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The present study examined whether 20-hydroxyeicosatetraenoic acid (HETE) contributes to the vasoconstrictor effect of angiotensin II (ANG II) in renal microvessels by preventing activation of the large conductance Ca(2+)-activated K(+) channel (KCa) in vascular smooth muscle (VSM) cells. ANG II increased the production of 20-HETE in rat renal microvessels. This response was attenuated by the 20-HETE synthesis inhibitors, 17-ODYA and HET0016, a phospholipase A2 inhibitor AACOF3, and the AT1 receptor blocker, Losartan, but not by the AT2 receptor blocker, PD123319. ANG II (10(-11) to 10(-6) M) dose-dependently decreased the diameter of renal microvessels by 41 ± 5%. This effect was blocked by 17-ODYA. ANG II (10(-7) M) did not alter KCa channel activity recorded from cell-attached patches on renal VSM cells under control conditions. However, it did reduce the NPo of the KCa channel by 93.4 ± 3.1% after the channels were activated by increasing intracellular calcium levels with ionomycin. The inhibitory effect of ANG II on KCa channel activity in the presence of ionomycin was attenuated by 17-ODYA, AACOF3, and the phospholipase C (PLC) inhibitor U-73122. ANG II induced a peak followed by a steady-state increase in intracellular calcium concentration in renal VSM cells. 17-ODYA (10(-5) M) had no effect on the peak response, but it blocked the steady-state increase. These results indicate that ANG II stimulates the formation of 20-HETE in rat renal microvessels via the AT1 receptor activation and that 20-HETE contributes to the vasoconstrictor response to ANG II by blocking activation of KCa channel and facilitating calcium entry.

Melatonin inhibits nitric oxide (NO)-induced relaxation of coronary arteries. We tested the hypothesis that melatonin increases the phosphorylation of phosphodiesterase 5 (PDE5), which increases the activity of the enzyme and thereby decreases intracellular cGMP accumulation in response to NO and inhibits NO-induced relaxation. Sodium nitroprusside (SNP) and 8-Br-cGMP caused concentration-dependent relaxation of isolated coronary arteries suspended in organ chambers for isometric tension recording. In the presence of melatonin, the concentration-response curve to SNP, but not 8-Br-cGMP, was shifted to the right. The effect of melatonin on SNP-induced relaxation was abolished in the presence of the PDE5 inhibitors zaprinast and sildenafil. Melatonin markedly inhibited the SNP-induced increase in intracellular cGMP in coronary arteries, an effect that was also abolished by zaprinast. Treatment of coronary arteries with melatonin caused a nearly fourfold increase in the phosphorylation of PDE5, which increased the catalytic activity of the enzyme and thereby increased the degradation of cGMP to inactive 5′-GMP. Melatonin-induced PDE5 phosphorylation was markedly attenuated in the presence of the PKG1 inhibitors DT-2 or Rp-8-Br-PET-cGMPS and in those arteries in which PKG1 expression was first downregulated by 24-h incubation with SNP before exposure to melatonin. The selective MT2receptor antagonist 4-phenyl-2-propionamidotetralin completely blocked the stimulatory effect of melatonin on PDE5 phosphorylation as well as the inhibitory effect of melatonin on SNP-induced relaxation and intracellular cGMP. Thus, in coronary arteries, melatonin acts via MT2receptors and PKG1 to increase PDE5 phosphorylation, resulting in decreased cGMP accumulation in response to NO and impaired NO-induced vasorelaxation.

Cardiomyocyte apoptosis has been documented involved in a variety of cardiac stresses, including ischemia-reperfusion injury, heart failure, and cardiomyopathy. Both angiotensin II (Ang II) and 20-Hydroxyeicosatetraenoic Acid (20-HETE), a hydroxylation product of arachidonic acid catalyzed by CYP450-ω hydroxylase, induce apoptosis in cardiomyocyte. However, the crosstalk between 20-HETE and Ang II in cardiomyocytes apoptosis process is unclear. In the current study, we examined apoptosis using flow cytometry in primary cultured neonatal rat ventricular myocytes treated with control, Ang II (100nM), Ang II plus HET0016 (10μM), HET0016, or 20-HETE (10nM) along. The results demonstrated that treatment with Ang II or 20-HETE significantly increased apoptosis and that Ang II-induced apoptosis were markedly attenuated by HET0016, a 20-HETE agonist. In addition, Ang II-induced increases of caspase-3 activity were significantly attenuated by 20.9±3.4% after co-treatment with HET0016. Our results also demonstrated that HET0016 significantly suppressed Ang II-induced increases of superoxide production by 27.52.3% and mitochondrial membrane potential by 64.5±6.3%. Finally, Ang II-induced nuclei crenation, chromatin condensation and fractionation were attenuated by 73.6±8.5% with HET0016 pre-treatment. In addition, treatment cardiomyocytes with Ang II increased CYP4A1 expression and 20-HETE production, measured by Western blot, real-time RT-PCR, and mass spectrometric analysis. All results suggest that 20-HETE may play a key role in Ang II-induced apoptosis in cardiomyocytes. 20-HETE and 20-HETE-producing enzymes could be novel targets for the treatment of Ang II related cardiac diseases.

