bluuebunny/crossref_metadata_2025 huggingface dataset:
Hyperglycemic switch from mitochondrial nitric oxide to superoxide production in endothelial cells
Authors: Brodsky Sergey V. , Gao Shujuan , Li Hong , Goligorsky Michael S.
Published: March 2015

The accumulated ultrastructural and biochemical evidence is highly suggestive of the existence of mitochondrial nitric oxide (NO) synthase (mtNOS), where local production of NO regulates the electron transport along the respiratory chain. Here, the functional competence of mtNOS in situ in a living cell was examined using an intravital fluorescent NO indicator, 4,5-diaminofluorescein, employing a new procedure for loading it into the mitochondria to demonstrate local NO generation in undisrupted endothelial cells and in isolated mitochondria as well as in human embryonic kidney cells stably expressing endothelial NOS. With the use of this approach, we showed that endothelial cells incubated in the presence of high concentration of d-glucose (but not l-glucose) are characterized by the reduced NO synthetic function of mitochondria despite the unaltered abundance of the enzyme. In parallel, mitochondrial generation of superoxide was augmented in endothelial cells incubated in the presence of a high concentration of d-glucose. Both the NO generation and superoxide production in hyperglycemic environment could be restored to control levels by treating cells with a cell-permeable superoxide dismutase mimetic. In addition, enhanced mitochondrial superoxide production could be suppressed with an inhibitor of NOS in stimulated endothelial cells. In conclusion, the data 1) provide direct evidence of mitochondrial NO production in endothelial cells, 2) demonstrate its suppression and enhanced superoxide generation in hyperglycemic environment, and 3) provide evidence that “uncoupled” mtNOS represents an important source of superoxide anions in endothelial cells incubated in high glucose-containing medium.
Early autonomic malfunction in normotensive individuals with a genetic predisposition to essential hypertension
Authors: Davrath L. R. , Goren Y. , Pinhas I. , Toledo E. , Akselrod S.
Published: March 2015

One of the primary pathologies associated with hypertension is a complex autonomic dysfunction with evidence of sympathetic hyperactivity and/or vagal withdrawal. We investigated the possibility for early detection of essential hypertension on the basis of the analysis of heart rate (HR) and blood pressure fluctuations, which reflect autonomic control. Young adult normotensive offspring of one hypertensive parent (KHT; n = 12) and normotensive offspring of two normotensive parents (YN; n = 14) participated in this study. ECG, continuous blood pressure, and respiration were recorded during steady-state conditions and under various autonomic challenges. Time-frequency decomposition of these signals was performed with the use of a continuous wavelet transform. The use of the wavelet transform enables the extension of typical HR variability analysis to non-steady-state conditions. This time-dependent spectral analysis of HR allows time-dependent quantification of different spectral components reflecting the sympathetic and parasympathetic activity during rapid transitions, such as an active change in posture (CP). During an active CP from the supine to standing position, KHT demonstrated a significantly greater increase in the low-frequency fluctuations in HR than YN, indicating enhanced sympathetic involvement in the HR response to CP, and a reduced α-index, indicating decreased baroreceptor sensitivity. On recovery from handgrip, vagal reactivation was more sluggish in KHT. These results indicate the early existence of malfunctions in both branches of autonomic control in individuals at increased risk of hypertension.
Arterial intimal-medial permeability and coevolving structural responses to defined shear-stress exposures
Authors: Fry Donald L.
Published: March 2015

The purpose of this research was to examine the evolution of arterial shear stress-induced intimal albumin permeability and coevolving structural responses in swine arteries. Uniform laminar shear-stress responses were compared with those of a simulated “flow separation” stress field. These fields were created using specially designed flow-configuring devices in an experimentally controlled, metabolically supported, ex vivo thoracoabdominal aorta preparation. The Evans blue dye-albumin complex (EBD-alb) permeability patterns that evolved were measured by a reflectometric method. The corresponding tissue structural responses were evaluated by histological, immunostaining, and ultrastructural microscopic techniques. It was shown that when a previously in vivo-adapted artery is challenged by a new mechanochemical environment, it undergoes a sequence of adaptive processes over the ensuing 95 h. Intimal regions of laminar shear-stress exposure (∼16 dyn/cm2) responded initially (23 h) with an increase in permeability. With continued stress exposure, intimal-medial structural changes ensued that restored the artery to a physiologically normal permeability. Over this same period, adjacent endothelial regions exposed to simulated flow separation stress fields (∼0.03–0.27 dyn/cm2) developed early and progressively increasing permeability. This was associated with formation of local intimal edema, loss of intimal matrix material, and development of distinctively raised, gelatinous-appearing intimal lesions having a potentially preatheromatous architecture.
Functional and pharmacological properties of canine ERG potassium channels
Authors: Wang Jixin , Della Penna Kimberly , Wang Hao , Karczewski Jerzy , Connolly Thomas M. , Koblan Kenneth S. , Bennett Paul B. , Salata Joseph J.
Published: March 2015

