bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of VEGFR-2 and PLD2 in endothelial caveolae is involved in VEGF-induced phosphorylation of MEK and ERK
Authors: Cho Chung-Hyun , Lee Chang Sup , Chang Mikyung , Jang Il-Ho , Kim Soo Jin , Hwang Inhwan , Ryu Sung Ho , Lee Chin O. , Koh Gou Young
Published: February 2004

To clarify the role of caveolae in VEGF/VEGF receptor-2 (VEGFR-2)-mediated signaling cascades, primary cultured human umbilical vein endothelial cells (HUVECs) were fractionated to isolate caveolae-enriched cell membranes. Interestingly, VEGFR-2, phospholipase D2 (PLD2), and Ras were enriched in caveolae-enriched fractions. Moreover, VEGF increased PLD activity in a time- and dose-dependent manner in HUVECs, whereas a ligand specific for VEGFR-1 placental growth factor did not change PLD activity. A PLD inhibitor, 1-butanol, almost completely suppressed VEGF-induced ERK phosphorylation and cellular proliferation, whereas the negative control for 1-butanol, 3-butanol, did not produce significant changes. Addition of phosphatidic acid negated the 1-butanol-induced suppression. Pharmacological analyses using several inhibitors indicated that PKC-δ regulates the VEGF-induced activation of PLD/ERK. Thus PLD2 could be involved in MEK/ERK signaling cascades that are induced by the VEGF/VEGFR-2/PKC-δ pathway in endothelial cells. Pretreatment with the cholesterol depletion agent methyl-β-cyclodextrin (MβCD) almost completely disassembled caveolar structures, whereas the addition of cholesterol to MβCD-treated cells restored caveolar structures. Pretreatment with MβCD largely abolished phosphorylation of MEK/ERK by VEGF, whereas the addition of cholesterol restored VEGF-induced MEK/ERK phosphorylations. These results indicate that intact caveolae are required for the VEGF/VEGFR-2-mediated MEK/ERK signaling cascade.
Direct effect of ethanol on human vascular function
Authors: Tawakol Ahmed , Omland Torbjørn , Creager Mark A.
Published: February 2004

Epidemiological studies indicate that moderate ethanol consumption reduces cardiovascular mortality. Cellular and animal data suggest that ethanol confers beneficial effects on the vascular endothelium and increases the bioavailability of nitric oxide. The purpose of this study was to assess the effect of ethanol on endothelium-dependent, nitric oxide-mediated vasodilation in healthy human subjects. Forearm blood flow (FBF) was determined by venous occlusion plethysmography in healthy human subjects during intra-arterial infusions of either methacholine (0.3, 1.0, 3.0, and 10.0 mcg/min, n = 9), nitroprusside (0.3, 1.0, 3.0, and 10.0 mcg/min, n = 9), or verapamil (10, 30, 100, and 300 mcg/min, n = 8) before and during the concomitant intra-arterial infusions of ethanol (10% ethanol in 5% dextrose). Additionally, a time control experiment was conducted, during which the methacholine dose-response curve was measured twice during vehicle infusions ( n = 5). During ethanol infusion, mean forearm and systemic alcohol levels were 227 ± 30 and 6 ± 0 mg/dl, respectively. Ethanol infusion alone reduced FBF (2.5 ± 0.1 to 1.9 ± 0.1 ml·dl−1·min−1, P < 0.05). Despite initial vasoconstriction, ethanol augmented the FBF dose-response curves to methacholine, nitroprusside, and verapamil ( P < 0.01 by ANOVA for each). To determine whether this augmented FBF response was related to shear-stress-induced release of nitric oxide, FBF was measured during the coinfusion of ethanol and NG-nitro-l-arginine (l-NAME; n = 8) at rest and during verapamil-induced vasodilation. The addition of l-NAME did not block the ability of ethanol to augment verapamil-induced vasodilation. Ethanol has complex direct vascular effects, which include basal vasoconstriction as well as potentiation of both endothelium-dependent and -independent vasodilation. None of these effects appear to be mediated by an increase in nitric oxide bioavailability, thus disputing findings from preclinical models.
Excess nitric oxide decreases cytochromeP-450 2J4 content andP-450-dependent arachidonic acid metabolism in lungs of rats with acute pneumonia
Authors: Yaghi Asma , Bend John R. , Webb Christopher D. , Zeldin Darryl C. , Weicker Sean , Mehta Sanjay , McCormack David G.
Published: February 2004

Recently, we demonstrated that pulmonary CYP2J4 content, a prominent source of EETs and HETEs formation in rat lungs, is reduced in pneumonia. Therefore, the purpose of this study was to determine the role of iNOS-derived NO in reduced pulmonary CYP2J4 protein content and decreased CYP metabolites in pneumonia. Rats were randomized to control, control plus 1400W (iNOS inhibitor), pneumonia, and pneumonia plus 1400W groups. Pseudomonas organisms were injected into lungs of pneumonia rats. At 40 h after surgery, rats were treated with either saline or 1400W for 4 h before death. Venous plasma samples were obtained for measuring nitrites/nitrates (NOx). There was no significant effect of 1400W on blood pressure measured in control or pneumonia rats, whereas 1400W reduced the elevated plasma NOx levels in pneumonia rats by half. CYP primary metabolites of AA formed at significantly lower rates in pulmonary microsomes from pneumonia rats compared with control rats. Treatment of pneumonia rats with 1400W resulted in a significant increase in the rate of formation of pulmonary EETs and ω-terminal HETEs compared with untreated pneumonia rats. The reduction in CYP2J4 protein content in pneumonia lung microsomes was also partially prevented by 1400W. Therefore, excess NO from iNOS decreases the pulmonary production of EETs and ω-HETEs in acute pneumonia. Inhibition of iNOS restores CYP2J4 protein content and CYP activity in acute pneumonia, indicating an important NO-CYP interaction in pulmonary responses to infection. We speculate CYP2J4 and its AA metabolites are involved in the modulation of pulmonary function in health and disease.
Impaired NO signaling in small pulmonary arteries of chronically hypoxic newborn piglets
Authors: Fike Candice D. , Aschner Judy L. , Zhang Yongmei , Kaplowitz Mark R.
Published: February 2004