Apelin-13 has been reported to act directly on APJ receptors in vascular smooth muscle (VSM) to induce vasocontraction. However, the ionic mechanisms underlying this action at the cellular level remain unclear. Large-conductance Ca2+-activated K+ (BKca) channels in VSM cells are critical regulators of membrane potential and vascular tone. Thus, in the present study, we examined the effect of apelin-13 on BKca channel activity in VSM cells, freshly isolated from rat middle cerebral arteries. In whole-cell patch clamp mode, apelin-13 (0.001μM-1μM) caused dose-dependent inhibition of BKca in VSM cells. Apelin-13 (0.1μM) significantly decreased the BKca current density from 71.25±8.14 pA/pF to 44.52±7.10 pA/pF (n=14 cells, P0.05), suggesting that the inhibitory effect of apelin-13 on BKca is not mediated by its direct action on BKca. In whole cell patches, pretreatment of VSM cells with LY-294002 (10 μM), a PI3-kinase inhibitor, markedly attenuated the apelin-13-induced decrease in BKca current density by 52%. Next, we examined the effect of apelin-13 on PI3- kinase activity in rat cerebral arteries using western blot analysis. Treatment with apelin-13 (0.1μM) significantly increased the ratio of phosphorylated-Akt/total Akt by 40%, indicating that apelin-13 significantly increases PI3-kinase activity. In summary, apelin-13 inhibits BKca channel current density via a PI3-kinase-dependent signaling pathway, which may contribute to its regulatory action in the control of vascular tone.

The objective of this study is to examine the effects of extracts from raw and cooked lentil on angiotensin II (Ang II)-induced cardiac hypertrophy, fibrosis, and hypertension in normotensive rats. Subcutaneous infusion of Ang II (200 ng/kg/min) using osmotic minipump significantly resulted in the elevation of blood pressure measured using telemetry in conscious rats. Histological examination revealed that Ang II infusion for 4 weeks induced significant cardiac hypertrophy, perivascular fibrosis in the heart and kidney. Rats received lentil extracts (oral administration for 4 weeks) significantly attenuated Ang II-induced elevation in blood pressure, cardiac hypertrophy, perivascular fibrosis. To examine whether the protective effect of lentil extracts on cardiac hypertrophy is mediated by attenuation of blood pressure or directly act on the cardiomyocytes, we examined the effect of lentil extracts on Ang II-induced hypertrophy in cultured cardiomyocytes. The result demonstrated that pretreatment of cardiomyocytes with cooked or raw lentil extract significantly attenuated the Ang II-induced increases in the size of cells. In addition, these lentil extracts also attenuated Ang II-induced increases in the ROS levels in cardiomyocytes. In summary, the results demonstrate that extracts from cooked or raw lentil prevent Ang II-induced elevation in blood pressure, perivascular fibrosis, and cardiac hypertrophy. The cardiac protective effect on Ang II-induced cardiac hypertrophy may be mediated by a direct action in cardiac tissue via reduction of oxidative stress.

We report bifunctional liposomal delivery system, combining transferrin (Tf)-mediated receptor targeting and poly-L-arginine (PR)-facilitated cell penetration, which overcomes the drawback of saturation of delivery. PR was conjugated to the distal end of distearoyl phosphoethanolamine-polyethylene glycol (PEG) 2000 and was incorporated with other phospholipids in chloroform/methanol (2:1) to form PR liposomes using thin-film hydration technique. Tf-PEG phospholipid micelles were incorporated into PR liposomes using postinsertion technique to form Tf-PR liposomes. The bifunctional liposomes demonstrated significantly (p < 0.05) higher cellular uptake by brain endothelial cells (bEnd.3) and about eightfold higher transfection in primary culture of glial cells as compared with the Tf liposomes. Cell viabilities of Tf-conjugated and bifunctional liposomes were not markedly different; however, transport across in vitro blood-brain barrier model improved considerably after dual modification. The study underlines the potential of bifunctional liposomes as high-efficiency and low-toxicity gene delivery system for the treatment of central nervous system disorders.