We established HEK-293 cell lines that stably express functional canine ether-à-go-go-related gene (cERG) K+ channels and examined their biophysical and pharmacological properties with whole cell patch clamp and35S-labeled MK-499 ([35S]MK-499) binding displacement. Functionally, cERG current had the hallmarks of cardiac delayed rectifier K+ current ( I Kr). Channel opening was time- and voltage dependent with threshold near −40 mV. The half-maximum activation voltage was −7.8 ± 2.4 mV at 23°C, shifting to −31.9 ± 1.2 mV at 36°C. Channels activated with a time constant of 13 ± 1 ms at +20 mV, showed prominent inward rectification at depolarized potentials, were highly K+ selective (Na+-to-K+permeability ratio = 0.007), and were potently inhibited by I Kr blockers. Astemizole, terfenadine, cisapride, and MK-499 inhibited cERG and human ERG (hERG) currents with IC50 values of 1.3, 13, 19, and 15 nM and 1.2, 9, 14, and 21 nM, respectively, and competitively displaced [35S]MK-499 binding from cERG and hERG with IC50 values of 0.4, 12, 35, and 0.6 nM and 0.8, 5, 47, and 0.7 nM, respectively. cERG channels had biophysical properties appropriate for canine action potential repolarization and were pharmacologically sensitive to agents known to prolong QT. A novel MK-499 binding assay provides a new tool to detect agents affecting ERG channels.
Jejunal tissue oxygenation and microvascular flow motion during hemorrhage and resuscitation
Authors: Pajk Werner , Schwarz Birgit , Knotzer Hans , Friesenecker Barbara , Mayr Andreas , Dünser Martin , Hasibeder Walter
Published: March 2015

The relationship between flow motion and tissue oxygenation was investigated during hemorrhage/retransfusion with and without dopamine in 14 pigs. During 45% bleed, jejunal microvascular hemoglobin O2saturation (HBjO2) and mucosal tissue Po2(Po2muc) were recorded in seven control and seven dopamine-treated animals. Mean arterial pressure and systemic O2delivery decreased during hemorrhage and returned to baseline after retransfusion. Hemorrhage decreased Po2mucfrom 33 ± 2.8 to 13 ± 1.6 mmHg and HBjO2from 53 ± 4.9% to 32 ± 3.9%, respectively, in control animals. During reperfusion, Po2mucand HBjO2remained low. Dopamine increased Po2mucfrom 28 ± 4.3 to 45 ± 4.6 mmHg and HBjO2from 54 ± 5.7% to 69 ± 1.5% and attenuated the decrease in Po2mucand HBjO2during hemorrhage. After retransfusion, dopamine restored Po2mucand HBjO2to baseline. Control animals developed rhythmic HBjO2oscillations with increasing amplitude (frequency, 4.5 to 7.6 cycles/min) and showed an inverse relationship between Po2mucand HBjO2oscillation amplitude. Dopamine prevented regular flow motion. The association between decreased Po2mucand increased oscillations in HBjO2after normalization of systemic hemodynamics and O2transport in control animals suggests a cause-and-effect relationship between low tissue Po2and flow motion activity within the jejunal microcirculation.
Superoxide-NO interaction decreases flow- and agonist-induced dilations of coronary arterioles in Type 2 diabetes mellitus
Authors: Bagi Zsolt , Koller Akos , Kaley Gabor
Published: March 2015

Type 2 diabetes mellitus (T2-DM) markedly increases the incidence of ischemic heart disease (IHD) and, consequently, mortality. However, the underlying mechanisms leading to IHD in T2-DM are not completely understood. We hypothesized that in T2-DM the regulation of coronary microvascular resistance by local mechanisms is altered. Thus, in coronary arterioles (diameter: ∼ 80 μm) isolated from male mice with T2-DM (C57BL/KsJ- db/ db) and control littermates, responses to changes in intraluminal pressure, flow, and agonists with known mechanisms of action were studied. Increases in pressure (from 20 to 120 mmHg) resulted in similar myogenic responses of coronary arterioles of control and db/ db mice, whereas dilations in response to cumulative concentrations of ACh and the nitric oxide (NO) donor NONOate were significantly decreased compared with those of control vessels. On the other hand, responses to adenosine were not different between vessels of control and db/ db mice. Increases in flow (0–20 μl/min) resulted in dilations of control vessels (maximum: 38 ± 4%) that were inhibited by the NO synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME). In contrast, arterioles of db/ db mice exhibited greatly reduced dilations to flow (maximum: 4 ± 6%) that were unaffected by l-NAME. In carotid arteries of db/ db mice, superoxide dismutase (SOD)-sensitive, enhanced superoxide production was detected by dihydroethydine staining and lucigenin enhanced chemiluminescence. Correspondingly, intraluminal administration of SOD significantly augmented flow-, ACh-, and NONOate-induced dilations of diabetic arterioles, and then flow- and ACh-induced responses could be inhibited by l-NAME. Collectively, these findings suggest that in T2-DM, due to an enhanced superoxide production, NO mediation of agonist- and flow-induced dilations of coronary arterioles is reduced. This alteration in the regulation of coronary microvascular resistance may contribute to the development of IHD in T2-DM.
Relationship among hyperinsulinemia, insulin resistance, and hypertension is dependent on sex
Authors: Galipeau Denise M. , Yao Linfu , McNeill John H.
Published: March 2015

Hyperinsulinemia and insulin resistance have been linked to hypertension; however, the influence of sex on this relationship has not been well studied. The purpose of this experiment was to compare the effects of chronic insulin treatment on insulin sensitivity and blood pressure in male and female rats. Male and female Wistar rats were treated with insulin (2 U/day) via subcutaneous sustained release implants for 5 wk. Systolic blood pressure was measured via the tail-cuff method before and after treatment, and insulin sensitivity was assessed with an oral glucose tolerance test. The insulin sensitivity of female rats was 4.5-fold greater than male rats. Chronic insulin treatment impaired insulin sensitivity in both sexes; however, this occurred to a greater degree in male rats. Blood pressure increased in male rats treated with insulin only. The results demonstrate that hyperinsulinemia and insulin resistance are associated with hypertension in male rats only. Therefore, the link between these conditions appears to depend on sex.
nNOS gene transfer to RVLM improves baroreflex function in rats with chronic heart failure
Authors: Wang Yu , Patel Kaushik P. , Cornish Kurtis G. , Channon Keith M. , Zucker Irving H.
Published: March 2015