We performed studies to determine whether chronic hypoxia impairs nitric oxide (NO) signaling in resistance level pulmonary arteries (PAs) of newborn piglets. Piglets were maintained in room air (control) or hypoxia (11% O2) for either 3 (shorter exposure) or 10 (longer exposure) days. Responses of PAs to a nonselective NO synthase (NOS) antagonist, Nω-nitro-l-arginine methylester (l-NAME), a NOS-2-selective antagonist, aminoguanidine, and 7-nitroindazole, a NOS-1-selective antagonist, were measured. Levels of NOS isoforms and of two proteins involved in NOS signaling, heat shock protein (HSP) 90 and caveolin-1, were assessed in PA homogenates. PAs from all groups constricted to l-NAME but not to aminoguanidine or 7-nitroindazole. The magnitude of constriction to l-NAME was similar for PAs from control and hypoxic piglets of the shorter exposure period but was diminished for PAs from hypoxic compared with control piglets of the longer exposure period. NOS-3, HSP90, and caveolin-1 levels were similar in hypoxic and control PAs. These findings indicate that NOS-3, but not-NOS 2 or NOS-1, is involved with basal NO production in PAs from both control and hypoxic piglets. After 10 days of hypoxia, NO function is impaired in PAs despite preserved levels of NOS-3, HSP90, and caveolin-1. The development of NOS-3 dysfunction in resistance level PAs may contribute to the progression of chronic hypoxia-induced pulmonary hypertension in newborn piglets.
Chemical activation of C1-C2 spinal neurons modulates intercostal and phrenic nerve activity in rats
Authors: Lu Fang , Qin Chao , Foreman Robert D. , Farber Jay P.
Published: February 2004

Chemical activation of upper cervical spinal neurons modulates activity of thoracic respiratory interneurons in rats. The aim of the present study was to examine the effects of chemical activation of C1-C2 spinal neurons on thoracic spinal respiratory motor outflows. Electroneurograms of left phrenic ( n = 23) and intercostal nerves (ICNs, n = 93) between T3 and T8 spinal segments were recorded from 36 decerebrated, vagotomized, paralyzed, and ventilated male rats. To activate upper cervical spinal neurons, glutamate pledgets (1 M, 1 min) were placed on the dorsal surface of the C1-C2 spinal cord. Glutamate on C1-C2 increased ICN tonic activity in 56/59 (95%) ICNs. The average maximal tonic activity of ICN was increased by 174% ( n = 59). After spinal transection at rostral C1, glutamate on C1-C2 still increased ICN tonic activity in 33/35 ICNs. However, the effects of C1-C2 glutamate on ICN phasic activity were highly variable, with observations of augmentation or suppression of both inspiratory and expiratory discharge. C1-C2 glutamate augmented the average amplitude of phrenic burst by 20%, whereas the increases in amplitude of ICN inspiratory activity, when they occurred, averaged 120%. The burst rate of phrenic nerve discharge was decreased from 34.2 ± 1.6 to 26.3 ± 2.0 (mean ± SE) breaths/min during C1-C2 glutamate. These data suggested that upper cervical propriospinal neurons might play a role in descending modulation of thoracic respiratory and nonrespiratory motor activity.
Effects of dietary calcium restriction and acute exercise on the antioxidant enzyme system and oxidative stress in rat diaphragm
Authors: Itoh Masayuki , Oh-ishi Shuji , Hatao Hidekazu , Leeuwenburgh Christiaan , Selman Colin , Ohno Hideki , Kizaki Takako , Nakamura Hiroyuki , Matsuoka Takeshi
Published: February 2004

Calcium deficiency is considered to increase intracellular calcium level; thus the aim of the current study was to elucidate whether dietary calcium restriction enhanced exercise-induced oxidative stress in rat diaphragm. Twenty male Wistar rats were randomly assigned to either a control group or a group subjected to 1 mo of calcium restriction. In addition, each group was subsequently subdivided into rested or acutely exercised group. Dietary calcium restriction significantly ( P < 0.05) upregulated the activities of manganese-superoxide dismutase (Mn-SOD), copper-zinc-superoxide dismutase (Cu-Zn-SOD), and glutathione peroxidase (Gpx) but not catalase. Acute exercise, in addition to calcium restriction, decreased both SOD isoenzymes in the diaphragm of calcium-restricted rats ( P < 0.05). On the other hand, calcium restriction resulted in increased Gpx mRNA expression ( P < 0.05). In control rats, acute exercise significantly ( P < 0.05) increased the expressions of both SOD mRNAs, whereas in the calcium-restricted rats, it increased that of Mn-SOD mRNA ( P < 0.05) but decreased that of Gpx mRNA ( P < 0.05). Furthermore, reactive carbonyl derivative, a marker of protein oxidation, was significantly greater in the calcium-restricted rats than in the control rats after acute exercise ( P < 0.05). The results suggest that antioxidant enzymes in rat diaphragm were upregulated in response to an increased oxidative stress by dietary calcium restriction but that upregulation is not enough to cope with exercise-induced further increase of oxidative stress.
Acute hemodynamic responses in the head during microgravity induced by free drop in anesthetized rats
Authors: Gotoh Taro Miyahara , Fujiki Nobuhiro , Tanaka Kunihiko , Matsuda Tomoko , Gao Shuang , Morita Hironobu
Published: February 2004