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.

20-Hydroxyeicosatetraenoic acid (20-HETE), a [omega]-hydroxylation product of arachidonic acid catalyzed by cytochrome P450 4A, may play a role in the cardiovascular system. It is well known that cytochrome P450 [omega]-hydroxylase inhibitors markedly reduced the cardiac ischemia reperfusion injury. However, the direct effect of 20-HETE on cardiomyocytes is still poorly investigated. Here, we studied the effect of 20-HETE on cardiomyocyte apoptosis and the apoptosis-associated signaling pathways.The cardiomyocyte apoptosis was measured by fluorescein isothiocyanate conjugated annexin V/propidium iodide double staining cytometry, indicating that the percentage of early apoptotic cells increased from 15.6% +/- 2.6% to 25.5% +/- 2.5% in control and 20-HETE-treated cells, respectively. The mitochondrial membrane potential ([DELTA][PSI]m) was measured by detecting the ratio of JC-1 green/red emission intensity. A significant decrease in the ratio was observed after treatment with 20-HETE for 24 hours in comparison with control group, suggesting the disruptive effect of 20-HETE on mitochondrial [DELTA][PSI]m. In addition, 20-HETE stimulated caspase-3 activity and Bax mRNA expression in cardiomyocytes. In contrast, the Bcl-2 mRNA levels were significantly decreased by 20-HETE treatment.These results demonstrate that 20-HETE induces cardiomyocyte apoptosis by activation of several intrinsic apoptotic pathways. The 20-HETE-induced apoptosis could contribute to the cytochrome P450 [omega]-hydroxylase-dependent cardiac injure during cardiac ischemia-reperfusion.

Microinjection of apelin-13 into the rostral ventrolateral medulla (RVLM) in the brainstem increases blood pressure in rats. In the present study, we tested the hypotheses that apelin-13 directly stimulates neuronal activity in neurons cultured from the brainstem and that NAD(P)H oxidase-derived reactive oxygen species are involved in this action of apelin-13. Microinjection of apelin-13 into the RVLM resulted in increases in arterial pressure and in renal sympathetic nerve activity in Sprague-Dawley rats. The pressor effect of apelin-13 was attenuated by the specific NAD(P)H-oxidase inhibitor gp91ds-tat. In neurons cultured from the ventral brainstem, spontaneous action potentials were recorded using current-clamp recording. Superfusion of neurons with apelin-13 (100 nM) increased the neuronal firing rate from 0.79 ± 0.14 to 1.45 ± 0.26 Hz (n = 7, P < 0.01) in angiotensin II receptor-like 1-positive neurons, identified with single-cell reverse transcriptase-polymerase chain reaction. Neither the angiotensin II type 1 receptor antagonist losartan nor the angiotensin II type 2 receptor antagonist 1-[[4-(dimethylamino)-3-methylphenyl[methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate (PD123319) altered the positive chronotropic effect of apelin-13. Pretreatment of cells with either the reactive oxygen species scavenger superoxide dismutase [polyethylene glycol-superoxide dismutase (PEG-SOD), 25 U/ml] or with gp91ds-tat significantly attenuated the chronotropic action of apelin-13. PEG-SOD and gp91ds-tat alone had no effect on basal neuronal firing. In addition, apelin-13 significantly increased NAD(P)H oxidase activity and elevated intracellular superoxide levels in neuronal cultures. The superoxide generator xanthine-xanthine oxidase also increased neuronal activity in neurons, mimicking the neuronal response to apelin-13. These observations provide the first evidence that apelin-13 directly increases neuronal activity via stimulation of NAD(P)H oxidase-derived superoxide, a cellular signaling mechanism that may be involved in the pressor effect of apelin-13 in the RVLM.