We hypothesized that gene transfer of neuronal nitric oxide synthase (nNOS) into the rostral ventrolateral medulla (RVLM) improves baroreflex function in rats with chronic heart failure (CHF). Six to eight weeks after coronary artery ligation, rats showed hemodynamic signs of CHF. A recombinant adenovirus, either Ad.nNOS or Ad.β-Gal, was transfected into the RVLM. nNOS expression in the RVLM was confirmed by Western blot analysis, NADPH-diaphorase, and immunohistochemical staining. We studied baroreflex control of the heart rate (HR) and renal sympathetic nerve activity (RSNA) in the anesthetized state 3 days after gene transfer by intravenous injections of phenylephrine and nitroprusside. Baroreflex sensitivity was depressed for HR and RSNA regulation in CHF rats (2.0 ± 0.3 vs. 0.8 ± 0.2 beats · min–1 · mmHg–1, P < 0.01 and 3.8 ± 0.3 vs. 1.2 ± 0.1% max/mmHg, P < 0.01, respectively). Ad.nNOS transfer into RVLM significantly increased the HR and RSNA ranges (152 ± 19 vs. 94 ± 12 beats/min, P < 0.05 and 130 ± 16 vs. 106 ± 5% max/mmHg, P < 0.05) compared with the Ad.β-Gal in CHF rats. Ad.nNOS also improved the baroreflex gain for the control of HR and RSNA (1.8 ± 0.2 vs. 0.8 ± 0.2 beats · min–1 · mmHg–1, P < 0.01 and 2.6 ± 0.2 vs. 1.2 ± 0.1% max/mmHg, P < 0.01). In sham-operated rats, we found that Ad.nNOS transfer enhanced the HR range compared with Ad.β-Gal gene transfer (188 ± 15 vs. 127 ± 14 beats/min, P < 0.05) but did not alter any other parameter. This study represents the first demonstration of altered baroreflex function following increases in central nNOS in the CHF state. We conclude that delivery of Ad.nNOS into the RVLM improves baroreflex function in rats with CHF.
Imaging hemoglobin oxygen saturation in sickle cell disease patients using noninvasive visible reflectance hyperspectral techniques: effects of nitric oxide
Authors: Zuzak Karel J. , Gladwin Mark T. , Cannon Richard O. , Levin Ira W.
Published: March 2015

Sickle cell disease is characterized by microvascular occlusion and hemolytic anemia, factors that impair tissue oxygen delivery. We use visible reflectance hyperspectral imaging to quantitate skin tissue hemoglobin oxygen saturation (HbO2) and to determine whether changes in blood flow during nitric oxide (NO) stimulation or gas administration (therapies proposed for this disease) improve skin tissue oxygen saturation in five patients with sickle cell disease. Compared with six healthy African-American subjects, sickle cell patients exhibited higher forearm blood flows (7.4 ± 1.8 vs. 3.2 ± 0.4 ml·min–1·100 ml tissue–1, P = 0.037) but significantly reduced percentages of skin HbO2 (61.0 ± 0.2 vs. 77.5 ± 0.2%, P < 0.001). Administration of acetylcholine to patients increased blood flow by 15.1 ± 3.8 ml·min–1·100 ml tissue–1 and the percentage of skin HbO2 by 4.1 ± 0.3% ( P = 0.02, P < 0.001, respectively, from baseline values). Sodium nitroprusside, a direct NO donor, increased blood flow by 3.9 ± 1.1 ml/min and the percentage of skin HbO2 by 2.9 ± 0.3% ( P = 0.02, P < 0.001, respectively). NO inhalation had no effect on forearm blood flow, yet increased the percentage of skin HbO2 by 2.3 ± 0.3% ( P < 0.001). Percentages of skin HbO2 were exponentially related to blood flow ( R = 0.97, P < 0.001), indicating a limit to skin tissue oxygen saturation at high blood flows. Thus, for acetylcholine infusion leading to blood flows sevenfold greater than those of healthy resting African-American subjects, patients still exhibited lower percentages of skin HbO2 (65.2 ± 0.2 vs. 77.5 ± 0.2%, P < 0.001). Visible reflectance hyperspectral imaging demonstrates that either the stimulation or the administration of NO pharmacologically or by gas inhalation improves, but does not normalize, skin tissue oxygen saturation in patients with sickle cell disease.
A1 adenosine receptor overexpression attenuates ischemia-reperfusion-induced apoptosis and caspase 3 activity
Authors: Regan Sara E. , Broad Michael , Byford Anne M. , Lankford Amy R. , Cerniway Rachael J. , Mayo Marty W. , Matherne G. Paul
Published: March 2015

We tested the hypothesis that myocardial ischemia-reperfusion (I/R)-induced apoptosis is attenuated in transgenic mice overexpressing cardiac A1 adenosine receptors. Isolated hearts from transgenic (TG, n = 19) and wild-type (WT, n = 22) mice underwent 30 min of ischemia and 2 h of reperfusion, with evaluation of apoptosis, caspase 3 activity, function, and necrosis. I/R-induced apoptosis was attenuated in TG hearts. TG hearts had less I/R-induced apoptotic nuclei (0.88 ± 0.10% vs. 4.22 ± 0.24% terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in WT, P < 0.05), less DNA fragmentation (3.30 ± 0.38-fold vs. 4.90 ± 0.39-fold over control in WT, P < 0.05), and less I/R-induced caspase 3 activity (145 ± 25% over nonischemic control vs. 234 ± 31% in WT, P < 0.05). TG hearts also had improved recovery of function and less necrosis than WT hearts. In TG hearts pretreated with LY-294002 (3 μM) to evaluate the role of phosphosinositol-3-kinase in acute signaling, there was no change in the functional protection or apoptotic response to I/R. These data suggest that cardioprotection with transgenic overexpression of A1 adenosine receptors involves attenuation of I/R-induced apoptosis that does not involve acute signaling through phosphoinositol-3-kinase.
Palmitate-induced cardiac apoptosis is mediated through CPT-1 but not influenced by glucose and insulin
Authors: Kong Jennifer Y. , Rabkin Simon W.
Published: March 2015

