Stephen T. O'Rourke

Apelin Gene Transfer Into the Rostral Ventrolateral Medulla Induces Chronic Blood Pressure Elevation in Normotensive Rats

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.

Endothelin-1 Regulates Cardiac L-Type Calcium Channels via NAD(P)H Oxidase-Derived Superoxide

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 ETA receptor antagonist cyclo(D-Trp-D-Asp-Pro-D-Val-Leu) [BQ-123 (1 μM)] but was not altered in the presence of an ETB receptor antagonist N-cis-2,6-dimethylpiperidinocarbonyl-b-tBu-Ala-D-Trp(1-methoxycarbonyl)-D-Nle-OH [BQ-788 (1 μM)]. Pretreatment of cells with the ROS scavenger tempol (100 μM), polyethylene glycol-superoxide dismutase (SOD, 25 U/ml), or the NAD(P)H-oxidase inhibitor gp91ds-tat ([H]RKKRRQRRR-CSTRIRRQL[NH3]) (5 μM) 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 Ca2+ channels via stimulation of NAD(P)H-derived superoxide production in cardiac myocytes.

20-HETE increases NADPH oxidase-derived ROS production and stimulates the L-type Ca2+ channel via a PKC-dependent mechanism in cardiomyocytes

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 Ca(2+) currents (I(Ca,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 I(Ca,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 I(Ca,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 I(Ca,L) and in NADPH oxidase activity. In summary, 20-HETE stimulates NADPH oxidase-derived superoxide production, which activates L-type Ca(2+) 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.

MT2 Receptors Mediate the Inhibitory Effects of Melatonin on Nitric Oxide-Induced Relaxation of Porcine Isolated Coronary Arteries

Previous studies from our laboratory demonstrated that melatonin inhibits nitric oxide (NO)-induced relaxation in porcine coronary arteries. The present study was designed to further characterize the mechanisms underlying this inhibitory effect of melatonin. Western immunoblot studies identified the presence of melatonin type 2 (MT2) receptors, but not MT1 or MT3 receptors, in porcine coronary arteries. Immunohistochemical analysis revealed that MT2 receptors colocalized with α-actin in the smooth muscle cell layer. In coronary arterial rings suspended in organ chambers for isometric tension recording, melatonin (10−7 M) inhibited relaxations induced by the exogenous NO donor sodium nitroprusside (SNP; 10−9 to 10−5 M) and by the α2-adrenoceptor agonist 5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline (UK14,304; 10−9 to 10−5 M), an endothelium-dependent vasodilator. The inhibitory effect of melatonin on SNP- and UK14,304-induced relaxations was abolished in the presence of the selective MT2 receptor antagonists 4-phenyl-2-propionamidotetralin (4P-PDOT; 10−7 M) and luzindole (10−7 M). In contrast to melatonin, the selective MT3 receptor agonist 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT; 10−7 M) had no effect on the concentration-response curves to either SNP or UK14,304. Melatonin (10−7 M) had no effect on coronary artery relaxation induced by 8-bromoguanosine 3′,5′-cyclic monophosphate, but it significantly attenuated the increase in intracellular cyclic GMP levels in response to SNP (10−5 M). This effect of melatonin was abolished in the presence of 4P-PDOT (10−7 M). Taken together, these data support the view that melatonin acts on MT2 receptors in coronary vascular smooth muscle cells to inhibit NO-induced increases in cyclic GMP and coronary arterial relaxation, thus demonstrating a novel function for MT2 receptors in the vasculature.

Angiotensin II increases GABAB receptor expression in nucleus tractus solitarii of rats

Increasing evidence indicates that both the angiotensin II (ANG II) and gamma-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.

2011 and 2012 Early Careers Achievement Awards: Placental programming: how the maternal environment can impact placental function.

Proper establishment of the placenta is important for fetal survival; however, placental adaptations to inadequate maternal nutrition or other stressors are imperative for fetal growth to be optimal. The effects of maternal nutritional status and activity level on placental vascular function and uteroplacental blood flows are important to understand as improper placental function leads to reduced growth of the fetus. In environments where fetal growth can be compromised, potential therapeutics may augment placental function and delivery of nutrients to improve offspring performance during postnatal life. Factors that could enhance placental function include supplementation of specific nutrients, such as protein, hormone supplements, such as indolamines, and increased activity levels of the dam. To understand the mechanism of how the maternal environment can impact uterine or umbilical blood flows, assessment of placental vascular reactivity has been studied in several large animal models. As we begin to understand how the maternal environment impacts uterine and umbilical blood flows and other uteroplacental hemodynamic parameters, development of management methods and therapeutics for proper fetal growth can be achieved.

Effects of potassium channel blockers on resting tone in isolated coronary arteries.

