Angiotensin-(1-7) attenuates the chronotropic response to angiotensin II via stimulation of PTEN in the spontaneously hypertensive rat neurons.

Several studies have focused on the beneficial effects of peripheral angiotensin-(1-7) [Ang-(1-7)] in the regulation of cardiovascular function, showing its counterregulatory effect against the actions of angiotensin II (ANG II). However, its actions in the central nervous system are not completely understood. In the present study, we investigated the intracellular mechanisms underlying the action of ANG-(1-7) using the patch-clamp technique in neurons cultured from the hypothalamus of neonatal spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Superfusion of neurons with ANG II (100 nM) significantly increased neuronal firing in both strains of rats, and this chronotropic effect of ANG II was significantly enhanced in prehypertensive SHR neurons compared with WKY rat neurons. The enhanced chronotropic effect of ANG II was attenuated by a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, LY 294002 (10 µM). Superfusion of neurons with ANG-(1-7) (100 nM) did not alter the neuronal firing rate in either SHR or WKY neurons; however, it significantly attenuated the chronotropic action of ANG II exclusively in prehypertensive SHR neurons. This counterregulatory effect of ANG-(1-7) on ANG II action in prehypertensive SHR neurons was attenuated by cotreatment with either A-779, a Mas receptor antagonist, or bisperoxovanadium, a phosphatase and tensin homologue deleted on chromosome ten (PTEN) inhibitor. In addition, incubation of WKY and prehypertensive SHR neurons with ANG-(1-7) significantly increased PTEN activity. The data demonstrate that ANG-(1-7) counterregulates the chronotropic action of ANG II via a PTEN-dependent signaling pathway in prehypertensive SHR neurons.

Lentil polyphenol extract prevents angiotensin II-induced hypertension, vascular remodelling and perivascular fibrosis.

The objective of the study was to investigate whether chronic administration of the Morton lentil polyphenol extract (MLPE), which possesses rich phenolic compounds and a high antioxidant activity, had any protective effects on angiotensin II-induced hypertension. After four weeks of subcutaneous infusion of angiotensin II (200 ng kg(-1) min(-1)) in male SD rats, the water intake and mean artery pressure was significantly increased by 39.8% and 48.3%, respectively, as compared with the control. The media/lumen ratio of the small arteries in the heart and kidneys were increased by 117% and 168% by angiotensin II infusion. The perivascular fibrosis was increased by 65% and 32% in the heart and kidneys, respectively. Levels of the reactive oxygen species in the aorta was enhanced by 115.8%. In another group of rats, which received four weeks of lentil extract administration (1% freeze-dried MLPE in the drinking water), followed by another four weeks of extract administration plus angiotensin II infusion, the angiotensin II-induced enhancement in water intake and mean artery pressures decreased by 12.7% and 8.2%, respectively, as compared with the rats that received angiotensin II infusion alone. The angiotensin II-induced rats showed increases in the media/lumen ratios which were attenuated by 43.6% and 47.2% in the small arteries of heart and kidneys, respectively. Angiotensin II-induced perivascular fibrosis was attenuated by 30% and 26% in the rats that received the extract. Angiotensin II-induced rats showed reactive oxygen species levels in the aorta was reduced by 48.9%. These findings demonstrated that lentil extract attenuated angiotensin II-induced hypertension and associated pathological changes, including remodelling and perivascular fibrosis in the small resistant arteries of heart and kidneys.

Morton lentil extract attenuated angiotensin II-induced cardiomyocyte hypertrophy via inhibition of intracellular reactive oxygen species levels in vitro.

The objective was to investigate whether a lentil (Morton) extract had any protective effect on cardiac hypertrophy, which is one of the most significant sequelae of cardiovascular diseases. High phenolic compounds (43.4 mg of GAE/g), including thirteen phenolic acid and two flavonoids, were detected in the acetone/water/acetic acid lentil extract. The extract showed strong antioxidant ability (105 µmol of TE/g). The effect of lentil extract on angiotensin (Ang) II-induced cardiac hypertrophy was examined. Results showed that pretreatment with lentil extract (25, 50, 100 µg/mL) significantly attenuated Ang II (0.1 µM)-induced hypertrophy by 18, 28, and 36% in rat cardiomycytes, respectively; lentil extract (12.5, 25, 50 µg/mL) attenuated Ang II (0.1 µM)-induced hypertrophy by 9, 17, and 25% in human cardiomycytes, respectively. Intracellular reactive oxygen species (ROS) levels were enhanced by Ang II treatment, and this stimulatory action was significantly attenuated (33% inhibition) by lentil extract (100 µg/mL) in rat cardiomyocytes and attenuated by 22% by 50 µg/mL lentil extract in human cardiomyocytes. In conclusion, Morton lentil extracts attenuated Ang II-induced rat and human cardiomyocytes hypertrophy via decreasing intracellular ROS levels.