To examine acute hemodynamic responses to microgravity (μG) in the head, we measured carotid artery pressure (CAP) and jugular vein pressure (JVP) to calculate cephalic perfusion pressure (CPP = CAP − JVP) and recorded images of microvessels in the iris to evaluate capillary blood flow velocity (CBFV) and capillary diameter (CD) in anesthetized rats during 4.5 s of μG induced by free drop. Rats were placed in 30° head-up whole body-tilted (HU, n = 7) or horizontal (flat, n = 6) position. In the flat group, none of the measured variables was significantly affected by μG, whereas in the HU group, CAP, JVP, and CPP increased, respectively, by 23.4 ± 2.6, 1.3 ± 0.2, and 22.9 ± 3.1 mmHg, and CBFV and CD increased, respectively, by 33 ± 8 and 9 ± 3%, showing an increase in capillary blood flow. To further examine the mechanisms underlying these CAP and JVP increases, another experiment was performed in which CAP and JVP were measured in anesthetized rats ( n = 6) during a postural change from HU to flat. In these animals, the change in JVP was similar to that observed during actual μG, but no change in CAP was seen, indicating that the JVP increase during actual μG is caused by disappearance of the gravitational pressure gradient in the head-to-foot axis, whereas the CAP increase is not. In conclusion, actual μG elicits an increase in CPP due to a greater increase in CAP than JVP, resulting in increased capillary blood flow. Although the increase in JVP is explained by the disappearance of gravitational pressure gradient in the head-to-foot axis as a result of μG, the larger increase in CAP is not.
Effect of 2′-phosphophloretin on renal function in chronic renal failure rats
Authors: Peerce B. E. , Weaver L. , Clarke R. D.
Published: February 2004

Hyperhosphatemia and secondary hyperparathyroidism are common and severe complications of chronic renal failure. Therapies to reduce serum phosphate have been shown to reduce serum parathyroid hormone (PTH) and slow the progression of renal failure. The effect of the inhibitor of intestinal phosphate absorption, 2′-phosphophloretin (2′-PP), on serum and urine chemistry, renal histology, and cardiac structure in the uremic rat model of renal failure, 5/6 nephrectomy (5/6 NX), was examined. The effect of 2′-PP on serum phosphate, serum PTH, serum total Ca2+, and ionized Ca2+, Ca2+× Piproduct, urine protein, urine osmolality, and creatinine clearance in 5/6 NX rats was examined. Uremic rats in chronic renal failure were gavaged daily with 25 μM 2′-PP. Over the course of a 5-wk experiment, serum chemistry in untreated uremic rats, 2′-PP-treated uremic rats, and age-matched control rats with normal renal function was determined twice a week. Urine creatinine, urine osmolality, urine phosphate, and urine protein were determined once a week from 24-h collections. 2′-PP reduced serum phosphate 40 ± 3% compared with a 17% increase in untreated uremic control rats. 2′-PP did not alter total serum Ca2+. During 5-wk experiments, serum PTH increased 65 ± 25% in untreated uremic rats and decreased 70 ± 7% in uremic rats treated with 25 μM 2′-PP. Creatinine clearance decreased 20% in untreated uremic rats compared with a 100% increase in 2′-PP-treated uremic rats. Urine protein decreased and urine osmolality increased in uremic rats treated with 2′-PP. The mechanism of the effect of 2′-PP on serum phosphate was inhibition of intestinal phosphate absorption. 2-PP inhibited intestinal phosphate absorption 50% without altering dietary protein absorption or intestinal Ca2+absorption. Over the course of the 5-wk treatment with 2′-PP, uremic animals treated with 2′-PP had a 2–4% weight gain/wk, similar to the weight gain seen in age-matched control rats with normal renal function.
Human cutaneous vascular responses to whole-body tilting, Gzcentrifugation, and LBNP
Authors: Watenpaugh Donald E. , Breit Gregory A. , Buckley Theresa M. , Ballard Richard E. , Murthy Gita , Hargens Alan R.
Published: February 2004

We hypothesized that gravitational stimuli elicit cardiovascular responses in the following order with gravitational stress equalized at the level of the feet, from lowest to highest response: short-(SAC) and long-arm centrifugation (LAC), tilt, and lower body negative pressure (LBNP). Up to 15 healthy subjects underwent graded application of the four stimuli. Laser-Doppler flowmetry measured regional skin blood flow. At 0.6 Gz(60 mmHg LBNP), tilt and LBNP similarly reduced leg skin blood flow to ∼36% of supine baseline levels. Flow increased back toward baseline levels at 80–100 mmHg LBNP yet remained stable during 0.8–1.0 Gztilt. Centrifugation usually produced less leg vasoconstriction than tilt or LBNP. Surprisingly, SAC and LAC did not differ significantly. Thigh responses were less definitive than leg responses. No gravitational vasoconstriction occurred in the neck. All conditions except SAC increased heart rate, according to our hypothesized order. LBNP may be a more effective and practical means of simulating cardiovascular effects of gravity than centrifugation.
Age dependency of cardiovascular autonomic responses to head-up tilt in healthy subjects
Authors: Laitinen Tomi , Niskanen Leo , Geelen Ghislaine , Länsimies Esko , Hartikainen Juha
Published: February 2004