The production of 20-hydroxyeicosatetraenoic acid (20-HETE) is increased during ischemia-reperfusion, and inhibition of 20-HETE production has been shown to reduce infarct size caused by ischemia. This study was aimed to discover the molecular mechanism underlying the action of 20-HETE in cardiac myocytes. The effect of 20-HETE on L-type Ca2+ currents ( ICa,L) was examined in rat isolated cardiomyocytes by patch-clamp recording in the whole cell mode. Superfusion of cardiomyocytes with 20-HETE (10–100 nM) resulted in a concentration-dependent increase in ICa,L, and this action of 20-HETE was attenuated by a specific NADPH oxidase inhibitor, gp91ds-tat (5 μM), or a superoxide scavenger, polyethylene glycol-superoxide dismutase (25 U/ml), suggesting that NADPH-oxidase-derived superoxide is involved in the stimulatory action of 20-HETE on ICa,L. Treatment of cardiomyocytes with 20-HETE (100 nM) increased both NADPH oxidase activity and superoxide production by approximately twofold. To study the molecular mechanism mediating the 20-HETE-induced increase in NADPH oxidase activity, PKC activity was measured in cardiomyocytes. Incubation of the cells with 20-HETE (100 nM) significantly increased PKC activity, and pretreatment of cardiomyocytes with a selective PKC inhibitor, GF-109203 (1 μM), attenuated the 20-HETE-induced increases in ICa,L and in NADPH oxidase activity. In summary, 20-HETE stimulates NADPH oxidase-derived superoxide production, which activates L-type Ca2+ channels via a PKC-dependent mechanism in cardiomyocytes. 20-HETE and 20-HETE-producing enzymes could be novel targets for the treatment of cardiac ischemic diseases.

Rationale: Central angiotensin (Ang) II inhibits baroreflex and plays an important role in the pathogenesis of hypertension. However, the underlying molecular mechanisms are still not fully understood. Objective: Our objective in the present study was to characterize the signal transduction mechanism of phosphatidylinositol 3-kinase (PI3K) involvement in Ang II–induced stimulation of central neuronal activity in cultured neurons and Ang II–induced inhibition of baroreflex in spontaneously hypertensive rats (SHR) versus WKY rats. Methods and Results: Application of Ang II to neurons produced a 42% greater increase in neuronal firing in cells from the SHR than the WKY rat. Although the Ang II–mediated increase in firing rate was abolished entirely by the protein kinase (PK)C inhibitor GF109230 in the WKY, blockade of both PKC and PI3K activity was necessary in the SHR. This was associated with an increased ability of Ang II to stimulate NADPH oxidase–reactive oxygen species (ROS)–mediated signaling involving phosphorylation of the p47phox subunit of the NADPH oxidase and was dependent on the activation of PI3K in the SHR. Inhibition of PI3K resulted in the reduction of levels of p47phox phosphorylation, NADPH oxidase activity, ROS levels, and ultimately neuronal activity in cells from the SHR but not the WKY rat. In addition, in working heart–brainstem preparations, inhibition of PKC activity in the nucleus of the solitary tract in situ abolished the Ang II–mediated depression of cardiac and sympathetic baroreceptor reflex gain in the WKY. In contrast, PKC inhibition in the nucleus of the solitary tract of SHR only partially reduced the effect of Ang II on the baroreceptor reflex gain. Conclusions: These observations demonstrate that PI3K in the cardiovascular brainstem regions of the SHR may be selectively involved in Ang II–mediated signaling that includes a reduction in baroreceptor reflex function, presumably via a NADPH-ROS mediated pathway.

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.

The peripheral apelin system plays a significant role in cardiovascular homeostasis and in the pathophysiology of cardiovascular diseases. However, the central effect of this neurohormonal system in neural control of cardiovascular function remains poorly understood. Thus, this study was undertaken to evaluate the effect of apelin in the rostral ventrolateral medulla (RVLM) on blood pressure, cardiac function, and sympathetic nerve activity. Apelin mRNA and protein levels were detected with real-time RT-PCR and Western blots, respectively. Expression of apelin was significantly enhanced in the RVLM of spontaneously hypertensive rat (SHR) compared with normotensive Wistar–Kyoto (WKY) rats. To study the functional consequence of upregulated apelin expression, apelin was overexpressed by bilateral microinjection of the AAV2-apelin viral vector into the RVLM of WKY rats. Immunofluorescence staining and Western blots demonstrated that microinjection of AAV2-apelin into the RVLM resulted in a significant increase in apelin expression, which was associated with a chronic elevation in blood pressure and cardiac hypertrophy. In addition, direct microinjection of exogenous apelin-13 (200 pmol in 50 nL) into the RVLM caused a 20 mm Hg elevation in blood pressure and a 24% increase in sympathetic nerve activity. The present study is the first to show that apelin expression is enhanced in the RVLM of SHR versus WKY rats and that overexpression of this gene in the RVLM results in chronic blood pressure elevation and cardiac hypertrophy in normotensive rats. Thus, the apelin system in the RVLM may play a very important role in central blood pressure regulation and in the pathogenesis of hypertension.

