Antihypertensive action of 2-hydroxyoleic acid in SHRs via modulation of the protein kinase A pathway and Rho kinase.

Olive oil consumption leads to high monounsaturated fatty acid intake, especially oleic acid, and has been associated with a reduced risk of hypertension. However, the molecular mechanisms and contribution of its different components to lower blood pressure (BP) require further evaluation. Here, we examined whether a synthetic, non-β-oxidation-metabolizable derivative of oleic acid, 2-hydroxyoleic acid (2-OHOA), can normalize BP in adult spontaneously hypertensive rats (SHRs) and whether its antihypertensive action involves cAMP-dependent protein kinase A (PKA) and Rho kinase, two major regulators of vascular smooth muscle contraction. Oral administration of 2-OHOA to SHRs induced sustained systolic BP decreases in a time-dependent (1–7 days) and dose-dependent (100–900 mg/kg every 12 h) manner. After 7 days of treatment with 2-OHOA (600 mg/kg), the systolic BP of SHRs was similar to that of normotensive Wistar Kyoto rats, returning to its initial hypertensive level after withdrawal of 2-OHOA. This treatment strongly increased the protein expression of the catalytic and regulatory RIα and RIIα PKA subunits as well as PKA activity in aortas from SHRs. Consistently, administration of the PKA inhibitor 8-bromo adenosine-3′,5′-cyclic monophosphorothioate, Rp isomer, to 2-OHOA-treated SHRs induced a pronounced reversal (up to 59%) of the antihypertensive effect of 2-OHOA. Additionally, 2-OHOA completely reversed the pathological overexpression of aortic Rho kinase found in SHRs, suppressing the vasoconstrictory Rho kinase pathway.

The Mediterranean diet is characterized by a high consumption of virgin olive oil, which contains a great proportion of the natural MUFA oleic acid (z80%). Epidemiological studies suggest that this diet provides cardiovascular benefits, including the reduction of blood pressure (BP), both in normal and hypertensive subjects (1,2). Moreover, it has been shown that long-term intake of high doses of olive oil has an inverse association with the risk of developing hypertension (3,4). There has been considerable research effort directed toward analyzing the effects of MUFA-rich diets, focusing on plasma lipids and the lipid/FA composition of cell membranes (5)(6)(7)(8), but the specific molecular mechanisms underlying the beneficial effects of oleic acid and related compounds are still not completely understood.
The increase of BP in essential hypertension is associated with increased peripheral vascular resistance, which is caused by functional and structural alterations of resistance arteries (9,10). During agonist-induced contraction of vascular smooth muscle cells, phosphorylation of myosin light chain (MLC) is a crucial step for force development (11). In this process, the extent of MLC phosphorylation depends on the equilibrium between the activity of Ca 21 -calmodulin-dependent MLC kinase and the MLC phosphatase. Moreover, contraction can also occur independently of changes in intracellular Ca 21 by regulation of the activity of MLC phosphatase, a phenomenon called Ca 21 sensitization (12). Activation of G s -coupled receptors (e.g., b 2 -adrenergic and prostacyclin receptors) induces smooth muscle relaxation through stimulation of adenylyl cyclase (AC) and an increase in cAMP levels. Subsequent activation of cAMP-dependent protein kinase A (PKA) phosphorylates pivotal regulatory proteins, which then act to decrease intracellular Ca 21 levels, finally leading to vasodilation (13). Furthermore, PKA also mediates Ca 21 -desensitizing effects in smooth muscle by blocking the inhibitory phosphorylation mechanism for MLC phosphatase (14). Accordingly, defects in receptor systems that mediate vasorelaxation via the activation of PKA are considered to play an important role in the pathogenesis and maintenance of increased peripheral vascular resistance, which is characteristic for the hypertensive state in humans (15)(16)(17) and in adult spontaneously hypertensive rats (SHRs) with established hypertension (18,19).
