bluuebunny/crossref_metadata_2025 huggingface dataset:
l-Arginine limits myocardial cell death secondary to hypoxia-reoxygenation by a cGMP-dependent mechanism
Authors: Agulló Luis , García-Dorado David , Inserte Javier , Paniagua Amaya , Pyrhonen Pasi , Llevadot Joan , Soler-Soler Jordi
Published: December 2017

The objective of this study was to investigate the effect ofl-arginine supplementation on myocardial cell death secondary to hypoxia-reoxygenation. Isolated rat hearts ( n = 51) subjected to 40 min of hypoxia and 90 min of reoxygenation received 3 mMl-arginine and/or 1 μM 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ; a selective inhibitor of soluble guanylyl cyclase) throughout the experiment or during the equilibration, hypoxia, or reoxygenation periods. The incorporation ofl-[3H]arginine into myocytes during energy deprivation was investigated in isolated adult rat myocytes. The addition ofl-arginine to the perfusate throughout the experiment resulted in higher cGMP release ( P < 0.05), reduced lactate dehydrogenase release ( P < 0.05), and increased pressure-rate product ( P < 0.05) during reoxygenation. These effects were reproduced whenl-arginine was added only during equilibration, but addition ofl-arginine during hypoxia or reoxygenation had no effect. Addition of ODQ either throughout the experiment or only during reoxygenation reversed the beneficial effects of l-arginine.l-[3H]arginine was not significantly incorporated into isolated myocytes subjected to energy deprivation. We conclude thatl-arginine supplementation protects the myocardium against reoxygenation injury by cGMP-mediated actions. To be effective during reoxygenation,l-arginine must be added before anoxia.
Altered reflex control of cutaneous circulation by female sex steroids is independent of prostaglandins
Authors: Charkoudian Nisha , Johnson John M.
Published: December 2017

We tested the hypothesis that the shift in the cutaneous vasodilator response to hyperthermia seen with elevated female reproductive hormones is a prostaglandin-dependent resetting of thermoregulation to higher internal temperatures, similar to that seen in the febrile response to bacterial infection. Using water-perfused suits to control body temperature, we conducted heat stress experiments in resting women under conditions of low and high progesterone and estrogen and repeated these experiments after an acute dose of ibuprofen (800 mg). In six women the hormones were exogenous (oral contraceptives); three women had regular menstrual cycles and were tested in the early follicular and midluteal phases. Resting oral temperature (Tor) was significantly elevated with high hormone status ( P < 0.05); this was not affected by ibuprofen treatment ( P > 0.2). The Tor threshold for cutaneous vasodilation was significantly increased by high hormone status (+0.27 ± 0.07°C, P < 0.02); the shift was not affected by ibuprofen treatment (with ibuprofen: +0.29 ± 0.08°C, P > 0.2 vs. control experiments). The Torthreshold for sweating was similarly increased by high hormone status (+0.22 ± 0.05°C, P < 0.05); this shift was not influenced by ibuprofen (with ibuprofen: +0.35 ± 0.05, P > 0.1 vs. control experiments). Thus the shift in thermoregulatory control of skin blood flow and sweating mediated by female reproductive steroids is not sensitive to ibuprofen; it therefore appears that this shift is independent of prostaglandins.
Hypoperfusion-induced contractile failure does not require changes in cardiac energetics
Authors: Saupe Kurt W. , Eberli Franz R. , Ingwall Joanne S. , Apstein Carl S.
Published: December 2017

Decreasing coronary perfusion causes an immediate decrease in contractile function via unknown mechanisms. It has long been suspected that this contractile dysfunction is caused by ischemia-induced changes in cardiac energetics. Our goal was to determine whether changes in cardiac energetics necessarily precede the contractile dysfunction as one would expect if a causal relationship exists. In 14 isolated rat hearts, we gradually decreased coronary perfusion using a coronary perfusate with a normal hematocrit and normal concentrations of the major metabolic substrates. Using31P NMR spectroscopy to measure ATP, phosphocreatine (PCr), Pi, and ADP concentrations ([ATP], [PCr], [Pi], [ADP]), pH, and amount of free energy released from ATP hydrolysis (‖Δ GATP‖), we found that none of these variables changed significantly until several minutes after systolic pressure had significantly decreased. Even when developed pressure had decreased by over one-third, only very slight changes in [Pi], pH, and ‖Δ GATP‖ had occurred, with no significant changes in [ATP], [PCr], or [ADP]. Additionally, the rate of high-energy phosphate transfer between ATP and PCr did not decrease enough during hypoperfusion to explain the contractile dysfunction. We conclude that nonenergetic factors are the dominant cause of the initial decrease in systolic function when myocardial perfusion is decreased.
Cardiac microvascular endothelial cells express α-smooth muscle actin and show low NOS III activity
Authors: Ando Hiroshi , Kubin Thomas , Schaper Wolfgang , Schaper Jutta
Published: December 2017