To test the hypothesis that regulation of palmitate metabolism, through carnitine palmitoyl transferase-1 (CPT-1) or through alterations of glycolysis, was involved in the pathway of palmitate-mediated cell death, cardiomyocytes were cultured from 7-day-old chick embryos. Palmitate-induced cell death, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, was enhanced by carnitine, a cofactor needed for palmitate transport into mitochondria via CPT-1. Carnitine co-incubation with palmitate significantly ( P < 0.01) increased the amount of apoptotic cells, assessed by propidium iodine staining and fluorescent-activated cell sorting analysis compared with treatment with either palmitate or carnitine alone. The CPT-1 inhibitor oxfenicine significantly ( P < 0.05) blocked the cell death induced by the combination of palmitate and carnitine. The short-chain saturated fatty acid capric acid (100 μM), which is not likely transported by CPT-1, did not significantly affect cell viability, whereas the C18 saturated fatty acid stearic (100 μM) significantly ( P < 0.01) reduced cell viability and to a similar extent as palmitate. In contrast, there was no significant alteration of palmitate-induced cell death by cotreatment with 100 nM insulin + 2 g/l glucose or 1 mM lactate, which promote ATP generation by glycolysis rather than fatty acid oxidation. Fumonisin did not alter palmitate-induced cell death or apoptosis, suggesting that the effect of palmitate was not operative through increased ceramide synthesis. These results suggest that oxidation of palmitate through CPT-1 is involved in the production of apoptosis in cardiomyocytes.
Contribution of laminar myofiber architecture to load-dependent changes in mechanics of LV myocardium
Authors: Takayama Yasuo , Costa Kevin D. , Covell James W.
Published: March 2015

The ventricular myocardium consists of a syncytium of myocytes organized into branching, transmurally oriented laminar sheets approximately four cells thick. When systolic deformation is expressed in an axis system determined by the anatomy of the laminar architecture, laminar sheets of myocytes shear and laterally extend in an approximately radial direction. These deformations account for ∼90% of normal systolic wall thickening in the left ventricular free wall. In the present study, we investigated whether the changes in systolic and diastolic function of the sheets were sensitive to alterations in systolic and diastolic load. Our results indicate that there is substantial reorientation of the laminar architecture during systole and diastole. Moreover, this reorientation is both site and load dependent. Thus as end-diastolic pressure is increased and the left ventricular wall thins, sheets shorten and rotate away from the radial direction due to transverse shearing, opposite of what occurs in systole. Both mechanisms of thickening contribute substantially to normal left ventricular wall function. Whereas the relative contributions of shear and extension are comparable at the base, sheet shear is the predominant factor at the apex. The magnitude of shortening/extension and shear increases with preload and decreases with afterload. These findings underscore the essential contribution of the laminar myocardial architecture for normal ventricular function throughout the cardiac cycle.
Thrombin increases permeability only in venules exposed to inflammatory conditions
Authors: Curry F. E. , Zeng M. , Adamson R. H.
Published: March 2015

Thrombin is widely used to stimulate a variety of responses in cultured endothelial cell monolayers as a model of acute vascular endothelial reponse to inflammatory mediators. However, preliminary results indicated that rat mesenteric venules did not respond acutely to thrombin. We tested the hypothesis that rat venules would respond to thrombin 24 h after prior injury by microperfusion. Vessel responsiveness was measured as hydraulic conductivity ( Lp). When venules were exposed to rat thrombin (10 U/ml) within 2 h of initial perfusion with vehicle control, there was no increase in Lp of any vessel from a mean baseline of 1.2 ± 0.2 × 10–7 cm·s–1·cmH2O–1. In contrast, when perfused with thrombin at 25–27 h after initial perfusion, every venule responded to thrombin with a transient increase in Lp. The mean peak Lp on day 2 in response to thrombin was 24 ± 4.2 × 10–7 cm·s–1·cmH2O–1. Our results suggest that prior endothelial injury modifies the endothelial cell phenotype and alters the response of endothelial cells to thrombin after 24 h. Phenotypic plasticity of endothelial cells may play a key role in the regulation of permeability of some endothelial cells in culture and in intact venules, where localized leaky sites may form where there had been a previous inflammatory response.
Disruption of vagal efferent axon and nerve terminal function in the postischemic myocardium
Authors: Kawada Toru , Yamazaki Toji , Akiyama Tsuyoshi , Mori Hidezo , Uemura Kazunori , Miyamoto Tadayoshi , Sugimachi Masaru , Sunagawa Kenji
Published: March 2015

Despite the importance of vagal control over the ventricle, little is known regarding vagal efferent conduction and nerve terminal function in the postischemic myocardium. To elucidate postischemic changes in the cardiac vagal efferent neuronal function, we measured myocardial interstitial acetylcholine (ACh) levels by using in vivo cardiac microdialysis and examined the ACh responses to electrical stimulation of the vagi or local administration of ouabain in anesthetized cats. Sixty-minute occlusions of the left anterior descending coronary artery (LAD) followed by 60-min reperfusion abolished electrical stimulation-induced ACh release (20.4 ± 3.9 vs. 0.9 ± 0.4 nmol/l; means ± SE, P < 0.01). In different groups of animals, 60-min LAD occlusion followed by 60-min reperfusion decreased but did not completely abolish ouabain-induced release of ACh (9.2 ± 1.8 vs. 3.9 ± 0.7 nmol/l; P < 0.05). These results indicate that function of the vagal efferent axon was completely interrupted, whereas the local ACh release was partially suppressed in the postischemic myocardium. The postischemic disruption of vagal efferent neuronal function might exert deleterious effects on cardiac regulation.
Role of COX-1 and -2 in prostanoid generation and modulation of angiotensin II responses
Authors: Baber Syed R. , Hyman Albert L. , Kadowitz Philip J.
Published: March 2015