In elderly subjects, heart rate responses to postural change are attenuated, whereas their vascular responses are augmented. Altered strategy in maintaining blood pressure homeostasis during upright position may result from various cardiovascular changes, including age-related cardiovascular autonomic dysfunction. This exploratory study was conducted to evaluate impact of age on cardiovascular autonomic responses to head-up tilt (HUT) in healthy subjects covering a wide age range. The study population consisted of 63 healthy, normal-weight, nonsmoking subjects aged 23–77 yr. Five-minute electrocardiogram and finger blood pressure recordings were performed in the supine position and in the upright position 5 min after 70° HUT. Stroke volume was assessed from noninvasive blood pressure signals by the arterial pulse contour method. Heart rate variability (HRV) and systolic blood pressure variability (SBPV) were analyzed by using spectral analysis, and baroreflex sensitivity (BRS) was assessed by using sequence and cross-spectral methods. Cardiovascular autonomic activation during HUT consisted of decreases in HRV and BRS and an increase in SBPV. These changes became attenuated with aging. Age correlated significantly with amplitude of HUT-stimulated response of the high-frequency component ( r = -0.61, P < 0.001) and the ratio of low-frequency to high-frequency power of HRV ( r = -0.31, P < 0.05) and indexes of BRS (local BRS: r = -0.62, P < 0.001; cross-spectral baroreflex sensitivity in the low-frequency range: r = -0.38, P < 0.01). Blood pressure in the upright position was maintained well irrespective of age. However, the HUT-induced increase in heart rate was more pronounced in the younger subjects, whereas the increase in peripheral resistance was predominantly observed in the older subjects. Thus it is likely that whereas the dynamic capacity of cardiac autonomic regulation decreases, vascular responses related to vasoactive mechanisms and vascular sympathetic regulation become augmented with increasing age.
Hyperoxia impairs airway relaxation in immature rats via a cAMP-mediated mechanism
Authors: Mhanna Maroun J. , Haxhiu Musa A. , Jaber Marwan A. , Walenga Ronald W. , Chang Chang-Ho , Liu Shijian , Martin Richard J.
Published: February 2004

Hyperoxic exposure enhances airway reactivity in newborn animals, possibly due to altered relaxation. We sought to define the role of prostaglandinand nitric oxide-mediated mechanisms in impaired airway relaxation induced by hyperoxic stress. We exposed 7-day-old rat pups to either room air or hyperoxia (>95% O2) for 7 days to assess airway relaxation and cAMP and cGMP production after electrical field stimulation (EFS). EFS-induced relaxation of preconstricted trachea was diminished in hyperoxic vs. normoxic animals ( P < 0.05). Indomethacin (a cyclooxygenase inhibitor) reduced EFS-induced airway relaxation in tracheae from normoxic ( P < 0.05), but not hyperoxic, rat pups; however, in the presence of NG-nitro-l-arginine methyl ester (a nitric oxide synthase inhibitor) EFS-induced airway relaxation was similarly decreased in tracheae from both normoxic and hyperoxic animals. After EFS, the increase from baseline in the production of cAMP was significantly higher in tracheae from normoxic than hyperoxic rat pups, and this was accompanied by greater prostaglandin E2release only in the normoxic group. cGMP production after EFS stimulation did not differ between normoxic and hyperoxic groups. We conclude that hyperoxia impairs airway relaxation in immature animals via a mechanism primarily involving the prostaglandin-cAMP signaling pathway with an impairment of prostaglandin E2release and cAMP accumulation.
Evidence of sustained forearm vasodilatation after brief isocapnic hypoxia
Authors: Tamisier Renaud , Norman Daniel , Anand Amit , Choi Yoon , Weiss J. Woodrow
Published: February 2004

Healthy subjects exposed to 20 min of hypoxia increase ventilation and muscle sympathetic nerve activity (MSNA). After return to normoxia, although ventilation returns to baseline, MSNA remains elevated for up to an hour. Because forearm vascular resistance is not elevated after hypoxic exposure, we speculated that the increased MSNA might be a compensatory response to sustained release of endogenous vasodilators. We studied the effect of isocapnic hypoxia (mean arterial oxygen saturation 81.6 ± 4.1%, end-tidal Pco2 44.7 ± 6.3 Torr) on plethysmographic forearm blood flow (FBF) in eight healthy volunteers while infusing intra-arterial phentolamine to block local α-receptors. The dominant arm served as control. Forearm arterial vascular resistance (FVR) was calculated as the mean arterial pressure (MAP)-to-FBF ratio. MAP, heart rate (HR), and FVR were reported at 5-min intervals at baseline, then while infusing phentolamine during room air, isocapnic hypoxia, and recovery. Despite increases in HR during hypoxia, there was no change in MAP throughout the study. By design, FVR decreased during phentolamine infusion. Hypoxia further decreased FVR in both forearms. With continued phentolamine infusion, FVR after termination of the exposure (17.47 ± 6.3 mmHg·min·ml-1·100 ml of tissue) remained lower than preexposure baseline value (23.05 ± 8.51 mmHg·min·ml-1·100 ml of tissue; P < 0.05). We conclude that, unmasked by phentolamine, the vasodilation occurring during hypoxia persists for at least 30 min after the stimulus. This vasodilation may contribute to the sustained MSNA rise observed after hypoxia.
Muscle mechanoreceptor modulation of sweat rate during recovery from moderate exercise
Authors: Shibasaki Manabu , Sakai Mieko , Oda Mayumi , Crandall Craig G.
Published: February 2004

The objective of this study was to identify whether muscle mechanoreceptor stimulation is capable of modulating sweat rate. Seven healthy subjects performed two 20-min bouts of supine exercise on a tandem cycle ergometer (60 rpm at 65% of maximal heart rate). After one bout, the subject stopped exercising (i.e., no pedaling), whereas, after the other bout, the subject's legs were passively cycled (at 60 rpm) via a second person cycling the tandem ergometer. This allows for mechanical stimulation of muscle with minimal activation of central command. Esophageal temperature (Tes), mean skin temperature (T̄sk), heart rate, mean arterial blood pressure, oxygen consumption, cutaneous vascular conductance (CVC), and sweat rate were not different during the two exercise bouts. Regardless of the mode of exercise recovery, there were no differences in Tes, T̄sk, or CVC. In contrast, early in the recovery period, chest and forearm sweat rate were significantly greater in the passive cycling recovery mode relative to the no-pedaling condition (chest: 0.57 ± 0.13 vs. 0.39 ± 0.14, forearm: 0.30 ± 0.05 vs. 0.12 ± 0.02 mg·cm-2·min-1; both P < 0.05). These results suggested that muscle mechanoreceptor stimulation to the previously activated muscle is capable of modulating sweat rate.
Ocular Tracking of Moving Targets: Effects of Perturbing the Background
Authors: Kodaka Yasushi , Miura Kenichiro , Suehiro Kazuyo , Takemura Aya , Kawano Kenji
Published: February 2004