We established a culture system of porcine coronary microvascular endothelial cells (MVEC) with high cellular yield and purity >98%. Endothelial origin was confirmed by immunostaining, immunoblotting and fluorescence-activated cell sorter (FACS) analysis using low-density lipoprotein uptake, CD31, von Willebrand factor, and the lectin Dolichos biflorus agglutinin. MVEC were positive for α-smooth muscle actin in culture and in myocardium, as confirmed by FACS. Of the primary MVEC, ∼30% expressed nitric oxide synthase (NOS) III in numbers decreasing from the first passage (6 ± 1%) to the second passage (4 ± 1%; P < 0.001 vs. primary isolates), whereas ∼100% of aortic endothelial cells (AEC) expressed NOS III. In AEC, NOS III activity (pmol citrulline ⋅ mg protein−1⋅ min−1) was 80 ± 10 and was nearly abolished in the absence of calcium (5 ± 1, P < 0.001). In primary MVEC, however, NOS III activity in the presence and absence of calcium was 20 ± 4 and 25 ± 5, respectively. We conclude that cardiac MVEC, in contrast to AEC, contain α-smooth muscle actin, show low-grade NOS III activity, and provide a suitable in vitro system for the study of endothelial pathophysiology.
A new sample-processing unit for the fluorescent microsphere method
Authors: Raab S. , Thein E. , Harris A. G. , Messmer K.
Published: December 2017

The use of fluorescent-labeled microspheres (FM) for measurement of regional blood flow is an attractive alternative to the use of radioactive-labeled microspheres. In the FM method the FM have to be completely recovered from the tissue samples in a time- and labor-intensive process. For this reason, a considerable loss of FM is possible. The aim of this study was to develop a filtration device that allows the tissue sample to remain in a single container throughout the procedure to make the process easier and to avoid the loss of FM. The core of the sample-processing unit (SPU) is a single-tube filtration device with a polyamide wire mesh. The protocol for processing tissue from different organs (heart, kidney, liver, spleen, intestine, muscle, bone, lung, brain) was modified and thus shortened significantly. Furthermore, the SPU allows direct filtration of the blood reference sample without previous digestion. Different experiments showed that the SPU in combination with the new protocol excludes the loss of 15-μm FM. The modifications of the whole procedure render it faster and highly standardized.
ATP reduces macromolecule permeability of endothelial monolayers despite increasing [Ca2+]i
Authors: Noll T. , Hölschermann H. , Koprek K. , Gündüz D. , Haberbosch W. , Tillmanns H. , Piper H. M.
Published: December 2017

We investigated the relationship between the ATP-evoked rise of cytosolic Ca2+concentration ([Ca2+]i) and barrier function in porcine aortic endothelial monolayers. ATP (0.01–100 μM) induced a transient rise of [Ca2+]iand reduced permeability in a concentration-dependent manner. In contrast, the Ca2+ionophore ionomycin (1 μM) elicited a rise in [Ca2+]icomparable to that induced by ATP (10 μM), but it increased permeability. For the reduction of permeability, nucleotides were found to be in the following order of potency: ATP = ATPγS > ADP = UTP. Blockade of adenosine receptors by 8-phenyltheophylline (10 μM) did not affect ATP (10 μM)-induced reduction of permeability. ATP reduced permeability even in endothelial monolayers that had been loaded with the Ca2+chelator BAPTA to prevent the rise in [Ca2+]i. U-73122 (1 μM), an inhibitor of phospholipase C (PLC), completely abolished the effect of ATP (10 μM) on permeability. It also abolished the translocation of protein kinase C (PKC) in response to ATP, which could also be achieved by the PKC inhibitors Gö-6976 (100 nM) or bisindolylmaleimide I (1 μM). In the presence of PKC inhibitors, however, the permeability effect of ATP was not affected. The presence of inhibitors of adenylate or guanylate cyclase (50 μM SQ-22536 or 20 μM ODQ) prevented changes in cyclic nucleotides but did not affect the permeability effects of ATP. The study shows that ATP reduces macromolecule permeability via a PLC-mediated mechanism that is independent of the concomitant effects of ATP on cytosolic Ca2+, cyclic nucleotides, or PKC.
Activation of Akt/protein kinase B after stimulation with angiotensin II in vascular smooth muscle cells
Authors: Takahashi Tomosaburo , Taniguchi Takahiro , Konishi Hiroaki , Kikkawa Ushio , Ishikawa Yuichi , Yokoyama Mitsuhiro
Published: December 2017

Involvement of Akt/Protein kinase B (PKB), a serine/threonine kinase with a pleckstrin-homology domain, in angiotensin II (ANG II)-induced signal transduction was investigated in cultured vascular smooth muscle cells (VSMC). Stimulation of the cells with ANG II led to a marked increase in the kinase activity of Akt/PKB, which coincided with Ser-473 phosphorylation. ANG II-stimulated Akt/PKB activation was rapid, concentration dependent, and inhibited by the AT1-receptor antagonist CV-11974, but not by pertussis toxin. Akt/PKB activity was stimulated by the Ca2+ionophore ionomycin, suggesting the possible involvement of Ca2+in ANG II-stimulated Akt/PKB activation. However, blockade of Ca2+mobilization by BAPTA-AM only partially inhibited ANG II-stimulated Akt/PKB activation. ANG II-stimulated Akt/PKB activation was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A and by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY-294002. These results indicate that ANG II stimulates Akt/PKB activity via AT1receptors in VSMC and that the activities of tyrosine kinase and PI3K are required for this activation.
Vasorelaxing effects of atrial and brain natriuretic peptides on coronary circulation in heart failure
Authors: Matsumoto Tetsuya , Wada Atsuyuki , Tsutamoto Takayoshi , Omura Tomoko , Yokohama Hiroshi , Ohnishi Masato , Nakae Ichiro , Takahashi Masayuki , Kinoshita Masahiko
Published: December 2017