Alterations in the Rho A/Rho kinase pathway, leading to the inhibition of MLC phosphatase, also play a central role in the agonist-induced Ca 21 sensitization and hypercontraction of smooth muscle found in hypertensive rats (20) and humans (21). Expression and activity of Rho kinase were found augmented in blood vessels of SHRs (22,23), and administration of the Rho kinase inhibitor Y-27632 reduced BP in various rat models of hypertension without affecting normal BP (24). In addition, the Rho kinase inhibitor fasudil increased forearm blood flow and decreased forearm vascular resistance in hypertensive patients more strongly than in normotensive humans (21), clearly demonstrating that Rho A/Rho kinase-mediated signaling is involved in the pathogenesis of hypertension.
In this work, we examined for the first time whether the MUFA 2-hydroxyoleic acid (2-OHOA), a synthetic derivative of oleic acid with a hydroxyl group on the a-carbon that avoids its rapid degradation through the b-oxidation pathway (25), in fact exhibits antihypertensive properties in the adult SHR, an animal model with established hypertension, compared with its normotensive counterpart, the Wistar Kyoto (WKY) rat, and whether the potential mechanisms mediating its antihypertensive action involve PKA and Rho kinase.

Animals, treatments, and BP measurements
Male SHRs and age-matched normotensive WKY rats (16 weeks old, 250-300 g) were obtained from Charles River Laboratories (Barcelona, Spain). The rats were kept at a constant temperature (24 6 18C) with a 12 h dark/light cycle. SHRs and WKY rats were randomly assigned to a control group (vehicle; n 5 25) or a 2-OHOA group [racemic mixture of (R)-and (S)-2-OHOA (Avanti Polar Lipids); 100-900 mg/kg, resuspended in water; n 5 38]. Rats were treated orally by gavage every 12 h for 7 days. Body weight was recorded and BP was measured at least in triplicate 6 h after drug administration in warmed, restrained conscious rats, using the tail-cuff method with a computerized oscillometric system recorder (Nyprem system from Cibertec, Barcelona, Spain) (26). Eighteen hours after the last treatment, rats were euthanized by decapitation, and their aortas were removed and immediately frozen in liquid nitrogen and stored at 2808C. Another group of SHRs received a single intraperitoneal injection of 10-30 mg/kg 8-bromo adenosine-39,59-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-cAMP) (Biolog, Bremen, Germany), a specific membrane-permeant inhibitor of PKA (27), after 7 days of treatment with vehicle (n 5 9) or 2-OHOA (600 mg/kg every 12 h; n 5 11). Subsequent changes in systolic BP were monitored during 180 min. All experiments were carried out according to the Institutional Committee of Ethics in Animal Research (Comissió de Bioètica de la Universitat de les Illes Balears).

Preparation of aortic homogenates and membranes
Frozen aortas were pulverized in liquid nitrogen and then homogenized in a buffer containing ice-cold 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 mM MgCl 2 , 1 mM PMSF, and 5 mM iodoacetamide (1:10, w/v) in a tissue blender (Ultra-Turrax; Janke and Kunkel). The homogenate was centrifuged for 5 min at 200 g and 48C. An aliquot of 200 ml of the resulting supernatant was solubilized for 10 min at 758C upon addition of SDS (1% final). Then, it was mixed with electrophoresis loading buffer, boiled for 5 min, and used for immunoblotting experiments. The remaining supernatant was subsequently centrifuged for 20 min at 40,000 g and 48C. The resulting pellet (membrane) was washed once in homogenization buffer, resuspended in assay buffer, and used for the determination of AC activity.

AC activity assay
Basal and forskolin (10 mM)-stimulated AC activities were determined in aortic membranes as described previously (28).
Immunoblot analysis and quantification of PKA subunit and Rho kinase levels Quantitative immunoblotting of PKA subunits and Rho kinase in aortic homogenates was performed as described elsewhere (28). Primary monoclonal anti-PKAcata, anti-PKARIa, anti-PKARIIa, anti-PKARIIb, and anti-Rho kinase II antibodies were from BD Biosciences Transduction Laboratories (Heidelberg, Germany). Quantification was performed by image analysis, using standard curves with four points (i.e., total protein loaded vs. integrated optical density) of different protein contents loaded on the same gels as described (29). This quantification procedure was repeated at least three times for each sample on different gels. Values were normalized to the protein content of the vehicle-treated WKY rats (taken as 100%).