The role of cyclooxygenase (COX)-1 and -2 in prostanoid formation and modulation of pressor responses to ANG II was investigated in the pulmonary and systemic vascular beds in the rat. In the present study, selective COX-1 and -2 inhibitors attenuated increases in pulmonary arterial pressure and decreases in systemic arterial pressure in response to arachidonic acid but did not alter responses to PGE1 or U-46619. The selective COX-1 and -2 inhibitors did not modify systemic pressor responses to injections or infusions of ANG II or pulmonary pressor responses to injections of the peptide. COX-2 inhibitors did not alter, whereas a COX-1 inhibitor depressed, arachidonic acid-induced platelet aggregation. These data provide evidence in support of the hypothesis that prostanoid synthesis occurs by way of the COX-1 and -2 pathways in the pulmonary and systemic vascular beds but that pressor responses to ANG II are not mediated or modulated by these pathways in the rat.
SLControl: PC-based data acquisition and analysis for muscle mechanics
Authors: Campbell Kenneth S. , Moss Richard L.
Published: March 2015

SLControl is a computerized data acquisition and analysis system that was developed in our laboratory to help perform mechanical experiments using striated muscle preparations. It consists of a computer program (Windows 2000 or later) and a commercially available data acquisition board (16-bit resolution, DAP5216a, Microstar Laboratories, Bellevue, WA). Signals from the user's existing equipment representing force, fiber length (FL), and (if desired) sarcomere length (SL) are connected to the system through standard Bayonet Neill Concelman cables and saved to data files for later analysis. Output signals from the board control FL and trigger additional equipment, e.g., flash lamps. Windows dialogs drive several different experimental protocols, including slack tests and rate of tension recovery measurements. Precise measurements of muscle stiffness and force velocity/power characteristics can also be accomplished using SL and tension control, respectively. In these situations, the FL command signal is updated in real time (at rates ≥2.5 kHz) in response to changes in the measured SL or force signals. Data files can be exported as raw text or analyzed within SLControl with the use of built-in tools for cursor analysis, digital filtering, curve fitting, etc. The software is available for free download at http://www.slcontrol.com .
Brain sodium channels and ouabainlike compounds mediate central aldosterone-induced hypertension
Authors: Wang Hao , Huang Bing S. , Leenen Frans H. H.
Published: March 2015

Central nervous system (CNS) effects of mineralocorticoids participate in the development of salt-sensitive hypertension. In the brain, mineralocorticoids activate amiloride-sensitive sodium channels, and we hypothesized that this would lead to increased release of ouabainlike compounds (OLC) and thereby sympathetic hyperactivity and hypertension. In conscious Wistar rats, intracerebroventricular infusion of aldosterone at 300 or 900 ng/h in artificial cerebrospinal fluid (aCSF) with 0.145 M Na+for 2 h did not change baseline mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA), or heart rate (HR). Intracerebroventricular infusion of aCSF containing 0.16 M Na+(versus 0.145 M Na+in regular aCSF) did not change MAP or RSNA, but significant increases in MAP, RSNA, and HR were observed after intracerebroventricular infusion of aldosterone at 300 ng/h for 2 h. Intracerebroventricular infusion of aCSF containing 0.3 M Na+increased MAP, RSNA, and HR significantly more after intracerebroventricular infusion of aldosterone versus vehicle. After intracerebroventricular infusion of aldosterone, the MAP, RSNA, and HR responses to intracerebroventricular infusion of aCSF containing 0.16 M Na+were blocked by blockade of brain OLC with intracerebroventricular infusion of Fab fragments or of brain sodium channels with intracerebroventricular benzamil. Chronic intracerebroventricular infusion of aldosterone at 25 ng/h in aCSF with 0.15 M Na+for 2 wk increased MAP by 15–20 mmHg and increased hypothalamic OLC by 30% and pituitary OLC by 60%. Benzamil blocked all these responses to aldosterone. These findings indicate that in the brain, mineralocorticoids activate brain sodium channels, with small increases in CSF Na+leading to increases in brain OLC, sympathetic outflow, and blood pressure.
c-Fos expression in the midbrain periaqueductal gray after chemoreceptor and baroreceptor activation
Authors: Hayward Linda F. , Von Reitzenstein Marcela
Published: March 2015

The pattern of Fos-like immunoreactivity (FLI) in the periaqueductal gray (PAG) associated with activation of arterial chemoreceptors versus baroreceptor afferents was examined in urethane-anesthetized rats. Chemoreflex responses elicited by repeat intravenous injections of potassium cyanide (KCN; 90 μg/kg) significantly increased FLI in all columns of the PAG relative to saline-injected animals. Pressor responses elicited by intravenous phenylephrine (PE) produced a similar pattern of increased FLI throughout the PAG except in the dorsomedial and lateral columns of the caudal PAG, where FLI was minimal. Chemoreflex responses were unaltered by blockade of excitatory amino acid receptors in the dorsomedial PAG, and <10% of the neurons of the caudal PAG that expressed FLI after KCN stimulation were retrogradely labeled from the A5 region of the caudal ventrolateral pons. These results indicate that integration of chemoreceptor inputs occurs primarily in the dorsal and lateral columns of the caudal PAG, but these neurons have little direct descending influence over lower brain stem regions integral to the central arterial chemoreflex arc.
Structural, functional, and molecular characterization of the SHHF model of heart failure
Authors: Heyen Jonathan R. R. , Blasi Eileen R. , Nikula Kristen , Rocha Ricardo , Daust Heather A. , Frierdich Gregory , Van Vleet John F. , De Ciechi Pam , McMahon Ellen G. , Rudolph Amy E.
Published: March 2015