Natriuretic peptide (NP) receptor has been postulated to be downregulated under a high concentration of atrial NP (ANP) in congestive heart failure (CHF), but limited information is available on how the vascular functional responsiveness to NPs is altered in coronary circulation during CHF. We assessed the relaxant effects of ANP, brain NP (BNP), and other vasodilators in isolated coronary arteries obtained from dogs with and without severe CHF induced by rapid right ventricular pacing. In CHF dogs, plasma ANP and cGMP concentrations were elevated compared with control dogs. In CHF arteries the relaxant effects of ANP and BNP (10−8and 10−7mol/l) were suppressed compared with control arteries. Nitroglycerin, nitric oxide, 8-bromo-cGMP, and beraprost sodium produced similar concentration-response curves in both arteries. The addition of 10−7mol/l ANP increased the level of tissue cGMP in control arteries, but not in CHF arteries. We conclude that there was a specific reduction in the relaxant effects of ANP and BNP in isolated coronary arteries in severe CHF dogs, which suggests the possibility of the downregulation of NP receptors coupled to guanylate cyclase.
Nitric oxide in the regulation of vasomotor tone in human skeletal muscle
Authors: Rådegran G. , Saltin B.
Published: December 2017

The role of nitric oxide (NO) as a regulator of vasomotor tone has been investigated in resting and exercising human skeletal muscle. At rest, NO synthase (NOS) inhibition by intra-arterial infusion of N G-monomethyl-l-arginine decreased femoral artery blood flow (FABF, ultrasound Doppler) from 0.39 ± 0.08 to 0.18 ± 0.03 l/min ( P < 0.01), i.e., by ∼52%, and increased leg O2 extraction from 62.1 ± 9.8 to 100.9 ± 4.5 ml/l ( P < 0.004); thus leg O2 uptake (V˙o 2, 22 ± 4 ml/min, ∼0.75 ml ⋅ min−1 ⋅ 100 g−1) was unaltered [not significant ( P = NS)]. Mean arterial pressure (MAP) increased by 8 ± 2 mmHg ( P < 0.01). Heart rate (HR, 53 ± 3 beats/min) was unaltered ( P = NS). The NOS inhibition had, however, no effect on the initial rate of rise or the magnitude of FABF (4.8 ± 0.4 l/min, ∼163 ml ⋅ min−1 ⋅ 100 g−1), MAP (117 ± 3 mmHg), HR (98 ± 5 beats/min), or legV˙o 2 (704 ± 55 ml/min, ∼24 ml ⋅ min−1 ⋅ 100 g−1, P = NS) during submaximal, one-legged, dynamic knee-extensor exercise. Similarly, FABF (7.6 ± 1.0 l/min, ∼258 ml ⋅ min−1 ⋅ 100 g−1), MAP (140 ± 8 mmHg), and leg V˙o 2(1,173 ± 139 ml/min, ∼40 ml ⋅ min−1 ⋅ 100 g−1) were unaffected at termination of peak effort ( P = NS). Peak HR (137 ± 3 beats/min) was, however, lowered by 10% ( P < 0.01). During recovery, NOS inhibition reduced FABF by ∼34% ( P< 0.04), which was compensated for by an increase in the leg O2 extraction by ∼41% ( P < 0.04); thus legV˙o 2 was unaltered ( P = NS). In conclusion, these findings indicate that NO is not essential for the initiation or maintenance of active hyperemia in human skeletal muscle but support a role for NO during rest, including recovery from exercise. Moreover, changes in blood flow during rest and recovery caused by NOS inhibition are accompanied by reciprocal changes in O2 extraction, and thusV˙o 2 is maintained.
Heat shock protein expression protects against cerebral ischemia and monoamine overload in rat heatstroke
Authors: Yang Yi-Ling , Lin Mao-Tsun
Published: December 2017

This study attempted to ascertain whether the ischemic damage to neurons and monoamine overload in brain that occur during rat heatstroke can be attenuated by heat shock protein (HSP) 72 induction. Effects of heatstroke on mean arterial pressure (MAP), cerebral blood flow (CBF), brain dopamine (DA) and serotonin (5-HT) release, and neural damage score were assayed in rats 0, 16, or 48 h after heat shock (42°C for 15 min) or chemical stress (5 mg/kg sodium arsenite ip). Brain HSP 72 in rats after heat shock or chemical stress was detected by Western blot, and brain monoamine was determined by a microdialysis probe combined with high-performance liquid chromatography. Heatstroke was induced by exposing the animal to a high ambient temperature (43°C); the moment at which MAP and CBF decreased from their peak values was taken as the time of heatstroke onset. Prior heat shock or chemical stress conferred significant protection against heatstroke-induced hyperthermia, arterial hypotension, cerebral ischemia, cerebral DA and 5-HT overload, and neural damage and correlated with expression of HSP 72 in brain at 16 h. However, at 48 h, when HSP 72 expression returned to basal values, the above responses that occurred during the onset of heatstroke were indistinguishable between the two groups (0 h vs. 48 h). These results lead to the hypothesis that the brain can be preconditioned by thermal or chemical injury, that this preconditioning will induce HSP 72, and that HSP 72 induction will correlate quite well with anatomic, histochemical, and hemodynamic protection in rat heatstroke.
Cardiac myosin heavy chains lacking the light chain binding domain cause hypertrophic cardiomyopathy in mice
Authors: Welikson Robert E. , Buck Scott H. , Patel Jitandrakumar R. , Moss Richard L. , Vikstrom Karen L. , Factor Stephen M. , Miyata Setsuya , Weinberger Howard D. , Leinwand Leslie A.
Published: December 2017

