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Novel mechanism of brain soluble epoxide hydrolase-mediated blood pressure regulation in the spontaneously hypertensive rat

Kathleen W. Sellers^*, Chengwen Sun^*, Carlos Diez-Freire^*, Hidefumi Waki, Christophe Morisseau, John R. Falck, Bruce D. Hammock, Julian F. Paton and Mohan K. Raizada^*,1

^* Department of Physiology and Functional Genomics, University of Florida College of Medicine and McKnight Brain Institute, Gainesville, Florida, USA;
Department of Physiology, School of Medical Sciences, University of Bristol, Bristol, UK;
Department of Entomology and U.C.D. Cancer Center, University of California Davis, California, USA; and
Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

¹Correspondence: Department of Physiology and Functional Genomics, College of Medicine, University of Florida, McKnight Brain Institute, Gainesville, FL 32610, USA. E-mail: mraizada{at}phys.med.ufl.edu

SPECIFIC AIMS

Gene profiling using microarray technology revealed that soluble epoxide hydrolase (sEH) mRNA levels were elevated in the spontaneously hypertensive rat (SHR) vs. the Wistar Kyoto (WKY) rat brain. Peripheral sEH overexpression has been linked to animal models of hypertension, and inhibition of sEH activity by pharmacological agents decreased high blood pressure (BP). Whereas sEH in the vasculature is proposed to exert its actions by converting vasodilatory epoxyeicosatrienoic acids (EETs) to their relatively inactive diol forms, the role of this enzyme in the brain is unknown. Thus, our objective was to investigate the involvement of brain sEH in BP control.

PRINCIPAL FINDINGS

1. sEH is up-regulated in the hypothalamus and brainstem of the SHR
Our first objective was to validate sEH expression changes identified in microarray analysis. Real-time RT-PCR and Western blot analysis revealed that sEH expression was several fold higher in the brainstem and hypothalamus of adult SHR than in WKY rats. This increase seems to be genetically linked, as neuronal cultures from prehypertensive rats exhibited a similar increase in expression. Thus, sEH is overexpressed in cardiovascularly relevant brain regions of the SHR.

2. Central sEH inhibition increases BP and heart rate in the SHR
Intraperitoneal delivery of pharmacological inhibitors to sEH results in a depressor effect in the SHR. We sought to determine what effect central inhibition of sEH had on blood pressure in SHR and WKY rats. Rats were implanted with a radiotelemetric pressure transducer (Data Sciences International, Arden Hills, MN, USA) in the abdominal aorta to transmit blood pressure and heart rate (HR) data every minute for analysis. sEH inhibitors were delivered ICV through a cannula inserted into the right lateral ventricle. ICV administration of adamantyl dodecanoic acid urea (AUDA), a potent sEH inhibitor, caused a dose-dependent increase in BP and HR in the SHR (Fig. 1 ). An increase of 25 ± 0.9 mmHg in BP was seen with as little as 0.8 ng AUDA; a dose of 15 ng AUDA increased BP by 32 ± 6 mmHg. This increase was observed as early as 30 min post-AUDA administration, reached maximal levels in 3–4 h, and returned to basal levels in 7 h. The increase in BP was associated with increases in HR in WKY and SHR by 46 ± 12 bpm and 54 ± 10 bpm, respectively. Central sEH inhibition did not alter BP in the WKY rat (Fig. 1) .

View larger version (26K): [in this window] [in a new window]   Figure 1. Effect of ICV administration of AUDA on blood pressure/heart rate in the SHR and WKY rat. AUDA (15 ng) was injected into the right lateral ventricle of unrestrained rats; BP and HR were recorded each minute with radiotelemetry before and after injection. A) Time sequence traces of MAP. Arrow: AUDA injection. X-axis: time in minutes relative to AUDA injection. Representative time sequence traces of HR in WKY (B) and SHR (C). Quantitation of MAP in mmHg (D) or HR in beats/min (E). Open bars = basal values; shaded bars = peak MAP or HR (10 min average) after AUDA injection. Data are mean ±SE (n=5). *Significantly different from basal MAP or HR (P<0.05).