Characterization of novel radicals from COX-catalyzed arachidonic acid peroxidation.

The peroxidation of arachidonic acid (AA) catalyzed by cyclooxygenase (COX) is a well-known free radical-mediated process that forms many bioactive products. Because of a lack of appropriate methodologies, however, no comprehensive structural evidence has been found previously for the formation of COX-mediated and AA-derived free radicals. Here we have used a combination of LC/ESR and LC/MS with a spin trap, alpha-[4-pyridyl-1-oxide]-N-tert-butylnitrone (POBN), to characterize the carbon-centered radicals formed from COX-catalyzed AA peroxidation in vitro, including cellular peroxidation in human prostate cancer cells (PC-3). Three types of radicals with numerous isomers were trapped by POBN as ESR-active peaks and MS-active ions of m/z 296, 448, and 548, all stemming from PGF(2)-type alkoxyl radicals. One of these was a novel radical centered on the carbon-carbon double bond nearest the PGF ring, caused by an unusual beta-scission of PGF(2)-type alkoxyl radicals. The complementary nonradical product was 1-hexanol, another novel beta-scission product, instead of the more common aldehyde. The characterization of these novel products formed from in vitro peroxidation provides a new mechanistic insight into COX-catalyzed AA peroxidation in cancer biology.

Mechanical hyperalgesia is attenuated by local administration of octreotide in pristane-induced arthritis in Dark-Agouti rats.

AIMS: The Dark-Agouti (DA) rat is very susceptible to pristane-induced arthritis (PIA) and represents a suitable model for rheumatoid arthritis. In the present study, we examined the pain sensitivity and the effect of local administration of octreotide (OCT) on mechanical hyperalgesia in PIA DA rats. MAIN METHODS: Arthritis was induced by intradermal injection of pristane (300 microl). The mechanical withdrawal threshold (MWT) and heat withdrawal latency (HWL) were used to evaluate the pain sensitivity. In addition, we recorded the discharge firings in the tibial nerve sensory C-fibers innervating the inflamed toe joints of arthritic DA rats. KEY FINDINGS: Two weeks after injection of pristane, all DA rats developed severe arthritis. This symptom was associated with a decreased MWT (78.50+/-5.68 mN before pristane injection, 19.50+/-6.27 mN on day 14 after pristane injection), indicating a mechanical hyperalgesia in PIA. In contrast, HWL was comparable before and after pristane injection (10.25+/-0.70 s before injection; 9.45+/-1.23 s on day 14 after injection). Local injection of OCT markedly increased MWT and relieved the hyperalgesia in PIA. In addition, OCT significantly decreased the discharge rate of afferent C units evoked by both non-noxious and noxious joint movements. SIGNIFICANCE: Taken together, the results demonstrate that mechanical hyperalgesia, but not thermal hyperalgesia is associated with PIA and that the mechanical hyperalgesia and the discharge of afferent C units are attenuated by local administration of OCT. These observations provide evidence for a novel therapeutic strategy for pain control in rheumatoid arthritis.

Lack of macrophage migration inhibitory factor regulation is linked to the increased chronotropic action of angiotensin II in SHR neurons.

Macrophage migration inhibitory factor acts via its intrinsic thiol-protein oxidoreductase activity to negatively regulate the neuronal chronotropic actions of angiotensin II in normotensive rat neurons. Because the chronotropic action of angiotensin II is potentiated in spontaneously hypertensive rat neurons, we investigated whether this negative regulatory mechanism is absent in these rats. Angiotensin II (100 nM) elicited an approximately 89% increase in neuronal firing in Wistar-Kyoto rat hypothalamus and brain stem cultured neurons and an increase in intracellular macrophage migration inhibitory factor levels in the same cells. The chronotropic action of angiotensin II was significantly greater (approximately 212% increase) in spontaneously hypertensive rat neurons, but angiotensin II failed to alter macrophage migration inhibitory factor expression in these cells. Intracellular application of recombinant macrophage migration inhibitory factor (0.8 nM) or its specific neuronal overexpression via Ad5-SYN-MIF (1x10(7) infectious units) significantly attenuated the chronotropic action of angiotensin II in spontaneously hypertensive rat neurons, similar to results from Wistar-Kyoto rat neurons. In contrast, C60S-macrophage migration inhibitory factor (0.8 nM), which lacks thiol-protein oxidoreductase activity, failed to alter the chronotropic action of angiotensin II in neurons from either rat strain. Thus, whereas macrophage migration inhibitory factor has the potential to depress the chronotropic action of angiotensin II in spontaneously hypertensive rat neurons, it is unlikely that this regulatory mechanism occurs, because angiotensin II does not increase the expression of this protein. The lack of this regulatory mechanism may contribute to the increased chronotropic action of angiotensin II in spontaneously hypertensive rat neurons.

NAD(P)H oxidase inhibition attenuates neuronal chronotropic actions of angiotensin II.