Myosin is a chemomechanical motor that converts chemical energy into the mechanical work of muscle contraction. More than 40 missense mutations in the cardiac myosin heavy chain (MHC) gene and several mutations in the two myosin light chains cause a dominantly inherited heart disease called familial hypertrophic cardiomyopathy. Very little is known about the biochemical defects in these alleles and how the mutations lead to disease. Because removal of the light chain binding domain in the lever arm of MHC should alter myosin’s force transmission but not its catalytic function, we tested the hypothesis that such a mutant MHC would act as a dominant mutation in cardiac muscle. Hearts from transgenic mice expressing this mutant myosin are asymmetrically hypertrophied, with increases in mass primarily restricted to the cardiac anterior wall. Histological examination demonstrates marked cellular hypertrophy, myocyte disorganization, small vessel coronary disease, and severe valvular pathology that included thickening and plaque formation. Skinned myocytes and multicellular preparations from transgenic hearts exhibited decreased Ca2+sensitivity of tension and decreased relaxation rates after flash photolysis of diazo 2. These experiments demonstrate that alterations in myosin force transmission are sufficient to trigger the development of hypertrophic cardiomyopathy.
Greater erythrocyte deformability in world-class endurance athletes
Authors: Smith John A. , Martin David T. , Telford Richard D. , Ballas Samir K.
Published: December 2017

Because athletes during endurance events require rapid uptake of oxygen, the ability of red blood cells (RBC) to move through capillaries may limit performance. Using ektacytometry, we determined whether RBC deformability (RCD) differed between elite road cyclists ( n = 9) and sedentary controls ( n = 5). Density profiles and standard hematological measurements were also performed. The deformability index (DI) was higher in the cyclists (0.723 ± 0.027) compared with that in controls (0.619 ± 0.040, P < 0.001). Cyclists also had a larger percentage of low-density RBCs ( P < 0.001), and mean cell volume (MCV) was also higher ( P = 0.013). These findings are indicative of a larger proportion of “young” RBCs in the blood of elite cyclists and provide further evidence that the turnover of RBCs in endurance athletes is higher than in the general population. With a younger more deformable RBC population and providing the destruction does not exceed replacement, performance potential should be enhanced. Furthermore, examination of factors that contribute to increased RBC turnover in athletes may help us understand the mechanisms that cause RBC aging.
New analytic framework for understanding sympathetic baroreflex control of arterial pressure
Authors: Sato Takayuki , Kawada Toru , Inagaki Masashi , Shishido Toshiaki , Takaki Hiroshi , Sugimachi Masaru , Sunagawa Kenji
Published: December 2017

The sympathetic baroreflex is an important feedback system in stabilization of arterial pressure. This system can be decomposed into the controlling element (mechanoneural arc) and the controlled element (neuromechanical arc). We hypothesized that the intersection of the two operational curves representing their respective functions on an equilibrium diagram should define the operating point of the arterial baroreflex. Both carotid sinuses were isolated in 16 halothane-anesthetized rats. The vagi and aortic depressor nerves were cut bilaterally. Carotid sinus pressure (CSP) was sequentially altered in 10-mmHg increments from 80 to 160 mmHg while sympathetic efferent nerve activity (SNA) and systemic arterial pressure (SAP) were recorded simultaneously under various hemorrhagic conditions. The mechanoneural arc was characterized by the response of SNA to CSP and the neuromechanical arc by the response of SAP to SNA. We parametrically analyzed the relationship between input and output for each arc using a four-parameter logistic equation model. In baseline states, the two arcs intersected each other at the point at which the instantaneous gain of each arc attained its maximum. Severe hemorrhage lowered the gain and offset of the neuromechanical arc and moved the operating point, whereas the mechanoneural arc remained unchanged. The operating points measured under the closed-loop conditions were indistinguishable from those estimated from the intersections of the two arc curves on the equilibrium diagram. The average root mean square errors of estimate for arterial pressure and SNA were 2 and 3%, respectively. Such an analytic approach could explain a mechanism for the determination of the operating point of the sympathetic baroreflex system and thus helps us integratively understand its function.
Chronic central infusion of ANG II potentiates cardiac sympathetic afferent reflex in dogs
Authors: Ma Rong , Schultz Harold D. , Wang Wei
Published: December 2017