Heart failure is a complex multifactorial disease resulting in a myriad of progressive changes at the molecular, cellular, and physiological level. To better understand the mechanisms associated with the development of congestive heart failure, a comprehensive examination of the aging lean male spontaneously hypertensive, heart failure-prone rat (SHHF) was conducted. Myocardial function and structural integrity progressively diminished as evidenced by decreased ejection fraction and increased left ventricular volume measured using echocardiography. Functional and structural changes were accompanied by elevations in circulating inflammatory markers, including tumor necrosis factor-α (TNF-α), IL-6, and TNF receptors type 1 and 2. Increased systemic inflammatory marker levels were consistent with age-dependent changes in the expression pattern of genes that contribute to stress, inflammation, and the extracellular matrix in SHHF animals analyzed from age 4 to 18 mo. In summary, the SHHF rat shares many hallmark features of the human disease state and represents a key experimental model for the dissection of complex human heart failure pathophysiology.
Evidence for a membrane site of action for 14,15-EET on expression of aromatase in vascular smooth muscle
Authors: Snyder Gary D. , Krishna U. Murali , Falck J. R. , Spector Arthur A.
Published: March 2015

Epoxyeicosatrienoic acids (EETs) are synthesized in the endothelial cells of vascular tissues. They are released from the endothelial cells and produce relaxation of the smooth muscle cells by hyperpolarization. The present findings demonstrate that EETs also regulate aromatase activity in vascular smooth muscle cells. Exposure of cultured rat aortic smooth muscle cells to either 1 μM 14,15-EET or 1 μM 11,12-EET inhibits dibutyryl cAMP-induced aromatase activity by 80–100%. 11,12-Dihydroxyeicosatrienoic acid, the hydration product of 11,12-EET, has no effect on dibutyryl cAMP-induced vascular smooth muscle aromatase activity. In contrast to 14,15-EET, the N-methylsulfanilamide derivative of 14,15-EET (14,15-EET-SA) was neither metabolized nor incorporated into cell lipids, but it retained the ability to inhibit cAMP-induced aromatase activity. Furthermore, the 14,15-EET-SA inhibition of cAMP-induced aromatase activity persisted when the sulfanilamide derivative of 14,15-EET was covalently tethered to silica beads (average diameter, 0.5 μm), which restricted 14,15-EET-SA from entering the cell. These data are consistent with the presence of a receptor for EETs in the plasma membrane and support the hypothesis that the inhibition of aromatase by EETs is initiated by the interaction of EET with the putative plasma membrane receptor.
G protein modulates thyroid hormone-induced Na+ channel activation in ventricular myocytes
Authors: Sen Luyi , Sakaguchi Yoshihide , Cui Guanggen
Published: March 2015

To evaluate the effects of liothyronine (3,5,3′-triiodo-l-thyronine, T3) on Na+ channel current ( I Na) properties, I Na was recorded in adult guinea pig ventricular myocytes. T3 (1 nM) acutely increased whole cell I Na and shifted the steady-state I Na inactivation curve dose dependently. When the pipette solution contained 100 μM GTP or GTPγS, the effect of T3 on the whole cell I Na was increased two- to threefold. This effect was almost completely abolished by pertussis toxin preincubation. In the cell-attached patch, T3 increased the open probability of single I Na by reducing the null probability. In the inside-out patch, T3 effect was 10 times faster than that in whole cell and cell-attached patches while GTPγS was present and could be completely washed out. T3 alone slightly increased the channel open probability by increasing the closed state to open state rate constant ( k CO) and reducing the null probability. GTPγS exposure only increased the number of functional channels. T3 and GTPγS synergistically enhanced the channel open probability 5.8 ± 0.5-fold by increasing k CO, decreasing the open state to absorbing inactivated state rate constant, and greatly reducing the null probability. These results demonstrate that T3 acts on the cytosolic side of the membrane and acutely activates I Na. Pertussis toxin-sensitive G protein modulation greatly magnifies the T3 effects on the channel kinetics and null probability, thereby increasing the channel open probability.
Increased coronary perfusion augments cardiac contractility in the rat through stretch-activated ion channels
Authors: Lamberts R. R. , van Rijen M. H. P. , Sipkema P. , Fransen P. , Sys S. U. , Westerhof N.
Published: March 2015

The role of stretch-activated ion channels (SACs) in coronary perfusion-induced increase in cardiac contractility was investigated in isolated isometrically contracting perfused papillary muscles from Wistar rats. A brief increase in perfusion pressure (3–4 s, perfusion pulse, n = 7), 10 repetitive perfusion pulses ( n = 4), or a sustained increase in perfusion pressure (150–200 s, perfusion step, n = 7) increase developed force by 2.7 ± 1.1, 7.7 ± 2.2, and 8.3 ± 2.5 mN/mm2(means ± SE, P < 0.05), respectively. The increase in developed force after a perfusion pulse is transient, whereas developed force during a perfusion step remains increased by 5.1 ± 2.5 mN/mm2( P < 0.05) in the steady state. Inhibition of SACs by addition of gadolinium (10 μmol/l) or streptomycin (40 and 100 μmol/l) blunts the perfusion-induced increase in developed force. Incubation with 100 μmol/l Nω-nitro-l-arginine [nitric oxide (NO) synthase inhibition], 10 μmol/l sodium nitroprusside (NO donation) and 0.1 μmol/l verapamil (L-type Ca2+channel blockade) are without effect on the perfusion-induced increase of developed force. We conclude that brief, repetitive, or sustained increases in coronary perfusion augment cardiac contractility through activation of stretch-activated ion channels, whereas endothelial NO release and L-type Ca2+channels are not involved.
Isolation of interstitial fluid from rat mammary tumors by a centrifugation method
Authors: Wiig Helge , Aukland Knut , Tenstad Olav
Published: March 2015