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca(2+)](i)) in a dose-dependent manner and activated the L-type Ca(2+) channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca(2+)](i) elevation was abolished in the presence of the ET(A) receptor blocker BQ123, but was not affected by the ET(B) receptor blocker BQ788. ET-1-induced an increase in [Ca(2+)](i), which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 micromol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca(2+)](i) increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca(2+) channel current and an increase of open-state probability (NPo) of an L-type single Ca(2+) channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca(2+) channel activation and Ca(2+)-induced Ca(2+) release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway.

It has been shown that reactive oxygen species (ROS) are involved in the intracellular signaling response to G-protein coupled receptor stimuli in vascular smooth muscle cells and in neurons. In the present study, we tested the hypothesis that NAD(P)H oxidase-derived ROS are involved endothelin-1 (ET-1)-induced L-type calcium channel activation in isolated cardiac myocytes. ET-1 (10 nM) induced a 2-fold increase in L-type calcium channel open-state probability (NPo). This effect of ET-1 was abolished by the ET(A) receptor antagonist cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) [BQ-123 (1 microM)] but was not altered in the presence of an ET(B) receptor antagonist N-cis-2,6-dimethylpiperidinocarbonyl-b-tBu-Ala-D-Trp(1-methoxycarbonyl)-D-Nle-OH [BQ-788 (1 microM)]. Pretreatment of cells with the ROS scavenger tempol (100 microM), polyethylene glycol-superoxide dismutase (SOD, 25 U/ml), or the NAD(P)H-oxidase inhibitor gp91ds-tat ([H]RKKRRQRRR-CSTRIRRQL[NH(3)]) (5 microM) significantly attenuated ET-1-induced increases in calcium channel NPo. Tempol, SOD, and gp91ds-tat alone had no effect on basal calcium channel activity. In addition, ET-1 significantly increased NAD(P)H oxidase activity and elevated intracellular superoxide levels in cultured cardiac myocytes. The superoxide generator, xanthine-xanthine oxidase (10 mM, 20 mU/ml), also increased calcium channel NPo in cardiac myocytes, mimicking the effect of ET-1. These observations provide the first evidence that ET-1 induces the activation of L-type Ca(2+) channels via stimulation of NAD(P)H-derived superoxide production in cardiac myocytes.

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 µl). 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.

Increasing evidence indicates that both the angiotensin II (ANG II) and γ-aminobutyric acid (GABA) systems play a very important role in the regulation of blood pressure (BP). However, there is little information concerning the interactions between these two systems in the nucleus tractus solitarii (NTS). In the present study, we examined the effects of ANG II on GABAA and GABAB receptor (GAR and GBR) expression in the NTS of Sprague-Dawley rats. The direct effect of ANG II on GBR expression was determined in neurons cultured from NTS. Treatment of neuronal cultures with ANG II (100 nM, 5 h) induced a twofold increase in GBR1 expression, as detected with real-time RT-PCR and Western blots, but had no effect on GBR2 or GAR expression. In electrophysiological experiments, perfusion of neuronal cultures with the GBR agonist baclofen decreased neuronal firing rate by 39% and 63% in neurons treated with either PBS (control) or ANG II, respectively, indicating that chronic ANG II treatment significantly enhanced the neuronal response to GBR activation. In contrast, ANG II had no significant effect on the inhibitory action of the GAR agonist muscimol. In whole animal studies, intracerebroventricular infusion of ANG II induced a sustained increase in mean BP and an elevation of GBR1 mRNA and protein levels in the NTS. These results indicate that ANG II stimulates GBR expression in NTS neurons, and this could contribute to the central nervous system actions of ANG II that result in dampening of baroreflexes and elevated BP in the central actions of ANG II.