PKA activity assay
Frozen aortas were pulverized in liquid nitrogen and then homogenized in ice-cold 50 mM Tris-HCl, pH 7.5, 50 mM 2mercaptoethanol, 10 mM EGTA, 5 mM EDTA, 1 mM PMSF, and 10 mM benzamidine (1:10, w/v). The homogenate was then sonicated on ice four times for 10 s and centrifuged for 10 min at 8,000 g and 48C. An aliquot of 12 ml of the resulting supernatant was mixed with 25 mM Tris-HCl, pH 7.0, 3 mM MgCl 2 , 0.1 mM ATP, 0.5 mM EDTA, 1 mM EGTA, and 5 mM 2-mercaptoethanol, in the absence (blank) or presence of 2 mM cAMP, in a total volume of 120 ml. PKA activity was measured using a nonradioactive ELISA kit (Medical and Biological Laboratories, Ltd., Nagoya, Japan), which measures the phosphorylation of the pseudosubstrate, N-RFARKGS(P)LRQKNV-C, where S(P) indicates the serine residue phosphorylated, according to the manufacturer's instructions.

Data analysis
Data are means 6 SEM from the number of animals indicated (n). The statistical significance was calculated using GraphPad Software. One-way (where indicated) or two-way ANOVA followed by Bonferroni's test was used for statistical evaluations. Differences were considered statistically significant at P , 0.05.

Effects of 2-OHOA on BP in SHRs and WKY rats
Oral administration of 2-OHOA (600 mg/kg every 12 h) induced a time-dependent reduction of BP in SHRs. The effect of 2-OHOA on systolic BP reached statistical significance after the 3rd day of treatment and had its maximal effect after 1 week (reductions of 30, 48, and 54 mm Hg after 3, 5, and 7 days of treatment, respectively, compared with vehicle-treated SHRs) (Fig. 1A). Diastolic BP was equally diminished, with reductions of 29 6 4 (P , 0.001), 48 6 4 (P , 0.001), and 51 6 6 mm Hg (Fig. 1B) after 3, 5, and 7 days of treatment, respectively, compared   with vehicle-treated SHRs [mean diastolic BP in vehicletreated SHRs was 177 6 3, 187 6 5, and 184 6 6 mm Hg (Fig. 1B) at 3, 5, and 7 days, respectively]. In contrast to SHRs, 2-OHOA did not have any effect on BP in normotensive WKY rats (Fig. 1A). After treatment of SHRs for 7 days, 2-OHOA reduced systolic BP (254 mm Hg, 227 6 4%) (Fig. 1A), diastolic BP (251 mm Hg, 228 6 3%), and mean BP (252 mm Hg, 228 6 3%) (Fig. 1B) to values close to those measured in normotensive WKY rats. Oral administration of 100, 300, 600, and 900 mg/kg 2-OHOA for 7 days decreased systolic BP in SHRs by 16, 39, 53, and 65 mm Hg, respectively, compared with vehicle-treated SHRs (Fig. 2). At the end of the treatment, systolic BP of SHRs treated with 600 and 900 mg/kg was similar to that of normotensive WKY rats. Heart rate was significantly higher in vehicle-treated SHRs than in WKY rats (Fig. 1B). After 2-OHOA treatment (600 mg/kg every 12 h) for 7 days, the heart rate decreased significantly in SHRs, whereas it increased in WKY rats (Fig. 1B). Together, these results indicate that 2-OHOA administration is able to reduce BP to normotensive levels in SHRs without inducing hypotension in WKY rats. Treatment with 2-OHOA also reduced body weight in SHRs and WKY rats by z13.1 6 0.7% and 10.5 6 1.0%, respectively, which was mainly caused by a reduction of fat depots. However, the antihypertensive effect of 2-OHOA does not seem to be directly related to its ability to reduce body weight. Food restriction of untreated SHRs leading to a similar body weight loss as 2-OHOA treatment (12.2 6 0.3%) did not affect their BP (200 6 6 mm Hg before and 192 6 5 mm Hg after food restriction).