Access to interstitial fluid is of fundamental importance to understand tumor transcapillary fluid balance, including the distribution of probes and therapeutic agents. Tumors were induced by gavage of 9,10-dimethyl-1,2-benzanthracene to rats, and fluid was isolated after anesthesia by exposing tissue to consecutive centrifugations from 27 to 6,800 g. The observed51Cr-EDTA (extracellular tracer) tissue fluid-to-plasma ratio obtained from whole tumor or from superficial tumor tissue by centrifugation at 27–424 g was not significantly different from 1.0 (0.92–0.99), suggesting an extracellular origin only. However, fluid collected from excised central tumor parts had a significantly lower ratio (0.66–0.77) for all imposed G forces, suggesting dilution by fluid deriving from a space unavailable for51Cr-EDTA. The colloid osmotic pressure in tumor fluid was generally higher than in fluid isolated from the subcutis, attributable to less selective capillaries and impaired lymphatic drainage in tumors. HPLC analysis of tumor fluid showed that low-molecular-weight macromolecules not present in arterial plasma were present in tumor fluid obtained by centrifugation and in venous blood draining the tumor, most likely representing proteins derived from tumor cells. We conclude that low-speed centrifugation may be a simple and reliable method to isolate interstitial fluid from tumors.
Sustained exogenous administration of Met5-enkephalin protects against infarction in vivo
Authors: Kuzume Koh , Wolff Roger A. , Amakawa Kazuhiko , Kuzume Kazuyo , Van Winkle Donna M.
Published: March 2015

The opioid antagonist naloxone abolishes infarct limitation by myocardial ischemic preconditioning, suggesting that one or more endogenous opioid peptides can mediate cardiac protection against ischemic damage. We tested the hypothesis that the naturally occurring opioid peptide Met5-enkephalin (ME) modulates myocardial infarct size in vivo. Experiments were conducted in barbiturate-anesthetized open-chest rabbits subjected to regional myocardial ischemia-reperfusion. ME was administered via osmotic minipump for 24 h. Infarct size was assessed with tetrazolium and is expressed as a percentage of the area at risk. Exogenous ME reduced the amount of the risk zone infarcted by ∼60% compared with saline-treated controls. ME-induced protection was sensitive to opioid receptor blockade with naloxone [NAL 50 ± 2% vs. ME + NAL 39 ± 3%, P = not significant (NS)] and also to blockade of sarcolemmal and mitochondrial ATP-sensitive K+(KATP) channels [5-hydroxydecanoate (5-HD) 33 ± 3% vs. ME + 5-HD 43 ± 8%, P = NS; and HMR-1098 60 ± 3% vs. ME + HMR-1098 54 ± 7%, P = NS]. We conclude that ME limits ischemic injury in vivo by an opioid receptor-mediated mechanism that involves both sarcolemmal and mitochondrial KATPchannels.
Preconditioning blocks cardiocyte apoptosis: role of KATPchannels and PKC-ε
Authors: Liu Huiping , Zhang Hong Yan , Zhu Xiangdong , Shao Zuohui , Yao Zhenhai
Published: March 2015

The aims of this study were to determine whether preconditioning blocks cardiocyte apoptosis and to determine the role of mitochondrial ATP-sensitive K+(KATP) channels and the protein kinase C ε-isoform (PKC-ε) in this effect. Ventricular myocytes from 10-day-old chick embryos were used. In the control series, 10 h of simulated ischemia followed by 12 h of reoxygenation resulted in 42 ± 3% apoptosis ( n = 8). These results were consistent with DNA laddering and TdT-mediated dUTP nick-end labeling (TUNEL) assay. Preconditioning, elicited with three cycles of 1 min of ischemia separated by 5 min of reoxygenation before subjection to prolonged simulated ischemia, markedly attenuated the apoptotic process (28 ± 4%, n = 8). The selective mitochondrial KATPchannel opener diazoxide (400 μmol/l), given before ischemia, mimicked preconditioning effects to prevent apoptosis (22 ± 4%, n = 6). Pretreatment with 5-hydroxydecanoate (100 μmol/l), a selective mitochondrial KATPchannel blocker, abolished preconditioning (42 ± 2%, n = 6). In addition, the effects of preconditioning and diazoxide were blocked with the specific PKC inhibitors Gö-6976 (0.1 μmol/l) or chelerythrine (4 μmol/l), given at simulated ischemia and reoxygenation. Furthermore, preconditioning and diazoxide selectively activated PKC-ε in the particulate fraction before simulated ischemia without effect on the total fraction, cytosolic fraction, and PKC δ-isoform. The specific PKC activator phorbol 12-myristate 13-acetate (0.2 μmol/l), added during simulated ischemia and reoxygenation, mimicked preconditioning to block apoptosis. Opening mitochondrial KATPchannels blocks cardiocyte apoptosis via activating PKC-ε in cultured ventricular myocytes. Through this signal transduction, preconditioning blocks apoptosis and preserves cardiac function in ischemia-reperfusion.
Mathematical analysis of dynamics of cardiac memory and accommodation: theory and experiment
Authors: Watanabe Mari A. , Koller Marcus L.
Published: March 2015