Primates are able to track a moving target with their eyes, even when the target is seen against a stationary textured background. In this situation, the tracking eye movement induces motion of the background images on the retina (reafference) that competes with the motion of the target's retinal image, potentially disrupting the tracking of the target. Previous work on humans reported that brief perturbations of the background in the opposite direction to pursuit were much less disruptive than perturbations in the same direction as pursuit. Furthermore, if the background moved together with the pursuit target—so as to effectively eliminate the reafference—then the effects of a subsequent background perturbation showed less dependence on direction. This suggested that the direction selectivity to background perturbations during pursuit against a stationary background was due, at least in part, to the prior motion of the background secondary to the pursuit. We now report similar findings in monkeys, and in addition, have investigated the effect of moving the background while the animal was fixating a stationary target. In this situation, the ocular tracking responses to subsequent brief perturbations of the moving background were weaker when the perturbations were in the same direction as the prior background motion than when in the opposite direction. This suggests that the selective insensitivity to the reafferent visual input associated with pursuit across a stationary background is, at least in part, independent of pursuit per se and attributable to a progressive reduction in the sensitivity to sustained background motion.
Contextual Modulation of Central Thalamic Delay-Period Activity: Representation of Visual and Saccadic Goals
Authors: Wyder Melanie T. , Massoglia Dino P. , Stanford Terrence R.
Published: February 2004

This study examines the influence of behavioral context on the activity of visuomotor neurons in primate central thalamus. Neurons that combine information about sensory stimuli and their behavioral relevance are thought to contribute to the decision mechanisms that link specific stimuli to specific responses. We reported in a previous study that neurons in central thalamus carry spatial information throughout the instructed delay period of a visually guided delayed saccade task. The goal of the current study was to determine whether the delay-period activity of thalamic neurons is modulated by behavioral context. Single neurons were evaluated during performance of visually guided and memory-guided variants of a saccadic choice task in which a cue designated the response field stimulus as the target of a rewarded saccade or as an irrelevant distracter. The relative influence of the physical stimulus and context on delay-period activity suggested a minimum of 3 neural groups. Some neurons signaled the locations of visible stimuli regardless of behavioral relevance. Other neurons preferentially signaled the locations of current saccadic goals and did so even in the absence of the physical stimulus. A third group signaled only the locations of currently visible saccadic goals. For the latter 2 groups, activity was the product of both stimulus and context, suggesting that central thalamic neurons play a role in the context-dependent linkage of sensory signals and saccadic commands. More generally, these data suggest that the anatomical substrate of sensorimotor decision making may include the cortico-subcortical loops for which central thalamus serves as the penultimate synapse.
Starvation response in mouse liver shows strong correlation with life-span-prolonging processes
Authors: Bauer Matthias , Hamm Anne C. , Bonaus Melanie , Jacob Andrea , Jaekel Jens , Schorle Hubert , Pankratz Michael J. , Katzenberger Joerg D.
Published: February 2004

We have monitored global changes in gene expression in mouse liver in response to fasting and sugar-fed conditions using high-density microarrays. From ∼20,000 different genes, the significantly regulated ones were grouped into specific signaling and metabolic pathways. Striking changes in lipid signaling cascade, insulin and dehydroepiandrosterone (DHEA) hormonal pathways, urea cycle and S-adenosylmethionine-based methyl transfer systems, and cell apoptosis regulators were observed. Since these pathways have been implicated to play a role in the aging process, and since we observe significant overlap of genes regulated upon starvation with those regulated upon caloric restriction, our analysis suggests that starvation may elicit a stress response that is also elicited during caloric restriction. Therefore, many of the signaling and metabolic components regulated during fasting may be the same as those which mediate caloric restriction-dependent life-span extension.
Molecular heterogeneity of calcium channel β-subunits in canine and human heart: evidence for differential subcellular localization
Authors: Foell Jason D. , Balijepalli Ravi C. , Delisle Brian P. , Yunker Anne Marie R. , Robia Seth L. , Walker Jeffrey W. , McEnery Maureen W. , January Craig T. , Kamp Timothy J.
Published: February 2004

Multiple Ca2+channel β-subunit (Cavβ) isoforms are known to differentially regulate the functional properties and membrane trafficking of high-voltage-activated Ca2+channels, but the precise isoform expression pattern of Cavβ subunits in ventricular muscle has not been fully characterized. Using sequence data from the Human Genome Project to define the intron/exon structure of the four known Cavβ genes, we designed a systematic RT-PCR strategy to screen human and canine left ventricular myocardial samples for all known Cavβ isoforms. A total of 18 different Cavβ isoforms were detected in both canine and human ventricles including splice variants from all four Cavβ genes. Six of these isoforms have not previously been described. Western blots of ventricular membrane fractions and immunocytochemistry demonstrated that all four Cavβ subunit genes are expressed at the protein level, and the Cavβ subunits show differential subcellular localization with Cavβ1b, Cavβ2, and Cavβ3predominantly localized to the T-tubule sarcolemma, whereas Cavβ1aand Cavβ4are more prevalent in the surface sarcolemma. Coexpression of the novel Cavβ2csubunits (Cavβ2cN1, Cavβ2cN2, Cavβ2cN4) with the pore-forming α1C(Cav1.2) and Cavα2δ subunits in HEK 293 cells resulted in a marked increase in ionic current and Cavβ2cisoform-specific modulation of voltage-dependent activation. These results demonstrate a previously unappreciated heterogeneity of Cavβ subunit isoforms in ventricular myocytes and suggest the presence of different subcellular populations of Ca2+channels with distinct functional properties.
Oxidized Low-Density Lipoprotein Retards the Growth of Proliferating Cells by Inhibiting Nuclear Translocation of Cell Cycle Proteins
Authors: Zettler Marjorie E. , Prociuk Michele A. , Austria J. Alejandro , Zhong Guangming , Pierce Grant N.
Published: February 2004

Objective—

Our study tested the hypothesis that the mitogenic effect of oxidized low-density lipoprotein (oxLDL) on vascular cells may be further enhanced by the presence of cytokines and growth factors known to be present in the atherosclerotic environment.