The aims of this study were to determine whether ANG II is involved in the central integration of the cardiac sympathetic afferent reflex (CSAR), and if this central effect of ANG II is mediated by the AT1 receptor. Experiments were undertaken in dogs that were anesthetized with α-chloralose, sinoaortic denervated, and vagotomized. The renal sympathetic nerve activity (RSNA) responses to varying frequency and voltage stimulation of cardiac sympathetic afferent nerves were used to evaluate the central sensitivity of the CSAR. In two groups of dogs, two doses (50 and 100 ng/min icv) of ANG II were acutely infused. In a third group of dogs, ANG II was chronically infused for 3 days (100 ng/min, 1 μl/h icv). We found that acute infusion into the cerebroventricle of two doses of ANG II did not affect the central sensitivity of the CSAR or the baseline hemodynamics, but the baseline RSNA increased significantly during the infusion of the higher dose of ANG II. However, chronic intracerebroventricular infusion of ANG II enhanced the central sensitivity of the CSAR significantly. In addition, chronic intracerebrovetricular infusion of ANG II elicited a significant increase in water intake and in arterial pressure from the first and second day of infusion, respectively. In the group that received chronic intracerebroventricular infusion of ANG II, the administration of an AT1-receptor antagonist losartan (0.125 mg/kg icv) abolished ANG II-induced augmentation of the CSAR. These results suggest that chronic elevation of central ANG II can sensitize the CSAR via central AT1 receptors.
Concentration-dependent effects of bradykinin on leukocyte recruitment and venular hemodynamics in rat mesentery
Authors: Shigematsu Sakuji , Ishida Shuji , Gute Dean C. , Korthuis Ronald J.
Published: December 2017

The results of several recent studies indicate that bradykinin protects tissues against the deleterious effects of ischemia-reperfusion (I/R). However, other studies indicate that bradykinin can act as a proinflammatory agent, inducing P-selectin expression, the formation of chemotactic stimuli, and endothelial barrier disruption. In the present study, we used intravital microscopic techniques to examine the dose-dependent effects of bradykinin on leukocyte-endothelial cell interactions, the formation of platelet-leukocyte aggregates, and venular hemodynamics in rat mesentery in an attempt to explain these divergent findings. Superfusion of the mesentery with low concentrations of bradykinin (≤10−7M) increased venular erythrocyte velocity ( VRBC) without increasing the number of adherent leukocytes, whereas higher concentrations (≥10−6M) decreased VRBC, increased the number of platelet-leukocyte aggregates, and induced leukocyte adhesion in single postcapillary venules. The formation of platelet-leukocyte aggregates and increased leukocyte adhesion induced by high-dose bradykinin were attenuated by administration of a B2-receptor (HOE-140) or a platelet-activating factor (PAF, WEB-2086) antagonist. Thus these adhesive interactions induced by high-dose bradykinin appear to be mediated by a mechanism that is dependent on B2-receptor activation and the formation of PAF or PAF-like lipids. The effects of bradykinin on venular VRBCand blood flow were also concentration dependent, with low doses producing nitric oxide-mediated vasodilation, whereas high doses decreased VRBCby a mechanism that is PAF independent.
Role of PTP-1B in aortic smooth muscle cell motility and tyrosine phosphorylation of focal adhesion proteins
Authors: Hassid Aviv , Huang Shile , Yao Jian
Published: December 2017

Recent studies have focused attention on the role of protein tyrosine kinases in vascular smooth muscle cell biology, but similar information regarding protein tyrosine phosphatases (PTP) is sparse. PTP-1B is a ubiquitous nonreceptor phosphatase with uncertain function and substrates that are mostly unidentified. We used antisense oligodeoxynucleotides (ODN) against PTP-1B to investigate the role of endogenous PTP-1B in motility of primary cultures of rat aortic smooth muscle cells (RASMC). Antisense ODN decreased PTP-1B protein levels and activity in a concentration-dependent fashion, whereas sense, scrambled, or three-base mismatch antisense ODN had little or no effect. Treatment of cells with antisense ODN, but not sense, scrambled, or three-base mismatch antisense ODN, enhanced cell motility and increased tyrosine phosphorylation levels of focal adhesion proteins paxillin, p130cas, and focal adhesion kinase. Our findings indicate that PTP-1B is a negative regulator of RASMC motility via modulation of phosphotyrosine levels in several focal adhesion proteins and suggest the involvement of PTP-1B in events such as atherosclerosis and restenosis, which are associated with increased vascular smooth muscle cell motility.
Endothelial nitric oxide synthase gene transfer enhances dilation of newborn piglet pulmonary arteries
Authors: Aschner Judy L. , Kovacs Nora , Perciaccante James V. , Figueroa Jorge P. , Thrikawala Nishadi , Robins Gregory S. , Busija David W.
Published: December 2017