Decreasing the slope of the dynamic, but not conventional, restitution curves is antifibrillatory. Cardiac memory/accommodation underlies the difference. We measured diastolic interval (DI) and action potential duration (APD) in epicardial, endocardial, and Purkinje tissue from eight dogs. Consecutive 100-stimulus trains were given to study transitions between basic cycle lengths (BCL) ranging from 400 to 1,300 ms. (DI,APD) pairs aligned immediately on the line DI + APD = BCL (64/67) or oscillated (3/67). The shifting effect of up to 10 extrastimuli on restitution curves was also measured. These curves were fit with the equation APD = α + β exp(−DI/τ), where α is asymptote, β is drop, and τ is time constant. Linear regression of the parameters against the number of extrastimuli showed that premature and postmature stimuli decreased and increased α and β and increased and decreased τ, respectively. Analysis of a mathematical model treating memory as an exponentially decreasing shift of restitution curves shows that oscillatory DI,APD is expected with large ΔBCL, steep restitution slope, or increased cardiac accommodation. The model explains phase shifts and suggests a common mechanism for Purkinje and myocardial electrical alternans.
Inhibitory effect of glucocorticoid on coronary artery endothelial function
Authors: Rogers Kestrel M. , Bonar Christi A. , Estrella Jaymie L. , Yang Shumei
Published: March 2015

Acute and chronic stresses are implicated in cardiovascular diseases including coronary artery disease. The present study was designed to examine the direct effects of the stress hormone cortisol on nitric oxide (NO) release and endothelial NO synthase (eNOS) expression in cultured bovine coronary artery endothelial cells (BCAEC). Nitrate, nitrite, and NO (NOx) were measured by the chemiluminescence method. At 24 h after treatment, cortisol (1 nM–10 μM) produced a dose-dependent decrease in NOx release, which was attenuated in the presence of the 11β-hydroxysteroid dehydrogenase inhibitor carbenoxolone (3 μM). In accordance, eNOS protein levels were significantly decreased by cortisol in a dose-dependent manner. Cortisol pretreatment significantly increased the rate of eNOS protein degradation in the presence of cycloheximide. In addition, cortisol pretreatment decreased ATP-induced intracellular Ca2+elevation and NOx release in BCAEC. The presence of glucocorticoid receptors in BCAEC was demonstrated by Western blot. The results suggest that cortisol, through activation of glucocorticoid receptors, suppresses NOx release in BCAEC by downregulating eNOS proteins and inhibiting intracellular Ca2+ mobilization. Decreased NOx is likely to result in an increase in contraction of coronary arteries, leading to a decrease in coronary blood flow.
Exercise training attenuates coronary smooth muscle phenotypic modulation and nuclear Ca2+ signaling
Authors: Wamhoff B. R. , Bowles D. K. , Dietz N. J. , Hu Q. , Sturek M.
Published: March 2015

Physical inactivity is an independent risk factor for coronary heart disease, yet the mechanism(s) of exercise-related cardioprotection remains unknown. We tested the hypothesis that coronary smooth muscle after exercise training would have decreased mitogen-induced phenotypic modulation and enhanced regulation of nuclear Ca2+. Yucatan swine were endurance exercise trained (EX) on a treadmill for 16–20 wk. EX reduced endothelin-1-induced DNA content by 40% compared with sedentary (SED) swine ( P < 0.01). EX decreased single cell peak endothelin-1-induced cytosolic Ca2+ responses compared with SED by 16% and peak nuclear Ca2+ responses by 33% ( P < 0.05), as determined by confocal microscopy. On the basis of these results, we hypothesized that sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and intracellular Ca2+ stores in native smooth muscle are spatially localized to dissociate cytosolic Ca2+and nuclear Ca2+. Subcellular localization of SERCA in living and fixed cells revealed a distribution of SERCA near the sarcolemma and on the nuclear envelope. These results show that EX enhances nuclear Ca2+ regulation, possibly via SERCA, which may be one mechanism by which coronary smooth muscle cells from EX are less responsive to mitogen-induced phenotypic modulation.
Age-related sensitivity to nicotine for inducible atrial tachycardia and atrial fibrillation
Authors: Hayashi Hideki , Omichi Chikaya , Miyauchi Yasushi , Mandel William J. , Lin Shien-Fong , Chen Peng-Sheng , Karagueuzian Hrayr S.
Published: March 2015

The influence of nicotine in modulating vulnerability to atrial tachycardia and fibrillation (AT/AF) remains ill defined. The isolated hearts of six young (2–3 mo) and six old (22–24 mo) male Fischer 344 rats were Langendorff perfused at 5 ml/min with oxygenated Tyrode solution at 37°C, and the whole heart was also super-fused with warmed oxygenated Tyrode solution at 15 ml/min. Nicotine prolonged the interatrial conduction time and effective refractory period that were significantly ( P < 0.05) higher in the old than in the young rats in a concentration-dependent manner. Nicotine had a biphasic effect on burst atrial pacing-induced AT in both groups, increasing it at 10–30 ng/ml while decreasing it at 50–100 ng/ml ( P < 0.01). Nicotine at 10–100 ng/ml increased burst atrial pacing-induced AF in the young rats but suppressed it in the old rats ( P < 0.01). Optical mapping showed the presence of multiple independent wavefronts during AF and a single periodic large wavefront during AT in both groups. Nicotine, at concentrations found in the blood of smokers (30–85 ng/ml), exerts biphasic effects on inducible AT/AF in young rats and suppresses it in the old rats by causing high degrees of interatrial conduction block.