It is well established that the central cardiovascular effects of angiotensin II (Ang II) involve superoxide production. However, the intracellular mechanism by which reactive oxygen species (ROS) signaling regulates neuronal Ang II actions remains to be elucidated. In the present study, we have used neuronal cells in primary cultures from the hypothalamus and brain stem areas to study the role of ROS on the cellular actions of Ang II. Ang II increases neuronal firing rate, an effect mediated by the AT(1) receptor subtype and involving inhibition of the delayed rectifier potassium current (I(Kv)). This increase in neuronal activity was associated with increases in NADPH oxidase activity and ROS levels within neurons, the latter evidenced by an increase in ethidium fluorescence. The increases in NADPH oxidase activity and ethidium fluorescence were blocked by either the AT(1) receptor antagonist losartan or by the selective NAD(P)H oxidase inhibitor gp91ds-tat. Extracellular application of the ROS scavenger, Tempol, attenuated the Ang II-induced increase in neuronal firing rate by 70%. In addition, gp91ds-tat treatment resulted in a 50% inhibition of Ang II-induced increase in firing rate. In contrast, the ROS generator Xanthine-Xanthine oxidase significantly increased neuronal firing rate. Finally, Ang II inhibited neuronal I(Kv,) and this inhibition was abolished by gp91ds-tat treatment. These observations demonstrate, for the first time, that Ang II regulates neuronal activity via a series of events that includes ROS generation and inhibition of I(Kv). This signaling seems to be a critical cellular event in central Ang II regulation of cardiovascular function.

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

The role of soluble epoxide hydrolase (sEH) in the central control of blood pressure (BP) has not been elucidated in spite of peripheral sEH overexpression being linked to hypertension. Thus, our objective was to investigate the involvement of brain sEH in BP control. sEH expression in the hypothalamus and brain stem, two cardioregulatory brain areas, was increased in the spontaneously hypertensive rat (SHR) compared to the Wistar Kyoto (WKY) rat. Inhibition of the enzyme by intracerebroventricular (icv) delivery of AUDA further increased both BP and heart rate (HR) by 32 +/- 6 mmHg and 54 +/- 10 bpm, respectively, (P<0.05) in the SHR. Analysis of waveform telemetry data revealed a decrease in spontaneous baroreceptor reflex gain following sEH inhibition, indicating the sustained increase in BP may be due to a decrease in baroreceptor reflex function. The hypertensive effect of sEH inhibition is likely a result of an increase in epoxyeicosatrienoic acid (EET)-mediated generation of ROS. This view is supported by the following: 1) Inhibition of EET formation attenuates AUDA-induced increase in BP; 2) delivery of an EET agonist increases BP and HR in the WKY rat, and 3) inhibition of NAD(P)H oxidase by gp91ds-tat prevents AUDA-induced increases in BP and HR. Finally, electrophysiological studies demonstrate that AUDA increased neuronal firing rate exclusively in the SHR, an effect completely abolished by gp91ds-tat. These observations suggest that EETs and sEH inhibition are involved in increasing BP in the SHR. We suggest that an increased expression of sEH is a futile central nervous system response in protection against hypertension.

Hypertension-linked decrease in the expression of brain gamma-adducin.

Gene profiling data coupled with adducin polymorphism studies led us to hypothesize that decreased expression of this cytosolic protein in the brain could be a key event in the central control of hypertension. Thus, our objectives in the present study were to (1) determine which adducin subunit gene demonstrates altered expression in the hypothalamus and brainstem (two cardioregulatory-relevant brain areas) in two genetic strains of hypertensive rats and (2) analyze the role of adducins in neurotransmission at the cellular level. All three adducin subunits (alpha, beta, and gamma) were present in the hypothalamus and brainstem of Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats. However, only the gamma-adducin subunit expression was 40% to 60% lower in the SH rat compared with WKY rat. A similar decrease in gamma-adducin expression was observed in the hypothalamus and brainstem of the renin transgenic rat compared with its normotensive control. Losartan treatment of the SH rat failed to normalize gamma-adducin gene expression. A hypertension-linked decrease of gamma-adducin was confirmed by demonstrating a decrease in gamma-adducin expression in hypothalamic/brainstem neuronal cultures from prehypertensive SH rats. Neuronal firing rate was evaluated to analyze the role of this protein in neurotransmission. Perfusion of a gamma-adducin-specific antibody caused a 2-fold increase in the neuronal firing rate, an effect similar to that observed with angiotensin II. Finally, we observed that preincubation of neuronal cultures for 8 hours with 100 nmol/L angiotensin II caused a 60% decrease in endogenous gamma-adducin and was associated with a 2-fold increase in basal firing rate. These observations support our hypothesis that a decrease in gamma-adducin expression in cardioregulatory-relevant brain areas is linked to hypertension possibly by regulating the release of neurotransmitters.