxide production by decreasing the

interaction between endothelial nitric oxide synthase and calmodulin in human umbilical vein endothelial cells: A mechanism for uric

acid-induced cardiovascular disease development

Jung-Hyun Park, Yoon Mi Jin, Soojin Hwang, Du-Hyong Cho, Duk-Hee Kang, Inho Jo

Carolina Mora SánchezLaura María Moreno León

INTRODUCTION

Cardiovascular Disease CVD’s

Are a group of disorders of the heart and blood vessels

Are the number one cause of death globally

Can be prevented by addressing risk factors.

>Coronary heart disease. >Cerebrovascular disease.

INTRODUCTION

INTRODUCTION

Uric Acid

INTRODUCTION

Most uric acid dissolves in blood and travels to the kidneys, where it passes out in urine. We can use uric acid levels in the diagnosis different pathologies

Is the end product of purine metabolism in humans

In normouricemic humans, the daily production and ingestion of uric acid is well balanced by the daily excretion

INTRODUCTION

hyperuricemia is associated with hypertension cardiovascular diseases.

Uric Acid

INTRODUCTION

Nitric Oxide NO eNOS is responsible for most of the vascular NO produced.

 That increased blood flow by dilating blood vessels

Is a gas and is an important protective molecule in the

vasculature

INTRODUCTION

eNOS

Dysregulation contributes to the pathogenesis of certain diseases Dimerization.

Interaction with calmodulin (CaM) and tetrahydrobiopterin (BH4), Acetylation Phosphorylation.

eNOS is regulated by

INTRODUCTION

Calmodulin CaM

Mediates the control of a large number of enzymes, ion channels and other proteins by Ca2+.

Is a calcium-binding messenger protein expressed in all eukaryotic cells.

Among the enzymes to be stimulated by the calmodulin-Ca2+ complex are a number of protein kinases and phosphatases.

INTRODUCTION

Specific tissue

Bovine aortic endothelial cells (BAEC)

Human umbilical vein EC (HUVEC)

Human plasma and aortic EC – in vitro

Pulmonary artery EC

INTRODUCTION

eNOS CaM in EC

Uric Acid eNOS activity

NO production

Reducing the binding between

GENERAL OBJECTIVE

Explain how uric acid decreases NO production by direct modulations on eNOS, leading to causing cardiovascular problems.

MATERIALES & MÉTODOS

Reactivos

Ácido úrico

eONS bovina recombinante

eNOS, p-eNOS-Thr 495, p-eNOS-Ser1177, p-eNOS- Ser114

Calmodulina recombinante

Fluo-4 AM

Ig CaM

FBS

M200 Ig Lisina acetilada

LSGS DPBS NCS L-glutamina

Penicilina y estreptomicina

MEM

Tripsina EDTA

MATERIALES & MÉTODOS

Cultivo Celular

Método para el estudio del comportamiento de las células, muy adecuado como modelo para el estudio del desarrollo y la diferenciación, por lo que las líneas celulares que conservan la capacidad de diferenciarse

MATERIALES & MÉTODOS

Medición de ON

La producción de ON es medida como concentración de nitrito en la células cultivadas.

MATERIALES & MÉTODOS

Medición de la actividad de la eONS

La cuantificación de la actividad de eNOS se lleva a cabo midiendo la conversión de L-arginina en L citrulina, en presencia de concentraciones saturantes de los cofactores enzimáticos adecuados.

MATERIALES & MÉTODOS

Medición de la actividad de la Xantina oxidasa

Se mide mediante la aparición de

su producto ácido úrico.

MATERIALES & MÉTODOS

Western Blot

Proteínas procedentes de extractos celulares son separados por electroforesis en gel según su tamaño kDa

Técnica: SDS-PAGE policrámida en la que son disueltas en una solución, con el detergente sodio duodecil sulfato.

MATERIALES & MÉTODOS

Ensayo in vitro

Manipulación que se hace con células o sistemas aislados de

células, en el laboratorio con los que se trata de simular las condiciones

que tendrían en los organismo de los que fueron aislados

MATERIALES & MÉTODOS

Canmunoprecipitación

Es una técnica popular para identificar las interacciones proteína-proteína

fisiológicamente relevantes mediante el uso de anticuerpos para determinar la presencia de una proteína específica.

MATERIALES & MÉTODOS

Dimerización

The chemical reaction that

joins two

molecular subunits,

resulting in the formation of

a single dimer.

MATERIALES & MÉTODOS

Arginasa

Es una enzima del ciclo de la urea que cataliza la hidrólisis de la arginina,

produciendo ornitina y urea.

MATERIALES & MÉTODOS

RT-PCR

Amplificación de RNA a través de la síntesis previa de su cDNA (DNA copia),

utilizando una transcriptasa inversa, seguida de varios ciclos de PCR

convencional.

Determinar expresión genética en diversos tejidos.

RESULTADOS

RESULTADOS

RESULTADOS

RESULTADOS

RESULTADOS

RESEARCHER COMMENT AGREE DISAGREE

D.I Feing

¨Epidemiology studies showed that higher blood level of uricacid is significantly associated with the risk of cardiovascular diseases¨.

X

DISCUSSION

RESEARCHER

COMMENT AGREE DISAGREE

I. Papeziková

¨Most recentstudy revealed that uric acid decreased NO production, both indirectlyand directly; by increasing arginase activity and decreasingeNOS-Ser1177 phosphorylation, respectively¨.

X

DISCUSSION

RESEARCHER COMMENT AGREE DISAGREE

C. Nathan

¨CaM is one of the key proteins for eNOS activation. AlthoughCaM binds tightly to eNOS, even at the resting basal levels of intracellularCa2+ levels¨.

X

DISCUSSION

RESEARCHER COMMENT AGREE DISAGREE

J. White

¨increased intracellular Ca2+ byextrastimuli such as ionomycin induced more association of CaMwith eNOS¨.

X

DISCUSSION

• Uric acid inhibits the production of nitric oxide which is a vasodilator important for the prevention of heart disease

• This project can help people to understand how if they do not have healthy habits can cause cardiovascular disease.

CONCLUSIONS

• Ionomycin is used in research to raise the intracellular level of calcium, this help the interaction between eNOs y CaM.

• Uric acid reduced the interaction between CaM and eNOS this produces a disminusion of the production of NO.

CONCLUSIONS

Carolina Mora Sánchez