The effects of several potassium channel blockers on resting vasomotor tone were studied in porcine isolated coronary arteries. Coronary artery rings were suspended in organ baths for isometric tension recording. The nonselective potassium channel blockers tetraethylammonium (TEA 10(-5)-3 x 10(-2) M) and 4-aminopyridine (4-AP 10(-5)-10(-2) M) caused concentration-dependent contractions that were similar in rings with and without endothelium. The concentration-response curves to TEA and 4-AP were unaffected by treatment with phentolamine (3 x 10(-6) M),propranolol (10(-6) M), or atropine (10(-6) M). Diltiazem (10(-6) M) almost abolished the contractions evoked by TEA and 4-AP. Charybdotoxin (10(-9)-10(-7) M) and apamin (10(-8)-10(-6) M), selective blockers of large and small calcium-activated potassium channels, respectively, and glyburide (10(-8)-10(-6) M), a selective blocker of ATP-sensitive potassium channels, caused little or no contraction in rings with or without endothelium. Therefore, in isolated coronary arteries, TEA and 4-AP caused contractions that were independent of the release of vasoactive mediators from the endothelium or perivascular nerves. These effects are not mediated by ATP-sensitive potassium channels or by large and small conductance calcium-activated potassium channels. The data are consistent with an effect of TEA and 4-AP on resting membrane potassium conductance in coronary arteries, resulting in contractions that are sensitive to inhibition by diltiazem. This pattern of responsiveness of isolated coronary arteries to potassium channel blockers differs from that observed in vessels from other vascular beds.

Pressor Effect of Apelin-13 in the Rostral Ventrolateral Medulla: Role of NAD(P)H Oxidase-Derived Superoxide

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.

Angiotensin II enhances GABAB receptor-mediated responses and expression in nucleus tractus solitarii of rats

Angiotensin II (ANG II) increases GABA(B) receptor expression in neuronal cultures from the nucleus tractus solitarii (NTS). In the present study, the chronic effects of ANG II on GABA(B) receptor expression and activity were examined in the NTS of Sprague-Dawley rats. Intracerebroventricular infusion of ANG II caused a significant elevation in blood pressure (BP) and an increase in GABA(B) receptor expression in the NTS. Conversely, chronic N(G)-nitro-l-arginine methyl ester (l-NAME) treatment also increased BP, but had no effect on GABA(B) receptor expression in the NTS. Next, we examined the BP response to the GABA(B) receptor agonist baclofen microinjected into the NTS of ANG II- or artificial cerebrospinal fluid (aCSF)-infused rats. NTS microinjection of baclofen increased BP in both groups of rats. However, the pressor response to baclofen was enhanced in ANG II-infused rats compared with aCSF-infused rats. In addition, bilateral microinjection of the GABA(B) receptor antagonist CGP-35348 into the NTS evoked a decrease in BP in both group of rats, and the depressor responses to CGP-35348 were enhanced in the ANG II-infused rats. In contrast, the pressor responses to the GABA(A) receptor agonist muscimol and the depressor responses to the GABA(A) receptor antagonist bicuculline were comparable between aCSF- and ANG II-infused rats. These results indicate that chronic ANG II infusion stimulates GABA(B) receptor expression and augments GABA(B) receptor-mediated responses in the NTS. This effect could contribute to the central nervous system actions of ANG II that result in dampening of baroreflexes and elevation in arterial BP.

Maternal nutrient restriction during pregnancy impairs an endothelium-derived hyperpolarizing factor-like pathway in sheep fetal coronary arteries.

The mechanisms underlying developmental programming are poorly understood but may be associated with adaptations by the fetus in response to changes in the maternal environment during pregnancy. We hypothesized that maternal nutrient restriction during pregnancy alters vasodilator responses in fetal coronary arteries. Pregnant ewes were fed a control [100% U.S. National Research Council (NRC)] or nutrient-restricted (60% NRC) diet from days 50 to 130 of gestation (term = 145 days); fetal tissues were collected at day 130. In coronary arteries isolated from control fetal lambs, relaxation to bradykinin was unaffected by nitro-l-arginine (NLA). Iberiotoxin or contraction with KCl abolished the NLA-resistant response to bradykinin. In fetal coronary arteries from nutrient-restricted ewes, relaxation to bradykinin was fully suppressed by NLA. Large-conductance, calcium-activated potassium channel (BKCa) currents did not differ in coronary smooth muscle cells from control and nutrient-restricted animals. The BKCa openers, BMS 191011 and NS1619, and 14,15-epoxyeicosatrienoic acid [a putative endothelium-derived hyperpolarizing factor (EDHF)] each caused fetal coronary artery relaxation and BKCa current activation that was unaffected by maternal nutrient restriction. Expression of BKCa-channel subunits did not differ in fetal coronary arteries from control or undernourished ewes. The results indicate that maternal undernutrition during pregnancy results in loss of the EDHF-like pathway in fetal coronary arteries in response to bradykinin, an effect that cannot be explained by a decreased number or activity of BKCa channels or by decreased sensitivity to mediators that activate BKCa channels in vascular smooth muscle cells. Under these conditions, bradykinin-induced relaxation is completely dependent on nitric oxide, which may represent an adaptive response to compensate for the absence of the EDHF-like pathway.