The rostral ventrolateral medulla (RVLM) is a relay point that provides supraspinal excitatory input to sympathetic preganglionic neurons in the regulation of blood pressure. The importance of the RVLM is further highlighted by observations that an increase of RVLM sensitivity to angiotensin II and enhanced sympathetic activity are associated with hypertension. Angiotensin-converting enzyme 2 (ACE2) has been shown to be central in maintaining the balance between vasoconstrictor activity of angiotensin II with vasoprotective action of angiotensin-(1-7) in the peripheral system. However, its role in central control of blood pressure in the RVLM is yet to be investigated. Thus, our objective in this study was to compare ACE2 expression in the RVLM of Wistar–Kyoto rats and spontaneously hypertensive rats and to determine whether RVLM ACE2 is involved in blood pressure control. ACE2 immunoreactivity was diffusely distributed in many cardiovascular regulatory neurons, including the RVLM. Western blot analysis revealed a 40% decrease in ACE2 in the RVLM of spontaneously hypertensive rat compared with Wistar–Kyoto rats. Lentiviral-mediated overexpression of ACE2 (lenti-ACE2) was used to determine whether a decrease in ACE2 in the RVLM is associated with hypertensive state. Bilateral injection of lenti-ACE2 resulted in a long-term expression of transgenic ACE2. This was associated with a decrease in mean arterial pressure exclusively in the spontaneously hypertensive rat (141±4 mm Hg in lenti-GFP versus 124±5 mm Hg in lenti-ACE2) and heart rate (304±7 bpm in lenti-GFP versus 285±5 bpm in lenti-ACE2). These observations demonstrate that overexpression of ACE2 overcomes its intrinsic decrease in the RVLM and decreases high blood pressure in the spontaneously hypertensive rat.

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 ≈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 (≈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 (1×10 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.

The effects of natural and synthetic eicosanoids on the diameter of rat interlobular arteries studied in vitro were compared to that of the potent, endogenous vasoconstrictor 20-HETE. Vasoconstrictor activity was optimum for chain lengths of 20-22 carbons with at least one olefin or epoxide between located between C(13)-C(15) and an oxygen substituent at C(20)-C(22). The presence of delta (Zou et al. Am. J. Physiol. 1996, 270, R228; Gebremedhin, D. et al. Am. J. Physiol. 1998, 507, 771)-, delta (Carroll et al. Am. J. Physiol. 1996, 271, R863; Vazquez et al. Life Sci. 1995, 56, 1455)-, or delta (Imig et al. Hypertension 2000, 35, 307; Lopez et al. Amer. J. Physiol. 2001, 281, F420)-olefins had no influence on the vasoconstrictor response whereas the introduction of a C(7)-thiomethylene enhanced potency. A sulfonamide or alcohol, but not a lactone, could replace the C(1)-carboxylate. These data were used to construct a putative binding domain map of the 20-HETE receptor consisting of: (i) a comparatively open, hydrophilic binding site accommodating the C(1)-functionality; (ii) a hydrophobic trough spanning the olefins; (iii) a shallow pocket containing a critical pi-pi binding site in the vicinity of the pi (Ito et al. Am. J. Physiol. 1998, 274, F395; Quigley, R.; Baum, M.; Reddy, K. M.; Griener, J. C.; Falck, J. R. Am. J. Physiol. 2000, 278, F949)-olefin; and (iv) an oxyphilic binding site proximate to the omega-terminus.

Despite advances in transgenic and gene transfer technologies, in vivo structure–function studies of the angiotensin II type I receptor (AT 1 R) have revealed limited information on the diverse actions of angiotensin II. Our objective in the present study was to determine if protein transduction technology with the use of the HIV-Tat protein transduction domain could fill this gap. Recombinant HIV-Tat protein transduction domain fused to EGFP and to the third intracellular loop of the AT 1 R was expressed. Incubation of hypothalamus and brainstem neurons with this peptide indicated an efficient transport of the protein to most of the cells. This transduction was accompanied by an increase in neuronal firing rate, an effect similar to that observed with angiotensin II stimulation of the neuronal AT 1 R. The characteristics of the chronotropic effects of recombinant third intracellular loop and its synthetic counterpart were similar and comparable to the effects of angiotensin II on these neurons. In addition, in the presence of the protein kinase C inhibitor calphostin C, the peptide failed to increase firing rate. These observations demonstrated that transduction of neurons with the third intracellular loop of the AT 1 R produces chronotropic effects similar to those induced by angiotensin II. The data suggests that protein transduction technology could be useful for in vivo AT 1 R domain transduction.

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.

