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How Oleic Acid in Olive Oil Reduces Blood Pressure. Pablo V. Escribá, University of the Balearic Islands, John E. Halver, University.

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Presentation on theme: "How Oleic Acid in Olive Oil Reduces Blood Pressure. Pablo V. Escribá, University of the Balearic Islands, John E. Halver, University."— Presentation transcript:

1 How Oleic Acid in Olive Oil Reduces Blood Pressure. Pablo V. Escribá, University of the Balearic Islands, John E. Halver, University of Washington

2 VASCULAR SMOOTH MUSCLE AMP c GMP c PKA PKG MUSCULAR RELAXATION VASODILATION CROSS-TALK Endothelium- dependent REGULATION OF BLOOD PRESSURE AC AMP c PKA G proteins

3 How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 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 Ca 2+.

4 How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver G proteins: peripheral membrane proteins G proteins propagate messages from membrane receptors to effectors that control cytosolic levels of second messengers (cAMP, cGMP, IP 3, DAG, ions, etc.). Lipid-protein interactions regulate the localization and activity of G proteins and, therefore, cell signaling.

5 G PROTEIN-COUPLED RECEPTORS (GPCRs) INVOLVED IN THE REGULATION OF BP CENTRAL NERVOUS SYSTEM : ADRENOCEPTORS α 2A α 2B PERIPHERAL NERVOUS SYSTEM : CARDIAC MUSCLE: ADRENOCEPTORS α 1 β 1 β 2 MUSCARINIC RECEPTORS M 2 SMOOTH MUSCLE: ADRENOCEPTORS α 1 β 2 MUSCARINIC RECEPTORS M 3

6 CARDIAC CELL SIGNALING  1 adrenoceptor Gs protein (+) Adenylyl cyclase cAMP (+) PKA contractility Gq 11 protein (+) Phospholipase C DAG IP 3 (+) PKC (+) Ca 2+ contractility Muscarinic M 2 Gi/Go proteins (+) Ca 2+ channels contractility (-) Adenylyl cyclase ( + ) K + Channels Cell hyperpolarization  1 adrenoceptor

7 VASCULAR CELL SIGNALING  1 adrenoceptor Gs protein (+) Adenylyl cyclase cAMP (+) PKA Vasorelaxation Gq 11 protein (+) Phospholipase C DAG IP 3 (+) PKC (+) Ca 2+ vasoconstriction Muscarinic M 3 Nitric Oxide Intracellular Ca 2+ Vasorelaxation (+) Guanylyl cyclase  2 adrenoceptor cGMP (+) PKG Intracellular Ca 2+

8 Membrane lipid structure and G protein-membrane interactions How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver  G protein heterotrimers prefer nonlamellar-prone membrane regions  G protein  -monomers prefer lamellar-prone regions ( higher PC content ) Binding of G proteins to model membranes (liposomes) determined by immunoblotting

9 Membrane lipid structure and G protein-membrane interactions  G protein  -dimers have a huge preference for nonlamellar-prone membrane regions ( higher PE content ).  They take G protein  - monomers from nearby GPCRs. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver Binding of G proteins to model membranes (liposomes) determined by immunoblotting

10 G  protein-membrane interactions Membrane on G  structure Membrane and aqueous phases Only in membrane G  on membrane structure FTIR analysis X-ray analysisDSC analysis How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver  C-terminal region of G  : Main player in G protein-lipid interactions.  Membrane lipids affect G protein structure and vice versa.

11 How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 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 G . Upon activation, G  goes to lamellar-prone regions (L) to regulate the activity of effectors (E, E1).

12 G  protein-membrane interactions How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver  C-terminal region of G  : Main player in G protein-lipid interactions. GFP G  2 - GFP Nuclei Cells Merge WTMutC1 MutC3MutC4 Altered G  protein distribution after point mutations on the C-terminal region (G  2 -GFP fusion proteins) Cellular localization of G  protein-GFP constructs by confocal microscopy

13 Membrane fatty acids Oleic Acid Elaidic AcidStearic Acid 18:1 n-9 cis 18:1 n-9 trans 18:0 How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver OA EASA

14 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 DEPE DEPE:OA (20:1, mole:mole) DEPE:EA (20:1, mole:mole)

15 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

16 Acute effects of VOO, TO, and OA on systolic BP How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver Normotensive rats V, vehicle; VOO, virgin olive oil; TO, triolein; OA, oleic acid Olive oil also induces short- term reductions of BP. Triolein and oleic acid have similar effects

17 Chronic effects of OA, elaidic acid and stearic acid on systolic BP How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver Normotensive rats V, vehicle; OA, oleic acid; EA, elaidic acid; SA, stearic acid 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%).

18 Effects of VOO treatment on BP in SHRs How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver Hypertensive rats Olive oil and oleic acid (OA) reduce BP in hypertensive animals. Elaidic acid (EA) and stearic acid (SA) failed to reduce BP.

19 Effects of VOO, TO, and OA treatments on total G protein αi 2, αi 3 and α q/11 subunits and PKCβ1 levels in aorta How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 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

20 Correlations between BP reductions (mmHg) and decrease of L-to-H II phase transition temperature (°C; A) or the dose of cis-MUFA (g/kg) administered to animals (B) How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 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.

21 Consequences for human health 1.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. 2.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

22 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!!!

23 Chronic effects of soybean oil, VOO, or TO treatments on systolic BP How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver Normotensive rats V, vehicle; SO, soy oil; VOO, virgin olive oil; TO, triolein 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.

24 OLEIC ACID ANALOGUES FOR TREATMENT OF BLOOD PRESSURE How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

25 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).

26 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 2-Hydroxyoleic acid on BP in hypertensive animals Effect of olive oil

27 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 2-Hydroxyoleic acid on BP in hypertensive animals

28 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

29 2-Hydroxyoleic acid has no cytotoxicity Lung Kidney Liver Control 2OHOA Control 2OHOA Aorta Heart S.I.

30 How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver WKY veh WKY 2OHOA SHR veh SHR 2OHOA ** cGMP levels in aorta (pmol ·mg protein) cGMP is not involved in the effects of 2-hydroxyoleic acid The levels of cGMP in normotensive (WKY) and hypertensive (SHR) rats were unrelated to 2- hydroxyoleic acid effects on BP

31 2-Hydoxyoleic acid on Gi proteins Serum w/o PDGF Serum with PDGF G  i3 actin G  i3 actin 48 h BASAL 10%FBSOH50OH100 PDGF How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 2-Hydroxyoleic acid induces reduced expression of G protein. This effect is PDGF-independent.

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 How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 2-Hydroxyoleic acid regulates the expression of some PKA subunits

33 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 How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 2-Hydroxyoleic acid treatments induce restoration of Adenyly cyclase activity in rat aorta. Adenyly cyclase produces cAMP, which in turn regulates PKA.

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 How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 2-Hydroxyoleic acid treatments induce restoration of PKA activity in aorta of hypertensive rats and do not change PKA activity in normotensive rats.

35 How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver  -Adrenoceptor signaling pathway Epinephrine activates  -adrenoceptors, which in turn activate a Gs protein. Gs proteins activate adenylyl cyclase, that produce cAMP and activate PKA.

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 How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 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.

37 Effects of withdrawal of 2-OHOA on systolic BP and aortic PKA subunit levels in SHRs How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 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.

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 How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 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 cell’s cytoskeleton is also regulated by this compound.

39 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. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver For references see:PNAS 105;13811(208)


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