Methods and Results—

Quiescent fibroblasts and vascular smooth muscle cells were treated with 10 or 50 μg/mL minimally-oxidized LDL in combination with serum for 24 or 48 hours. Surprisingly, these cells showed inhibited release from growth arrest and a significant reduction in the number of cells completing the cell cycle when compared with cells treated with serum alone. This was not due to an induction of apoptosis. The antiproliferative effects were not closely associated with changes in the expression of cell cycle proteins. Instead, oxLDL inhibited the translocation of cell cycle proteins cell division cycle (Cdc) 2, cyclin-dependent kinase (Cdk) 2, Cdk 4, Cyclin A, Cyclin B1, Cyclin D1, and proliferative cell nuclear antigen (PCNA) into the nucleus, as compared with separate treatments with serum alone. Kinase activation associated with specific cell cycle proteins was also inhibited by oxLDL.

Conclusions—

oxLDL, in the presence of serum, has a surprising inhibitory effect on cell proliferation that occurs through an inhibition of import of cell cycle proteins into the cell nucleus.
Expansive Arterial Remodeling: Location, Location, Location
Authors: Pasterkamp Gerard , Galis Zorina S. , de Kleijn Dominique P.V.
Published: February 2004

The artery is a dynamic organ capable of changing its geometry in response to atherosclerotic plaque formation. Expansion of the vessel diameter retards luminal narrowing and is considered a compensatory response. However, the expansive remodeling response is a “wolf in sheep’s clothes,” because expansion is associated with the presence of inflammatory cells, proteolysis, and a thrombotic plaque phenotype. The prevalence and clinical presentation of expansively remodeled lesions may differ among vascular beds. However, it is evident that all types of atherosclerotic arterial expansive lesions share the presence of inflammatory cells and subsequent protease activities. The potential role of inflammation and protease activity in the development of the different remodeling modes is discussed.
PDGF C Is A Selective α Platelet-Derived Growth Factor Receptor Agonist That Is Highly Expressed in Platelet α Granules and Vascular Smooth Muscle
Authors: Fang Li , Yan Yibing , Komuves Laszlo G. , Yonkovich Shirlee , Sullivan Carol M. , Stringer Bradley , Galbraith Sarah , Lokker Nathalie A. , Hwang S. Stuart , Nurden Paquita , Phillips David R. , Giese Neill A.
Published: February 2004

Objective—

The platelet-derived growth factor (PDGF) family consists of four members, PDGF A, PDGF B, and 2 new members, PDGF C and PDGF D, which signal through the α and β PDGF receptor (PDGFR) tyrosine kinases. This study was performed to determine the receptor specificity and cellular expression profile of PDGF C.

Methods and Results—

PDGF C growth factor domain (GFD) was shown to preferentially bind and activate α PDGFR and activate β PDGFR when it is co-expressed with α PDGFR through heterodimer formation. An investigation of PDGF C mRNA and protein expression revealed that during mouse fetal development, PDGF C was expressed in the mesonephric mesenchyme, prefusion skeletal muscle, cardiac myoblasts, and in visceral and vascular smooth muscle, whereas in adult human tissues expression was largely restricted to smooth muscle. Microarray analysis of various cell types showed PDGF C expression in vascular smooth muscle cells, renal mesangial cells, and platelets. PDGF C mRNA expression in platelets was confirmed by real-time polymerase chain reaction, and PDGF C protein was localized in α granules by immuno-gold electron microscopy. Western blot analysis of platelets identified 55-kDa and 80-kDa PDGF C isoforms that were secreted on platelet activation.

Conclusions—

Taken together, our results demonstrated for the first time to our knowledge that like PDGF A and B, PDGF C is likely to play a role in platelet biology.
Role of p160 Coactivator Complex in the Activation of Liver X Receptor
Authors: Huuskonen Jarkko , Fielding Phoebe E. , Fielding Christopher J.
Published: February 2004

Objective—

Liver X receptor (LXR) is a member of a nuclear receptor family regulating the expression of several key proteins involved in lipid metabolism and inflammation. In contrast to several other nuclear receptors, very little is known about the coactivators needed for the agonist-mediated transactivation by LXR. In this study, we have investigated the role of p160 coactivator complex in the regulation of ATP-binding transporter A1 (ABCA1), a clinically important gene transcriptionally upregulated by LXR/RXR (retinoid X receptor) heterodimer.

Methods and Results—

Overexpression of LXRα, SRC-1, and p300, either alone or in combination, increased the luciferase activity driven by the wild-type ABCA1 promoter. The same coactivators bound to the ABCA1 promoter on oxysterol induction in chromatin immunoprecipitation assays. To the contrary, CARM-1 and P/CAF had no effect on ABCA1 transactivation, nor do they bind the promoter. When the DR-4 element was mutated from the ABCA1 promoter, only p300 was able to activate ABCA1 transcription in a ligand-independent manner.