Effect of 2-OHOA on AC activity in aorta
Increases of intracellular cAMP levels are involved in the vasorelaxant effect of a variety of vasoactive agonists, such as prostaglandins and b-adrenergic agonists (19). Therefore, AC activities were measured in aortic membranes from vehicle-and 2-OHOA-treated SHRs and WKY rats. In aortic membranes from vehicle-treated SHRs, forskolin (10 mM)-stimulated AC activity was lower (27 6 6%) compared with that in WKY rats (Fig. 3). Upon 7 days of treatment with 2-OHOA (600 mg/kg every 12 h), forskolin-stimulated AC activity was increased (by 39 6 5%). In aortic membranes from WKY rats, 2-OHOA treatment resulted in increased basal (by 153 6 12%) and forskolin-stimulated (by 45 6 19%) AC activities. These data indicate that 2-OHOA treatment induces an enhance- The amount of total protein loaded was 30 mg. Inmunoreactive bands are representative of five to nine animals per group. Columns show levels (means 6 SEM) of aortic PKA subunits quantified against standard curves and normalized to the protein content of vehicle-treated WKY rats (taken as 100%). PKAcata and PKARIa levels were increased, whereas PKARIIa was decreased, in vehicle-treated SHRs compared with vehicle-treated WKY rats ( # P , 0.05, ## P , 0.01, ### P , 0.001). *** P , 0.001 versus vehicle-treated SHRs. ment of aortic AC activity in both rat strains, which on the other hand responded very distinctly to this agent in terms of BP and heart rate changes.
Effects of 2-OHOA on the density and activity of PKA in aorta As the vasodilatory action of cAMP is mediated by PKA (13), we next studied the effect of 2-OHOA on both the expression and activity of PKA isoenzymes in aortas from vehicle-and 2-OHOA-treated SHRs and WKY rats. The concentrations of the catalytic (cata) and regulatory (RIa, RIIa, and RIIb) PKA subunits in aorta were determined by quantitative immunoblotting. This analysis showed that the amounts of aortic PKAcata and PKARIa subunits were significantly higher (27 6 2% for PKAcata and 39 6 10% for PKARIa) in vehicle-treated SHRs compared with normotensive WKY rats (Fig. 4A, C). Moreover, in vehicletreated SHRs, lower PKARIIa (decrease of 34 6 4%) and unchanged PKARIIb subunit levels were found (Fig. 4B,  D). Treatment of SHRs with 2-OHOA (600 mg/kg every 12 h) for 7 days further increased the levels of aortic PKAcata (19 6 3%) and PKARIa (45 6 13%) compared with vehicle-treated SHRs (Fig. 4A, C). Likewise, the level of PKARIIa, but not PKARIIb, in aortas from 2-OHOAtreated SHRs was also upregulated (increase of 82 6 7%) (Fig. 4B, D). In agreement with these protein expression data, we observed a significant increase of PKA activity (30 6 5%) in aortas from 2-OHOA-treated SHRs compared with vehicle-treated SHRs (Fig. 5). In contrast to SHRs, 2-OHOA treatment had no effect on the density of any of the PKA subunits studied (Fig. 4) or PKA activity (Fig. 5) in aortas from WKY rats. Thus, 2-OHOA treatment specifically increased the expression of PKA subunits and PKA activity in aortas from SHRs.
