How Oleic Acid in Olive Oil Reduces Blood Pressure'

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How Oleic Acid in Olive Oil Reduces Blood
Pressure.
Pablo V. Escribá, University of the Balearic
Islands, pablo.escriba_at_uib.es John E. Halver,
University of Washington halver_at_u.washington.edu
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REGULATION OF BLOOD PRESSURE
VASCULAR SMOOTH MUSCLE
PKA
MUSCULAR RELAXATION
VASODILATION
G proteins
PKG
Endothelium-dependent
PKA
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GPCR-associated signaling
GPCR-associated signaling
Blood Pressure is tightly controlled by several
signaling systems. Many of them involve
regulation of cell functions through G
protein-coupled receptors (GPCRs). This slide
shows a Gq protein-associated pathway involved in
the regulation of cytosolic Ca2.
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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G proteins peripheral membrane proteins
G proteins propagate messages from membrane
receptors to effectors that control cytosolic
levels of second messengers (cAMP, cGMP, IP3,
DAG, ions, etc.). Lipid-protein interactions
regulate the localization and activity of G
proteins and, therefore, cell signaling.
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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G PROTEIN-COUPLED RECEPTORS (GPCRs) INVOLVED IN
THE REGULATION OF BP

• CENTRAL NERVOUS SYSTEM ADRENOCEPTORS
  a 2A a2B
• PERIPHERAL NERVOUS SYSTEM
• CARDIAC MUSCLE ADRENOCEPTORS a1 ß1 ß2
• MUSCARINIC RECEPTORS M2
• SMOOTH MUSCLE ADRENOCEPTORS a1
  ß2
• MUSCARINIC
  RECEPTORS M3

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CARDIAC CELL SIGNALING
?1 adrenoceptor
Muscarinic M2
?1 adrenoceptor
Gs protein
Gq11 protein
Gi/Go proteins
() Adenylyl cyclase
() Phospholipase C
(-) Adenylyl cyclase
() K Channels
cAMP
DAG IP3
() Ca2
() PKA
() PKC
Cell hyperpolarization
() Ca2 channels
contractility
contractility
contractility
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VASCULAR CELL SIGNALING
?1 adrenoceptor
Muscarinic M3
?2 adrenoceptor
Nitric Oxide
Gs protein
Gq11 protein
() Guanylyl cyclase
() Adenylyl cyclase
() Phospholipase C
cGMP
cAMP
DAG IP3
() PKG
() Ca2
() PKA
() PKC
Intracellular Ca2
Intracellular Ca2
vasoconstriction
Vasorelaxation
Vasorelaxation
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Membrane lipid structure and G protein-membrane
interactions
Binding of G proteins to model membranes
(liposomes) determined by immunoblotting

• G protein heterotrimers prefer nonlamellar-prone
  membrane regions
• G protein a-monomers prefer lamellar-prone
  regions (higher PC content)

How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Membrane lipid structure and G protein-membrane
interactions

• G protein bg-dimers have a huge preference for
  nonlamellar-prone membrane regions (higher PE
  content).
• They take G protein a-monomers from nearby GPCRs.

Binding of G proteins to model membranes
(liposomes) determined by immunoblotting
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Gg protein-membrane interactions
X-ray analysis
DSC analysis
FTIR analysis
Membrane and aqueous phases
Only in membrane
Gg on membrane structure
Membrane on Gg structure

• C-terminal region of Gg Main player in G
  protein-lipid interactions.
• Membrane lipids affect G protein structure and
  vice versa.

How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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A new model of G protein activity based on their
interactions with lipids
Two views of the same process. From the membrane
(above) or from the cytosol (right) receptors
(R) are activated by agonists (a or AG) in
hexagonal phase-prone regions (H), which are
loaded with G proteins (G) thanks to Gbg. Upon
activation, Ga goes to lamellar-prone regions (L)
to regulate the activity of effectors (E, E1).
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Gg protein-membrane interactions
Altered Gg protein distribution after point
mutations on the C-terminal region (Gg2-GFP
fusion proteins)
Cellular localization of Gg protein-GFP
constructs by confocal microscopy

• C-terminal region of Gg Main player in G
  protein-lipid interactions.