To study the mechanism by which BP and HR are concomitantly increased in SHR, spontaneous baroreceptor reflex gain was determined from spontaneous changes in systolic blood pressure (SBP) and pulse interval (PI) using a time series method. Analysis of wave form telemetry data showed a 56 ± 5% decrease in baroreceptor reflex gain. High-frequency analysis of PI as an indicator of cardiac vagal tone decreased 33 ± 8% after ICV AUDA administration in the SHR. These data demonstrate that increases in BP are consistent with a decrease in baroreceptor reflex functions.

3. Increased EETs linked to increase in BP through ROS production
We next sought to elucidate the mechanism underlying the central sEH inhibitor-induced pressor effect in the SHR. Given that sEH inhibition leads to accumulation of EETs, we hypothesized that brain EETs may be key in this pressor response. This hypothesis was tested with the use of N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH), an inhibitor of the cytochrome P450 epoxygenase pathway that generates EETs and related PUFA epoxides, as well as EET agonist 11-nonyloxy-undec-8-enoic acid (11-NODA). MS-PPOH was delivered ICV 1 h before sEH inhibition with AUDA. Central treatment with MS-PPOH attenuated the AUDA-induced increase in BP by 65% in the SHR. MS-PPOH alone had no effect on BP. ICV delivery of the EET agonist 11-NODA in the WKY rat resulted in an increase in basal BP and HR by 13 ± 2 mmHg and 61 ± 19 bpm, respectively. Central AUDA-induced BP is therefore mediated by an increase in EETs in the brain.

We examined the role of ROS on sEH inhibitor-mediated increase in BP in the SHR. Cytochrome P-450 member 2C9 generates EETs and is linked to ROS, and since increase in central ROS is associated with an increase in BP, we argued one would be able to prevent AUDA-induced increases in BP by inhibitors of ROS. Pretreatment with gp91ds-tat, a blocker of NAD(P)H oxidase, caused an 85% attenuation of BP and a 77% decrease in HR induced by AUDA. In contrast, gp91ds-tat alone had no effect on BP and HR. Therefore, ROS production is involved in invoking a pressor response after central sEH inhibition in the SHR.

4. In vitro validation with neuronal cells in primary culture
Primary cocultures from hypothalamus-brainstem of WKY and SHR have been used to elucidate cellular and molecular mechanisms of physiological dysregulation in neural control of hypertension. We used these cultures to validate in vivo data and determine the cellular basis of an sEH inhibitor-induced increase in BP in the SHR. Western blot analysis demonstrated a 4-fold increase in sEH protein levels in neuronal culture of SHR vs. WKY rats (Fig. 2 A). The effect of AUDA on neuronal firing rate was studied to determine whether inhibition of sEH would increase neuronal activity in the SHR. 0.4 mg/mL AUDA resulted in a 2-fold increase in neuronal firing rate exclusively in SHR neurons (Fig. 2B, C ), attenuated 75% by pretreatment with 5 µM gp91ds-tat.

View larger version (22K): [in this window] [in a new window]   Figure 2. Effect of AUDA on neuronal activity in vitro. A) sEH protein levels in neuronal cultures from 1-day-old SHR and WKY rats. Top: representative audioradiograph; bottom: quantitation of sEH protein band. *Significantly different in SHR vs. WKY (P<0.05, n=4). B) Effect of AUDA and gp91ds-tat on neuronal firing of neurons cultured from SHR rat brain hypothalamus. Recordings of action potentials from representative neurons were recorded at control conditions and treatment with AUDA (0.4 mg/mL), AUDA + gp91ds-tat (5 µM), or AUDA + scrambled gp91ds-tat (5 µM). C) Bar graphs: means ± SE from 9 neurons showing that AUDA significantly increase neuronal firing rate; this effect was attenuated by gp92ds-tat. *P < 0.05 compared with control condition. P < 0.05 vs. AUDA.