Conclusions—

The p160 coactivator complex members SRC-1 and p300, but not CARM-1 and P/CAF, coactivate LXR-mediated transcription of ABCA1 gene. In addition, p300 activates ABCA1 transcription independently of DR-4 element and LXR/RXR.
Three-Dimensional Reconstruction of the Rabbit Atrioventricular Conduction Axis by Combining Histological, Desmin, and Connexin Mapping Data
Authors: Ko Yu-Shien , Yeh Hung-I , Ko Yu-Lin , Hsu Yu-Chun , Chen Chin-Fen , Wu Semon , Lee Ying-Shiung , Severs Nicholas J.
Published: February 2004

Background—

The 3D structure of the atrioventricular conduction axis incorporating detailed cellular and molecular composition, especially that relating to gap-junctional proteins, is still unclear, impeding mechanistic understanding of cardiac rhythmic disorders.

Methods and Results—

A 3D model of the rabbit atrioventricular conduction axis was reconstructed by combining histological and immunofluorescence staining on serial sections. The exact cellular boundaries, especially those between transitional cells and atrial myocardium, were demarcated by a dense and irregular desmin-labeling pattern in conductive myocardium. The model demonstrates that the atrioventricular conduction axis is segregated into 2 connecting compartments, 1 predominantly expressing connexin45 (compact node and transitional cells) and the other predominantly coexpressing connexin43 and connexin45 (His bundle, lower nodal cells, and posterior nodal extension). The transitional zone shows unique features of spatial complexity, including a bridging bilayer structure (a deep transitional zone connecting with a superficial atrial-transitional overlay) and asymmetrical continuity (wider atrial-transitional interfaces and shorter atrial-axial distances in the hisian portion than in the ostial portion). In the latter compartment, the His bundle, lower nodal cells, and posterior nodal extension form a continual axis and longitudinal transitional-axial interface.

Conclusions—

Key findings of the present study are the demonstration of a distinct anatomical border between transitional and atrial cells, connection between transitional cells and both lower nodal cells and posterior nodal extension, and distinctive connexin expression patterns in different compartments of the rabbit atrioventricular conduction axis. These features, synthesized in a novel 3D model, provide a structural framework for the interpretation of nodal function.
Oral Imatinib Mesylate (STI571/Gleevec) Improves the Efficacy of Local Intravascular Vascular Endothelial Growth Factor-C Gene Transfer in Reducing Neointimal Growth in Hypercholesterolemic Rabbits
Authors: Leppänen Olli , Rutanen Juha , Hiltunen Mikko O. , Rissanen Tuomas T. , Turunen Mikko P. , Sjöblom Tobias , Brüggen Josef , Bäckström Gudrun , Carlsson Marianne , Buchdunger Elisabeth , Bergqvist David , Alitalo Kari , Heldin Carl-Henrik , Östman Arne , Ylä-Herttuala Seppo
Published: February 2004

Background—

Platelet-derived growth factor (PDGF) antagonists have demonstrated beneficial effects on neointima formation, but in studies using PDGF inhibitors and extended follow-up, the lesions reoccur. These findings implicate a need to combine targeting of PDGF with other strategies. Stimulation of reendothelialization by treatment with endothelial cell mitogens of the vascular endothelial growth factor (VEGF) family counteracts restenosis, but there are also concerns regarding the durability of the effect with this approach.

Methods and Results—

To explore whether a combined use of PDGF antagonist and stimulation of reendothelialization confers better results than each therapy alone, we combined systemic administration of imatinib mesylate (STI571/Gleevec, 10 mg/kg
−1
per d
−1
), a tyrosine kinase inhibitor with activity against PDGF receptors, with local intravascular adenovirus-mediated VEGF-C gene transfer (1.15×10
10
pfu) in cholesterol-fed, balloon-injured rabbits. Throughout the course of the STI571 therapy, the circulating concentrations were able to suppress PDGF receptor phosphorylation. At 3 weeks, the treatment with STI571 led to a transient decrease in intralesion macrophages and to an increase in intimal smooth muscle cell apoptosis. VEGF-C application reduced neointima formation and accelerated reendothelialization. However, none of the therapies alone reduced intimal thickening at a 6-week time point, whereas the combined treatment led to a persistent reduction (55% versus control) in lesion size at this time point.

Conclusions—

Our study provides one of the first successful examples of gene therapy combined with a pharmacological treatment to modulate 2 distinct ligand-receptor signaling systems and suggests combination of local VEGF-C gene therapy with systemic inhibition of PDGF signaling as a novel principle to prevent intimal hyperplasia after vascular manipulations.
Role of Endothelium-Derived Hyperpolarizing Factor in ACE Inhibitor-Induced Renal Vasodilation in Vivo
Authors: Matsuda Hiroto , Hayashi Koichi , Wakino Shu , Kubota Eiji , Honda Masanori , Tokuyama Hirobumi , Takamatsu Ichiro , Tatematsu Satoru , Saruta Takao
Published: February 2004