How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Membrane fatty acids
OA
EA
SA
Oleic Acid Elaidic Acid Stearic
Acid 181 n-9 cis 181 n-9 trans
180
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Effect of OA and EA on membrane structure by
X-Ray diffraction

• Oleic acid but not the structurally related fatty
  acids, elaidic and stearic acid, regulates
  membrane lipid structure.

How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Effect of OA and EA on G protein function

• Oleic acid but not the structurally related fatty
  acids, elaidic and stearic acid, regulates G
  protein function.

How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Acute effects of VOO, TO, and OA on systolic BP
Normotensive rats
Olive oil also induces short-term reductions of
BP. Triolein and oleic acid have similar effects
V, vehicle VOO, virgin olive oil TO, triolein
OA, oleic acid
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Chronic effects of OA, elaidic acid and stearic
acid on systolic BP
Normotensive rats
Oleic acid, but not elaidic and stearic acids,
induces BP reductions. Oleic acid is the fatty
acid present in triolein and the main component
of olive oil (70-80).
V, vehicle OA, oleic acid EA, elaidic acid SA,
stearic acid
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Effects of VOO treatment on BP in SHRs
Olive oil and oleic acid (OA) reduce BP in
hypertensive animals. Elaidic acid (EA) and
stearic acid (SA) failed to reduce BP.
Hypertensive rats
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Effects of VOO, TO, and OA treatments on total G
protein ai2, ai3 and aq/11 subunits and PKCß1
levels in aorta
Olive oil, triolein and oleic acid regulate Gi
and Gq protein levels and those of downstream
effectors, such as phospholipase C (PLC).
V, vehicle VOO, virgin olive oil TO, triolein
OA, oleic acid
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Correlations between BP reductions (mmHg) and
decrease of L-to-HII phase transition temperature
(C A) or the dose of cis-MUFA (g/kg)
administered to animals (B)
Treatments 1, VOO 2, 2-hydroxyoleic acid 3,
TO 4 OA 5, stearic acid 6 vehicle 7,
elaidic acid 8, soybean oil.
Decrease of BP correlates with the dose of
cis-monounsaturated fatty acids (cis-MUFA). Fatty
acids, rather than triacylglycerides, are
involved in this effect.
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Consequences for human health

• Consumption of fats that contain
  cis-monounsaturated fatty acids (e.g., oleic
  acid) have positive effects on cardiovascular
  health, compared with saturated or trans-MUFA
  fats.
• Because this effect has structural molecular
  bases, we can design molecules to regulate BP or
  reverse other pathological processes.

How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Then, I can control cell functions by regulating
membrane lipid structure and composition!!!
Sure. You may even design nutritional or
pharmaceutical approaches to treat human
pathologies!!!
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Chronic effects of soybean oil, VOO, or TO
treatments on systolic BP
Olive oil, but not soy oil, reduces blood
pressure. Triolein, the main triacylglyceride in
olive oil reproduces this effect. VOO is the
oil obtained from olives by pressure at room
temperature.
Normotensive rats
V, vehicle SO, soy oil VOO, virgin olive oil
TO, triolein
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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OLEIC ACID ANALOGUES FOR TREATMENT OF BLOOD
PRESSURE
OLEIC ACID ANALOGUES FOR TREATMENT OF BLOOD
PRESSURE
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Rational design of 2-hydroxyoleic acid
Rational design of 2-hydroxyoleic acid
2-Hydroxyoleic acid (left) is an oleic acid
(center) analog that keeps most structural
properties of oleic acid and differs from elaidic
acid (pink) in its interaction with membranes
(right).
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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2-Hydroxyoleic acid on BP in hypertensive animals
2-Hydroxyoleic acid on BP in hypertensive animals
Effect of olive oil
Effects of 2-hydroxyoleic acid (2-OHOA) treatment
(600 mg/kg every 12 h) on blood pressure (BP) and
heart rate in spontaneously hypertensive rats
(SHRs) and Wistar Kyoto (WKY) rats
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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2-Hydroxyoleic acid on BP in hypertensive animals
2-Hydroxyoleic acid on BP in hypertensive animals
Dose-dependent effects of 2-OHOA on systolic BP
in SHRs
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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2OHOA increases elasticity in aorta rings
Rat aorta rings were preincubated 1 hour in the
presence (2OHOA) or absence (control) of
2-hydroxyoleic acid. Then, the contraction in
response to noradrenaline was determined.
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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2-Hydroxyoleic acid has no cytotoxicity
Control
Control
2OHOA
2OHOA
Lung
Aorta
Heart
Kidney
Liver
S.I.
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cGMP is not involved in the effects of
2-hydroxyoleic acid
8
6
protein)
levels in aorta
4
-1
mg