CONCLUSIONS AND SIGNIFICANCE

Our results suggest a novel role for sEH and EETs in the brain of the SHR. After ICV administration of sEH inhibitors, BP and HR both increase and are associated with a dampening of the baroreceptor reflex. We surmise that these effects of sEH inhibition occur through the accumulation of their substrates, the EETs and other epoxy lipids. This conclusion is supported by the finding that the selective inhibitor of lipid epoxidase MS-PPOH attenuates the AUDA-induced increase in BP. Administration of an EET agonist results in a transient increase in BP in the WKY rat. Thus, the role of central EETs appears to be distinct from that of its peripheral expression, which is characterized by its vasodilatory properties that are associated with a decrease in BP.

In contrast to the SHR, sEH inhibition had no significant effect on BP in the WKY rat. This observation is consistent with our Western blot data indicating the presence of little sEH protein in the WKY rat brain. HR, however, was increased in the WKY rat, though the duration of the effect was attenuated compared with that of the SHR. This finding may be related to differential levels of EETs that induce BP- and HR-specific effects. Further studies are needed to resolve these and other possibilities.

The mechanism linking central sEH inhibition to the increase in BP and HR appears to function through ROS. This hypothesis is supported by our data: 1) pretreatment with gp91ds-tat, an NAD(P)H oxidase blocker prevents AUDA-induced increase in BP and HR, and 2) AUDA treatment increased neuronal firing rate, an effect attenuated by gp91ds-tat. Thus, we propose that an increase in EETs by inhibition of sEH leads to an increase in ROS in the brain and hence an increase in BP. An alternate possibility is that ROS stimulates EETs, which leads to an increase in BP. The increase in BP is associated with dampening of the baroreceptor reflex. Whether this is a causative mechanism is unknown, but recent data indicates that baroreceptor reflex plays a critical role in chronic and long-term control of BP. Finally, our study suggests that overexpression of sEH in the brain may be a compensatory response that fails to override hypertensive conditions in the SHR. This would be important to prove with the use of other experimental models of hypertension. The SHR was exclusively chosen for this study because it exhibits many similarities to primary human hypertension.

This study provides a novel mechanism by which sEH regulates designated cardiovascular activity in the brain and raises important questions. For example, in what cell type in the brain is sEH localized? We believe it is the neuronal sEH that participates in EET-mediated BP control. This is supported by our findings that neuronal cells in primary culture from the prehypertensive SHR hypothalamus and brainstem demonstrate increased expression of sEH similar to that seen in the adult SHR brain. AUDA treatment caused an exclusive increase in neuronal firing in neurons from the SHR hypothalamus and brainstem. Finally, astroglial cultures from the same brain areas showed no significant differences in the sEH expression between WKY and SHR. While these data implicate neuronal sEH, they do not eliminate the role of cerebrovascular sEH, which we will investigate. It is important to determine what nucleus (or nuclei) in the hypothalamus and brainstem is/are key in EETs-mediated regulation of BP and how sEH-EET-ROS-mediated signals are transmitted to the peripheral system, which is translated into increases in HR and BR. We have developed a lentiviral vector-mediated gene delivery system in selective brain nuclei and transmitter-specific neurons in vivo in an attempt to answer these questions. Nonetheless, these studies establish that an increase gene expression for sEH appears to be a protective mechanism against neural control of hypertension, an increase that fails to override the hypertensive dysregulatory pathways.

View larger version (10K): [in this window] [in a new window]   Figure 3. Proposed mechanism of central sEH on blood pressure regulation in the SHR. An increase in central EETs leads to an increase in blood pressure via ROS mediation. We propose that EETs induce the production of ROS, including NAD(P)H oxidase. While high sEH expression in the SHR converts EETs, inhibition of this enzyme activates this cascade, leading to a further increase in blood pressure.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3128fje; doi: 10.1096/fj.04-3128fje

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