In vivo effect of the PKA inhibitor Rp-8-Br-cAMP on the antihypertensive effect of 2-OHOA in SHRs To demonstrate that the augmented expression and activity of vascular PKA induced by 2-OHOA treatment in SHRs is in fact involved in the observed antihypertensive action, the effect of the specific membrane-permeant PKA inhibitor Rp-8-Br-cAMP on systolic BP was investigated in vivo. For this purpose, SHRs treated for 7 days either with vehicle (control) or 2-OHOA (600 mg/kg every 12 h) received a single intraperitoneal injection of 10, 20, or 30 mg/kg Rp-8-Br-cAMP, and subsequent changes in systolic BP and heart rate were  monitored during 180 min. As described above (Fig. 1), treatment of SHRs for 7 days with 2-OHOA strongly reduced systolic BP (267 mm Hg; n 5 11). Administration of Rp-8-Br-cAMP to 2-OHOA-treated SHRs at doses of 20 and 30 mg/kg, but not 10 mg/kg, induced a pronounced rebound of systolic BP at 30 min after compound administration, with increases of 25 6 4 and 38 6 7 mm Hg, respectively (Fig. 6A), without affecting heart rate ( Table 1). The relative reversal of systolic BP reduction induced by 2-OHOA amounted to 57 6 11% and 59 6 17% at 20 and 30 mg/kg Rp-8-Br-cAMP, respectively (Fig. 6B). The BP-increasing effect of Rp-8-Br-cAMP was maintained for 60 min with the dose of 30 mg/kg (127 6 3 mm Hg) and disappeared later, with systolic BP values returning to initial levels at 90 min (Fig. 6A). In contrast to 2-OHOA-treated SHRs, administration of Rp-8-Br-cAMP affected neither systolic BP (Fig. 6A) nor heart rate ( Table 1) in vehicle-treated SHRs at any dose or time studied. These results clearly demonstrate the physiological involvement of PKA in the antihypertensive effect of 2-OHOA in SHRs.

Effects of withdrawal of 2-OHOA on BP and the density of aortic PKA in SHRs
We next studied the posttreatment evolution of BP and aortic PKA levels in SHRs after withdrawal of 2-OHOA. Treatment of SHRs for 7 days with 2-OHOA (600 mg/kg every 12 h) induced a time-dependent reduction of systolic BP (reductions of 33 and 59 mm Hg after 3 and 7 days of treatment, respectively, compared with basal systolic BP values) (Fig. 7A). At the end of treatment, withdrawal of 2-OHOA led to a time-dependent increase of systolic BP (increases of 31, 53, 63, and 74 mm Hg at 3, 5, 7, and 9 days after 2-OHOA withdrawal, respectively), returning to their initial hypertensive state after z5 days (Fig. 7A). The concentrations of the PKAcata, PKARIIa, and PKARIa subunits in aortic homogenates were quantified by immunoblotting after 9 days of 2-OHOA withdrawal. This analysis showed that the amounts of these PKA subunits in aortas from SHRs after withdrawal of 2-OHOA were comparable to the PKA levels found in vehicle-treated SHRs (Fig. 7B). Together, these results demonstrate that the antihypertensive effect of 2-OHOA is reversible, with BP levels returning to their usual hypertensive values, which are accompanied by aortic PKA levels similar to those found in control SHRs.

Effects of 2-OHOA on the density of Rho kinase in aorta
Moreover, we studied the effect of 2-OHOA on the expression of Rho kinase II, which plays a key role in the pathogenesis of hypertensive vascular disease (21,22). The levels of Rho kinase in aortas from vehicle-and 2-OHOAtreated SHRs and WKY rats were determined by quantitative immunoblotting. This analysis showed that the levels of aortic Rho kinase were significantly higher (60 6 15%) in vehicle-treated SHRs compared with normotensive WKY rats (Fig. 8). Treatment of SHRs with 2-OHOA (600 mg/kg every 12 h) for 7 days decreased aortic Rho kinase expression to levels close to those measured in normotensive WKY rats (Fig. 8). In contrast to SHRs, 2-OHOA treatment had no effect on the density of Rho kinase in aortas from WKY rats. Thus, 2-OHOA treatment specifically decreased the high expression of Rho kinase in aortas from SHRs.