2
(pmol
cGMP
0
WKY
WKY
SHR
SHR
2OHOA
veh
veh
2OHOA
The levels of cGMP in normotensive (WKY) and
hypertensive (SHR) rats were unrelated to
2-hydroxyoleic acid effects on BP
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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2-Hydoxyoleic acid on Gi proteins
48 h
10FBS
OH50
OH100
Serum w/o PDGF
G?i3
actin
BASAL
PDGF
G?i3
Serum with PDGF
actin
2-Hydroxyoleic acid induces reduced expression of
G protein. This effect is PDGF-independent.
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
32
Effects of 2-hydroxyoleic acid treatment (600
mg/kg every 12 h) for 7 days on protein kinase A
(PKA) subunit levels in aortas from SHRs and WKY
rats
2-Hydroxyoleic acid regulates the expression of
some PKA subunits
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Effects of 2-hydroxyoleic acid treatment (600
mg/kg every 12 h) for 7 days on adenylyl cyclase
(AC) activities in SHRs and WKY rat aortas
2-Hydroxyoleic acid treatments induce restoration
of Adenyly cyclase activity in rat aorta.
Adenyly cyclase produces cAMP, which in turn
regulates PKA.
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
34
Effects of 2-OHOA treatment (600 mg/kg every 12
h) for 7 days on PKA activity in aortas from SHRs
and WKY rats
2-Hydroxyoleic acid treatments induce restoration
of PKA activity in aorta of hypertensive rats and
do not change PKA activity in normotensive rats.
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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b-Adrenoceptor signaling pathway
Epinephrine activates b-adrenoceptors, which in
turn activate a Gs protein. Gs proteins activate
adenylyl cyclase, that produce cAMP and activate
PKA.
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
36
Effects of acute administration of the PKA
inhibitor 8-bromo adenosine-3',5'-cyclic
monophosphorothioate, Rp isomer (Rp-8-Br-cAMP),
on systolic BP in vehicle-treated (control) and
2-OHOA-treated SHRs
The use of a specific PKA inhibitor in rats
treated with 2-hydroxyoleic acid reversed the
hypotensive effect of this drug. This result
demonstrates that the proposed mechanism of the
hypotensive effects of 2-hydroxyoleic acid (via
Gs protein, adenyly cyclase and PKA) operates in
vivo. In normotensive animals (treated and
untreated) and hypertensive rats not treated with
2-hydroxyoleic acid, no blood pressure reductions
were observed (data not shown), indicating that
the effect cannot be attributed to the PKA
inhibitor itself. In addition, this effect was
time- and concentration-dependent.
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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Effects of withdrawal of 2-OHOA on systolic BP
and aortic PKA subunit levels in SHRs
After treatments with 2-hydroxyoleic acid, blood
pressure started to rise to reach their initial
high values about two weeks after the
administration of the last dose. This slow
recovery of blood pressure indicates that the
modification induced by the fatty acid occurs on
membrane lipids, whose turnover is slower.
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
38
Effects of 2-OHOA treatment (600 mg/kg every 12
h) for 7 days on Rho kinase II levels in aortas
from SHRs and WKY rats
Rho kinase also appeared to be markedly increased
in hypertensive rats. Treatment with 2-OHOA
reduced the expression of this protein to normal
levels. This result indicates that besides PKA,
vascular cells cytoskeleton is also regulated by
this compound.
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver
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CONCLUSION

• The CIS structure of Oleic Acid fits neatly into
  the phospholipid structure of biomembranes, and
  alters receptor sites on the membrane surface, to
  regulate blood pressure. Q.E.D.

For references seePNAS 10513811(208)
How Oleic Acid in olive oil reduces blood
pressure. P.V. Escribá J.E. Halver