Although the angiotensin-converting enzyme (ACE) inhibitor-induced bradykinin enhances nitric oxide (NO) release, bradykinin may also stimulate the production of an additional vasodilator, endothelium-derived hyperpolarizing factor (EDHF). This study examined the role of EDHF in mediating the NO-independent action of ACE inhibitors in canine renal microcirculation in vivo. We used intravital CCD camera videomicroscopy that allowed direct visualization of renal microcirculation in superficial and juxtamedullary nephrons in an in vivo, in situ, and relatively intact setting. In the presence of E4177 (an angiotensin receptor blocker), cilazaprilat (30 μg/kg) had no effect on diameter of superficial afferent arterioles (Aff), but it increased renal contents of bradykinin and nitrate plus nitrite, and it elicited dilation of juxtamedullary Aff (from 24.0±0.2 to 28.2±0.8 μm), juxtamedullary efferent arterioles (Eff) (from 24.2±0.2 to 28.0±0.8 μm), and superficial Eff (from 18.2±0.2 to 19.7±0.2 μm). These changes in diameters were prevented by N
α
-adamantaneacetyl-
d
-Arg-[Hyp
3
,Thi
5,8
,D-Phe
7
]bradykinin, a bradykinin receptor antagonist. The pretreatment with nitro-
l
-arginine methylester (
l
-NAME) plus E4177 eliminated the dilator response of juxtamedullary/superficial Eff and the increase in renal nitrate plus nitrite levels induced by cilazaprilat. In contrast, in the presence of E4177+
l-
NAME, cilazaprilat still caused 8%±3% dilation of juxtamedullary Aff, which was completely eliminated by proadifen, a cytochrome-P450 and K
Ca
channel blocker. Collectively, the ACE inhibitor exerts multiple vasodilator mechanisms, including the inhibition of angiotensin II formation; blockade of angiotensin II activity appears to be a dominant mechanism in superficial Aff, whereas the bradykinin-induced NO acts on superficial Eff and juxtamedullary Aff/Eff. Furthermore, a putative EDHF is an additional mechanism for the ACE inhibitor-induced vasodilation of juxtamedullary Aff in vivo.
Possible Contributions of Reactive Oxygen Species and Mitogen-Activated Protein Kinase to Renal Injury in Aldosterone/Salt-Induced Hypertensive Rats
Authors: Nishiyama Akira , Yao Li , Nagai Yukiko , Miyata Kayoko , Yoshizumi Masanori , Kagami Shoji , Kondo Shuji , Kiyomoto Hideyasu , Shokoji Takatomi , Kimura Shoji , Kohno Masakazu , Abe Youichi
Published: February 2004

Studies were performed to test the hypothesis that reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) contribute to the pathogenesis of aldosterone/salt-induced renal injury. Rats were given 1% NaCl to drink and were treated with one of the following combinations for 6 weeks: vehicle (0.5% ethanol, SC, n=6); aldosterone (0.75 μg/H, SC, n=8); aldosterone plus a selective mineralocorticoid receptor antagonist; eplerenone (0.125% in chow, n=8); aldosterone plus an antioxidant; and tempol (3 mmol/L in drinking solution, n=8). The activities of MAPKs, including extracellular signal-regulated kinases (ERK)1/2, c-Jun-NH
2
-terminal kinases (JNK), p38MAPK, and big-MAPK-1 (BMK1) in renal cortical tissues were measured by Western blot analysis. Aldosterone-infused rats showed higher systolic blood pressure (165±5 mm Hg) and urinary excretion of protein (106±24 mg/d) than vehicle-infused rats (118±3 mm Hg and 10±3 mg/d). Renal cortical mRNA expression of p22phox, Nox-4, and gp91phox, measured by real-time polymerase chain reaction, was increased in aldosterone-infused rats by 2.3, 4.3, and 3.0-fold, respectively. Thiobarbituric acid-reactive substances (TBARS) content in renal cortex was also higher in aldosterone (0.23±0.02) than vehicle-infused rats (0.09±0.01 nmol/mg protein). ERK1/2, JNK, and BMK1 activities were significantly elevated in aldosterone-infused rats by 3.3, 2.3, and 3.0-fold, respectively, whereas p38MAPK activity was not changed. Concurrent administration of eplerenone or tempol to aldosterone-infused rats prevented the development of hypertension (127±2 and 125±5 mm Hg), and the elevations of urinary excretion of protein (10±2 and 9±2 mg/day) or TBARS contents (0.08±0.01 and 0.11±0.01 nmol/mg protein). Furthermore, eplerenone and tempol treatments normalized the activities of ERK1/2, JNK, and BMK1. These data suggest that ROS and MAPK play a role in the progression of renal injury induced by chronic elevations in aldosterone.
Combined Echocardiographic Left Ventricular Hypertrophy and Electrocardiographic ST Depression Improve Prediction of Mortality in American Indians
Authors: Okin Peter M. , Roman Mary J. , Lee Elisa T. , Galloway James M. , Howard Barbara V. , Devereux Richard B.
Published: February 2004

Echocardiographic left ventricular hypertrophy (Echo-LVH) and ST segment depression (STD) on the ECG have each been demonstrated to predict cardiovascular (CV) and all-cause (AC) mortality. However, the prognostic value of combining Echo-LVH and ECG-STD has not been examined. ECGs and echocardiograms were examined in 2193 American Indian participants in the second Strong Heart Study examination. STD was measured by computer and was considered abnormal if ≥50 μV. Echo-LVH was defined by indexed LV mass >116 g/m
2
in men and >104 g/m
2
in women. After a mean follow-up of 3.1±0.7 years, there were 57 CV and 169 AC deaths. In univariate Cox analyses, Echo-LVH (χ
2
=54.2 and χ
2
=68.5) and ECG-STD (χ
2
=35.9 and χ
2
=46.3, all
P
<0.001) predicted CV and AC mortality, respectively. The combination of Echo-LVH and ECG-STD improved risk stratification compared with either alone for both CV death (χ
2
=74.4,
P
<0.001) and AC death (χ
2
=102.0,
P
<0.001), with presence of both ECG-STD and Echo-LVH associated with the greatest risks. After adjustment for age, sex, and relevant risk factors, combined Echo-LVH and ECG-STD remained predictive of CV mortality (χ
2
=19.7,
P
<0.001) and AC mortality (χ
2
=24.9,
P
<0.001), with the presence of both Echo-LVH and ECG-STD associated with a 6.3-fold increased risk of CV death (95% CI: 2.8 to 14.2) and a 4.6-fold increased risk of AC mortality (95% CI: 2.5 to 8.5). ECG-STD and Echo-LVH additively increase the risk of both CV mortality and AC mortality. These findings support the value of combining Echo-LVH and ECG-STD to improve risk stratification. These findings require verification in other populations.