To describe the usefulness of REMEDi HS in the diagnosis of acute drug poisoning patients.Samples from 1 200 drug poisoning patients were analysed with REMEDi HS. REMEDi HS is a broad spectrum rapid drug identification system, designed for emergency toxicology screening and forensic applications. The total analysis time is about 30 min. The current library of the system has 916 drugs and their metabolites.Among the 1 200 poisoning patients identified by REMIDi HS, the most frequently encountered drugs were benzodiazepines, phenothiazine anti-psychotics, non-phenothiazine anti-psychotics, tricyclic anti-depressants and opioid analgesics. Estazolam (10.25%)diazepam (9.25%)cimetidine (4.33%)chlorpromazine (3.50%)clozapine(2.75%)caffeine (2.58%)nikethamide (2.42%)morpine (1.92%)lidocaine (1.75%) and alprazolam (1.50%) are the ten drugs of highest frequency identified.Our study demonstrated that REMEDi HS is particularly useful for rapid measurement of drug concentration in the samples from acute poisoning patients.

Angiotensin II (Ang II) plays an important role in the central control of blood pressure and baroreflexes. These effects are initiated by stimulation of Ang II type 1 (AT 1 ) receptors on neurons within the hypothalamus and brain stem, and involve increasing the activity of noradrenergic, substance P, and glutamatergic pathways. The goal of this study is to investigate the intracellular signaling molecules, which are involved in mediating the Ang II-induced increases in neuronal activity. Using neurons in primary culture from newborn rat hypothalamus and brain stem, we have previously determined that Ang II elicits an AT 1 receptor-mediated inhibition of delayed rectifier K + current, a stimulation of Ca 2+ current, and a consequent increase in firing rate. In the present study we have demonstrated that this chronotropic action of Ang II in neuronal cultures involves activation of Ca 2+ -dependent signaling molecules. The Ang II-induced increase in firing rate was abolished by inhibition of phospholipase C with U73122 (10 μmol/L), and was attenuated by the protein kinase C inhibitor calphostin C (10 μmol/L) or by the calcium/calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 (10 μmol/L). A combination of calphostin C and KN-93 completely inhibited this Ang II action. These results indicate that the AT 1 receptor-mediated increase in neuronal firing rate involves activation of both PKC and CaMKII, and suggest that these enzymes are potential targets for manipulating the central actions of Ang II.

SUMMARY 1. Arachidonic acid (AA) is metabolized by cytochrome P450 (CYP)-dependent pathways to epoxyeicosatrienoic acids (EET) and 20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney and the peripheral vasculature. 2. The present short review summarizes the renal and cardiovascular actions of these important mediators. 3. Epoxyeicosatrienoic acids are vasodilators produced by the endothelium that hyperpolarize vascular smooth muscle (VSM) cells by opening Ca2+-activated K+ (KCa) channels. 20-Hydroxyeicosatetraenoic acid is a vasoconstrictor that inhibits the opening of KCa channels in VSM cells. Cytochrome P450 4A inhibitors block the myogenic response of small arterioles to elevations in transmural pressure and autoregulation of renal and cerebral blood flow in vivo. Cytochrome P450 4A blockers also attenuate the vasoconstrictor response to elevations in tissue PO2, suggesting that this system may serve as a vascular oxygen sensor. Nitric oxide and carbon monoxide inhibit the formation of 20-HETE and a fall in 20-HETE levels contributes to the activation of KCa channels in VSM cells and the vasodilator response to these gaseous mediators. 20-Hydroxyeicosatetraenoic acid also mediates the inhibitory actions of peptide hormones on sodium transport in the kidney and the mitogenic effects of growth factors in VSM and mesangial cells. A deficiency in the renal production of 20-HETE is associated with the development of hypertension in Dahl salt-sensitive rats. 4. In summary, the available evidence indicates that CYP metabolites of AA play a central role in the regulation of renal, pulmonary and vascular function and that abnormalities in this system may contribute to the pathogenesis of cardiovascular diseases.

The present study examined the contribution of elevations in cGMP versus inhibition of cytochrome P-4504A enzymes and the production of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) to the vasodilator actions of NO in renal arterioles. The NO donor sodium nitroprusside (SNP) at 10−5, 10−4, and 10−3 M reduced the production of 20-HETE in microsomes prepared from renal arterioles to 80 ± 2, 43 ± 5, and 7 ± 1% of control, respectively ( n = 4). In other experiments, the vasodilator response to SNP (10−7 to 10−3 M) was examined in rat renal interlobular arteries (