We determined the expression and functional correlate of in vitro transfection with a recombinant adenoviral vector encoding the gene for bovine endothelial nitric oxide synthase (AdCMVeNOS) or Escherichia coliβ-galactosidase (AdCMVLacZ) in pulmonary endothelial cells (EC), vascular smooth muscle cells (VSMC), and pulmonary arteries (PA) from newborn piglets. AdCMVeNOS and AdCMVeLacZ vectors, grown in 293-cell monolayers, were purified by double-cesium gradient ultracentrifugation. Cell cultures and PA were incubated with increasing vector titers for 30 or 60 min, followed by incubation in fresh medium for 18 h at 37°C. LacZ expression was assessed by histochemical staining; eNOS expression was evaluated by Western blot analysis. Functional eNOS expression was determined by measurement of cGMP and quantification of the relaxation response to bradykinin (BK). In PA, LacZ transgene expression was preferentially localized to the adventitia and endothelium. Increased eNOS protein expression was observed in EC and VSMC transfected with AdCMVeNOS. Functional studies revealed increased cGMP abundance in cultured cells and enhanced relaxation to BK in AdCMVeNOS-transfected PA. These studies demonstrate that gene transfer with AdCMVeNOS results in functional expression and altered vasoactive responses in the neonatal pulmonary vasculature. Gene transfer with replication-deficient adenovirus vectors is a useful tool for the study of targeted genes in vascular biology.
Cellular basis of ventricular arrhythmias and abnormal automaticity in heart failure
Authors: Nuss H. Bradley , Kääb Stefan , Kass David A. , Tomaselli Gordon F. , Marbán Eduardo
Published: December 2017

The high incidence of sudden death in heart failure may reflect an increased propensity to abnormal repolarization and long Q-T interval-related arrhythmias. If so, cells from failing hearts would logically be expected to exhibit a heightened susceptibility to early afterdepolarizations (EAD). We found that midmyocardial ventricular cells isolated from dogs with pacing-induced heart failure exhibited an increased action potential duration and many more EAD than cells from nonpaced controls; this was the case both under basal conditions ( P < 0.01) and after lowering external K+concentration ([K+]o) to 2 mM and exposing cells to cesium (3 mM; P < 0.05). An unexpected finding was the occurrence of spontaneous depolarizations (SD, >5 mV) from the resting potential that were not coupled to prior action potentials. These SD were observed in 20% of failing cells ( n = 5 of 25) under basal ionic conditions but in none of the normal cells ( n = 0 of 27, P < 0.05). The net inward current that underlies SD is not triggered by Ca2+ oscillations and thus differs fundamentally from the currents that underlie delayed afterdepolarizations. We conclude that cardiomyopathic canine ventricular cells are intrinsically predisposed to EAD and SD. Because EAD have been linked to the pathogenesis of torsade de pointes, our results support the hypothesis that sudden death in heart failure often arises from abnormalities of repolarization. The frequent occurrence of SD points to a novel cellular mechanism for abnormal automaticity in heart failure.
Detection of a ferrylhemoglobin intermediate in an endothelial cell model after hypoxia-reoxygenation
Authors: McLeod Laurie L. , Alayash Abdu I.
Published: December 2017

A cell culture model of bovine aortic endothelial cells attached to microcarrier beads was used to study the interaction of diaspirin cross-linked hemoglobin (an oxygen-carrying blood substitute) with hypoxia-reoxygenation. Hemoglobin (200 μM) and hypoxia-volume restriction (3–5 h), together and separately, caused toxicity in this model, as measured by decreased cellular replating efficiency. Hemoglobin (60 μM) caused a reduction in hydrogen peroxide concentration and an increase in lipid peroxidation above that induced by hypoxia alone. Incubation of hemoglobin with endothelial cells caused transient oxidation of hemoglobin to its highly reactive and toxic ferryl species after ≥3 h of hypoxia, followed by 1 h of reoxygenation. Lipid peroxidation, which may occur in the presence of ferrylhemoglobin, also occurred after 1 h of reoxygenation. Hemoglobin caused a dose-dependent decrease in intracellular glutathione concentration, suggesting that it caused an oxidative stress to the cells. However, addition of ascorbate, α-tocopherol, or trolox did not decrease hemoglobin oxidation in the presence of normal or hypoxic cells. It is concluded that diaspirin cross-linked hemoglobin forms a ferryl intermediate in the absence of any exogenously added oxidant and contributes to the oxidative burden experienced by endothelial cells after hypoxia-reoxygenation, a condition that is likely to be encountered during trauma and surgery when hemoglobin solutions are used as perfusion agents.
Differential effects of ovariectomy on calcium activation of cardiac and soleus myofilaments
Authors: Wattanapermpool Jonggonnee , Reiser Peter J.
Published: December 2017

The hypothesis that ovarian sex hormone deficiency affects cardiac myofilament activation was tested. Chemically skinned ventricular trabeculae and single soleus muscle fibers were prepared from 10- and 14-wk ovariectomized and control rats. Tension-pCa (−log [Ca2+]) relations of left ventricular trabeculae and soleus fibers were compared to test whether thin filament proteins are potential sites of modulated activation. Trabeculae from ovariectomized rats exhibited a significant increase in Ca2+sensitivity with no change in maximal tension-generating ability. In contrast, soleus fibers demonstrated no shift in Ca2+sensitivity but generated significantly less maximal tension. No changes in thin filament protein isoform expression or loss of thin filament proteins were apparent in the trabeculae or soleus fibers from ovariectomized rats. Although not directly tested, our results are consistent with a possible modulation of regulatory proteins (e.g., cardiac troponin I) to account for the observed change in myofilament responsiveness of hearts from ovariectomized rats. Other possible mechanisms for the altered myocardial Ca2+sensitivity after ovariectomy are discussed.
Activation of contraction in cat ventricular myocytes: effects of low Cd2+ concentration and temperature
Authors: Wasserstrom J. Andrew , Vites Ana-Maria
Published: December 2017