DISCUSSION
Recent epidemiological studies in Mediterranean areas have shown an inverse association between olive oil consumption, a natural source of the MUFA oleic acid, and the risk of hypertension (3,4). However, oleic acid and other fatty acids are good cellular fuels, and they can be degraded through b-oxidation when imported into the mitochondria. This metabolism impairs the potential pharmacological use of fatty acids, although it does seem that oleic acid-rich oils (e.g., olive oil) had a moderate beneficial effect on BP, but only when high doses were consumed for several months (30,31). Nevertheless, the specific molecular mechanisms and the contribution of the different components of olive oil to its beneficial effect on BP are not fully understood (1,2). Thus, to overcome this problem, we have synthesized a new oleic acid analog with a hydroxyl group on the a-carbon, 2-OHOA. This modification of the fatty acid avoids its direct metabolism via the rapid b-oxidation pathway and only allows a basal metabolism by passing first through the slow and noninducible a-oxidation pathway (25). For this reason, its use as a source of energy would be decreased and its cell/organ availability increased. Indeed, the presence of this additional hydroxy group markedly increased the biological activity of 2-OHOA with respect to oleic acid in human adenocarcinoma cells (32), demonstrating the efficiency of this modification. We have recently shown that 2-OHOA has low histological toxicity in several rat organs (29) and is able to induce decreases in systolic BP in normotensive Sprague-Dawley rats (28), but it was completely uncertain whether this new molecule is indeed able to decrease high BP in established hypertension. Therefore, we applied 2-OHOA to adult SHRs with established hypertension and analyzed the cardiovascular responses of these rats compared with normotensive WKY rats. Oral treatment of SHRs induced a time-and dose-dependent sustained reduction of systolic and diastolic BP in SHRs. In contrast, 2-OHOA treatment had no effect on BP in normotensive WKY rats. After 7 days of treatment with 2-OHOA (600 mg/kg every 12 h), BP values of SHRs were close to those in WKY rats. Thus, 2-OHOA is in fact an effective antihypertensive agent.
The reasons why 2-OHOA decreased systolic BP in normotensive Sprague-Dawley rats (28) but not WKY rats are not clear at present. Possible explanations are the different rat strains (Sprague-Dawley vs. WKY) used in the two studies and the different genders (female Sprague-Dawley vs. male WKY rats). In fact, in the studies on female Sprague-Dawley rats, the decrease in systolic BP induced by 2-OHOA was not associated with changes in heart rate (28), whereas in WKY rats, heart rate was increased significantly by treatment with 2-OHOA (Fig. 1B). This higher cardiac rate could be considered a compensatory mechanism to avoid BP reduction in these rats. However, this does not exclude the possibility that WKY rats may exhibit further mechanisms to counteract a hypotensive effect of 2-OHOA.
In this study, we found an enhanced AC responsiveness to forskolin in aortas from both SHRs and WKY rats, suggesting that 2-OHOA may increase the sensitivity to endogenous vasodilators acting via the AC/cAMP pathway. Treatment of 2-OHOA clearly restored forskolin-stimulated AC activity in SHRs to the level found in normotensive vehicle-treated WKY rats. Although forskolin-stimulated AC activity in 2-OHOA-treated WKY rats was further enhanced compared with that in vehicle-treated WKY rats, no hypotensive effect could be observed. In this regard, high levels of cAMP in heart are able to exert positive chronotropic effects and might be responsible for the above-mentioned increase of heart rate in 2-OHOA-treated WKY rats, a possible compensatory mechanism avoiding BP reduction in these rats. We next studied whether the effector system of cAMP (i.e., PKA) was altered by 2-OHOA treatment. Treatment of WKY rats with 2-OHOA had no effect on the expression of PKA subunits or PKA activity in aorta. In contrast, the expression of PKAcata and the regulatory subunits RIa and RIIa was increased significantly in aortas from SHRs treated with 2-OHOA. In agreement  with these protein expression data, PKA activity was upregulated in aortas from 2-OHOA-treated SHRs. Hypertension in SHRs is characterized by an impaired G s -coupled receptor-induced