The effects of Cd2+ (20 μM) and different bath temperatures were used to study the contributions of two separate triggering mechanisms, L-type Ca2+current ( I Ca) and reverse mode Na+/Ca2+exchange, to excitation-contraction (E-C) coupling in cat ventricular myocytes. Ionic currents and cell shortening were studied with patch pipettes filled with K+-containing internal solution and discontinuous (“switch”) voltage clamp. Superfusion with Cd2+ blocked cell shortening that closely mirrored the block of I Ca; the voltage dependence of Cd2+-induced reduction in contraction was bell-shaped, displaying minima at test potentials below −10 mV and above +50 mV and a maximum at about +20 mV. Cd2+-insensitive cell shortening was blocked by ryanodine (10 μM) and Ni2+ (4–5 mM). When an action potential was used as the command waveform for the voltage clamp (action potential clamp), Cd2+reduced contraction to ∼60 ± 7% of control cell shortening ( n = 7). The remaining contraction was blocked by ryanodine and Ni2+. Superfusion with nifedipine (10 μM) caused nearly identical effects to Cd2+. The voltage dependence of contraction was sigmoidal at temperatures above 34°C but bell-shaped below 30°C. When Cd2+ was added to superfusate, contraction was abolished at 25°C (to 6 ± 3% of control) but reduced only modestly at 34°C (to 65 ± 13% of control, test potential +10 mV, n = 4, P < 0.01). These results indicate that 1) there is a component of contraction that is sensitive to I Ca antagonists, and the block is equivalent with either organic or inorganic antagonists; 2) the contribution of Na+/Ca2+exchange to triggering of contraction under our experimental conditions is fairly linear throughout the entire voltage range tested; 3) the contribution of I Ca is superimposed on this background component contributed by the Na+/Ca2+exchanger; and 4) triggering via the exchanger is temperature-dependent, providing a major contribution at physiological temperatures but failing at temperatures below 30°C in a nearly all-or-none fashion.
Supine exercise restores arterial blood pressure and skin blood flow despite dehydration and hyperthermia
Authors: González-Alonso José , Mora-Rodríguez Ricardo , Coyle Edward F.
Published: December 2017

We determined whether the deleterious effects of dehydration and hyperthermia on cardiovascular function during upright exercise were attenuated by elevating central blood volume with supine exercise. Seven trained men [maximal oxygen consumption (V˙o 2 max) 4.7 ± 0.4 l/min (mean ± SE)] cycled for 30 min in the heat (35°C) in the upright and in the supine positions (V˙o 2 2.93 ± 0.27 l/min) while maintaining euhydration by fluid ingestion or while being dehydrated by 5% of body weight after 2 h of upright exercise. When subjects were euhydrated, esophageal temperature (Tes) was 37.8–38.0°C in both body postures. Dehydration caused equal hyperthermia during both upright and supine exercise (Tes = 38.7–38.8°C). During upright exercise, dehydration lowered stroke volume (SV), cardiac output, mean arterial pressure (MAP), and cutaneous vascular conductance and increased heart rate and plasma catecholamines [30 ± 6 ml, 3.0 ± 0.7 l/min, 6 ± 2 mmHg, 22 ± 8%, 14 ± 2 beats/min, and 50–96%, respectively; all P < 0.05]. In contrast, during supine exercise, dehydration did not cause significant alterations in MAP, cutaneous vascular conductance, or plasma catecholamines. Furthermore, supine versus upright exercise attenuated the increases in heart rate (7 ± 2 vs. 9 ± 1%) and the reductions in SV (13 ± 4 vs. 21 ± 3%) and cardiac output (8 ± 3 vs. 14 ± 3%) (all P< 0.05). These results suggest that the decline in cutaneous vascular conductance and the increase in plasma norepinephrine concentration, independent of hyperthermia, are associated with a reduction in central blood volume and a lower arterial blood pressure.
Inhaled NO impacts vascular but not extravascular compartments in postischemic peripheral organs
Authors: Kubes Paul , Payne Derrice , Grisham Matthew B. , Jourd-Heuil David , Fox-Robichaud Alison
Published: December 2017

Inhaled nitric oxide (NO) reduces pulmonary hypertension and dampens various aspects of lung inflammation; however, its effects are thought to be restricted to the lung because of its short half-life in biological systems. More recently, however, NO was shown to nitrosylate hemoglobin, albumin, and other plasma molecules to form stable nitrosothiol derivatives and could have an impact on the periphery. We examined whether inhaled NO could have an impact on the two compartments of distal organs, namely, the intravascular and extravascular spaces. The feline intestine was exposed to 1 h of ischemia and 1 h of reperfusion, and intestinal blood flow and mucosal dysfunction were measured in animals ventilated with room air and inhaling 0 or 80 ppm NO. A decrease in intestinal blood flow and an increase in mucosal barrier leakiness were noted in animals not exposed to inhaled NO. The intestinal blood flow impairment was entirely reversed in animals breathing 80 ppm NO, but the mucosal dysfunction was not affected. We further examined whether inhaled NO could reach the extravascular space by simply inhibiting NO in the intestine with the NO synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME) that causes an increase in mucosal permeability that is rapidly reversed with NO donors. However, inhaled NO had no effect on the rise in mucosal permeability. l-NAME reduced lymph nitrosothiol concentrations, but inhaled NO could not replenish these levels. To further explore the intravascular impact of inhaled NO, we used intravital microscopy to visualize the microvasculature and demonstrated that inhaled NO could be initiated after reperfusion and still reduced microvascular disturbances, including reversing the impairment in blood flow and increasing leukocyte adhesion. The effects of inhaled NO persisted for an additional hour after termination of NO inhalation, consistent with a dramatic increase in nitrate within 1 h of NO inhalation, which persisted for 1 h after the termination of NO inhalation. These data suggest that inhaled NO can reach distal organs to dramatically improve reperfusion-induced microvascular but not extravascular dysfunction.
Matrix metalloproteinase synthesis and expression in isolated LV myocyte preparations
Authors: Coker Mytsi L. , Doscher Melissa A. , Thomas Chadwick V. , Galis Zorina S. , Spinale Francis G.
Published: December 2017