vasorelaxation that is mediated by cAMP and PKA in smooth muscle (33)(34)(35). Thus, the enhanced AC responsiveness, together with the specific increase in PKA subunit expression observed only in 2-OHOA-treated SHRs, very likely explains the BP decrease in these hypertensive rats. To corroborate this hypothesis, we examined the effect on BP of a specific membrane-permeant inhibitor of PKA, the cAMP analog Rp-8-Br-cAMP (27), that was intraperitoneally administered in a single dose at the end of 2-OHOA treatment. In vehicle-treated SHRs, administration of Rp-8-Br-cAMP (10-30 mg/kg) altered neither systolic BP nor heart rate. In contrast, the acute administration of Rp-8-Br-cAMP to 2-OHOA-treated SHRs produced a pronounced reversal (up to 59% at 30 mg/kg Rp-8-Br-cAMP) of the antihypertensive action of 2-OHOA, strongly supporting the notion that augmented PKA expression and activity is one of the main molecular mechanisms responsible for the BP reduction induced by 2-OHOA in SHRs (Fig. 6A, B). On the other hand, withdrawal of 2-OHOA in SHRs resulted in a regression of decreased BP values to hypertensive levels, which was accompanied by aortic PKA levels comparable to those found in vehicle-treated SHRs, indicating that the antihypertensive effect of 2-OHOA is reversible.
It is known that Rho kinase, a pivotal regulatory protein of BP, is pathologically overexpressed in SHRs, thereby contributing to the high BP observed in this animal model of hypertension (22). For this reason, we investigated whether Rho kinase also could be implicated in the molecular mechanism by which 2-OHOA reduces BP in this rat strain. As reported previously, we found that expression of Rho kinase II in aorta from SHRs was increased significantly with respect to normotensive WKY rats. Administration of 2-OHOA decreased this overexpression of Rho kinase in SHRs to a level observed in normotensive WKY rats, whereas it did not alter the expression of Rho kinase in WKY rats. These results indicate that 2-OHOA normalizes supranormal Rho kinase expression rather than downregulates Rho kinase expression by a general inhibitory transcriptional mechanism. Thus, these data suggest that the molecular mechanisms by which 2-OHOA achieves a normalization of increased BP in SHRs involve, in addition to the upregulation of the vasodilatory PKA pathway, a downregulation of the vasoconstrictory Rho kinase.
Although these data do not exclude additional mechanisms involved in the antihypertensive action of 2-OHOA, such as direct effects of the fatty acid on Ca 21 or K 1 channels and contractile proteins, these are rather unlikely. First, such direct effects should also lead to vasodilation and a decrease in BP in WKY rats. Second, a direct inhibitory effect of 2-OHOA on proteins involved in vasoconstriction should result in a more rapid decrease in BP. In contrast, the decrease in BP caused by 2-OHOA, even at the highest effective dose, developed rather slowly, over days. It cannot be completely excluded that this time frame could reflect the necessity of reaching sufficiently high intracellular con-centrations of the compound to achieve its effects, including rapid mechanisms, such as kinases or phosphatases. However, it is also in agreement with an altered expression of proteins, as observed here for PKA subunits and Rho kinase and reported previously for Ga s (28). In agreement with our observations, during the past two decades it has become evident that fatty acids can act as signaling molecules involved in the regulation of gene expression, although all known target genes to date mainly encode proteins with roles in fatty acid transport or metabolism (36). Further investigations will provide more insights into the transcriptional and posttranscriptional mechanisms by which 2-OHOA is able to control specifically the expression of PKA and Rho kinase. As a result, the development of derivatives of oleic acid could represent a novel pharmacological approach to treat hypertension by controlling the vascular expression of BP-regulating proteins.
We conclude from our study that the MUFA 2-OHOA is an effective antihypertensive agent, which decreases high BP in SHRs to normotensive values. The sustained antihypertensive action of 2-OHOA is apparently caused by an upregulation of the vasodilatory AC/cAMP/PKA pathway and a downregulation of the vasoconstrictory Rho kinase pathway.