In several cardiac disease states, alterations in myocyte and extracellular matrix (ECM) structure occur with left ventricular (LV) remodeling and are associated with changes in matrix metalloproteinase (MMP) activity. Although nonmyocyte cell types have been implicated as sites for synthesis and expression of MMPs within the ECM, whether the LV myocyte itself expresses specific types and active forms of MMPs remains unknown. Accordingly, isolated Ca2+-tolerant LV porcine myocytes (105cells/ml) in which selective disaggregation and resuspension was performed (13 independent experiments) were plated on basement membrane substrates including Matrigel, collagen IV, laminin, and fibronectin as well as poly-l-lysine. After 24-h incubation, LV myocyte conditioned media were subjected to zymography, a specific MMP-2 proteolytic capture assay, immunoblotting, and ELISA for detection of MMP activity and relative content of the 72-kDa gelatinase MMP-2. Although robust zymographic activity [(pixels ⋅ mm2)/cell] was observed in conditioned media from LV myocytes plated on collagen IV (1,673 ± 297), fibronectin (1,530 ± 281), and poly-l-lysine (2,545 ± 560), proteolytic activity appeared to be lower in conditioned media from LV myocytes plated on Matrigel (842 ± 83) and laminin (1,329 ± 238). MMP-2 proteolytic activity was increased by approximately eightfold in conditioned media taken from LV myocytes plated on poly-l-lysine compared with that of Matrigel. With respect to each of the adhesion substrates, MMP-2 content was at least 50% lower in LV myocyte conditioned media taken from Matrigel and laminin. Immunofluorescent labeling of LV myocytes yielded a strong signal for MMP-2 within the myocyte and along the sarcolemmal surface. In conclusion, this study demonstrated for the first time that adult LV myocytes synthesize and express members of the MMP family and thus may potentially participate in the LV remodeling process through synthesis and secretion of MMPs.
Nitric oxide activity in the peripheral vasculature during normotensive and preeclamptic pregnancy
Authors: Anumba Dilly O. C. , Robson Stephen C. , Boys Richard J. , Ford Gary A.
Published: December 2017

We investigated the role of nitric oxide (NO) in the vascular resistance changes of normotensive and preeclamptic pregnancy. Forearm blood flow (FBF) responses to brachial artery infusion of N G-monomethyl-l-arginine (l-NMMA), an NO synthase inhibitor, and angiotensin II (ANG II), an NO-independent vasoconstrictor, were determined by plethysmography in 20 nonpregnant women, 20 normotensive primigravidae, and 15 primigravidae with untreated preeclampsia. In pregnant subjects, FBF was reduced to nonpregnancy levels by infusion of norepinephrine (NE), which was then coinfused with ANG II (2, 4, and 8 ng/min) and l-NMMA (200, 400, and 800 μg/min) each for 5 min. In separate studies, responses to NE (20, 50, and 100 ng/min) were determined in 8 nonpregnant women, with FBF elevated to pregnancy levels by concomitant infusion of glyceryl trinitrate, and 10 pregnant women. Vasoconstrictor responses tol-NMMA were increased in pregnant compared with nonpregnant subjects [mean ± SE summary measure (in arbitrary units): 60 ± 7 vs. 89 ± 8, respectively; P < 0.01], whereas responses to ANG II were blunted (125 ± 11 vs. 79 ± 7, respectively; P < 0.001). Compared with normotensive pregnant subjects, preeclamptic subjects had an enhanced response to ANG II (79 ± 7 vs. 103 ± 8, respectively; P < 0.05) but no difference in response to l-NMMA (89 ± 8 vs. 73 ± 10, respectively; P = 0.30). Responses to NE were similar in pregnant and nonpregnant subjects (110 ± 20 vs. 95 ± 33, respectively; P = 0.66). During the third trimester of pregnancy, forearm constrictor responses tol-NMMA are increased. The responses to NE are unchanged, whereas responses to ANG II are blunted. Increased NO activity contributes to the fall in peripheral resistance. In preeclampsia, forearm constrictor responses to ANG II but notl-NMMA are increased compared with those in normal pregnancy. Changes in vascular NO activity are unlikely to account for the increased vascular tone in this condition.
NO alters cell shape and motility in aortic smooth muscle cells via protein tyrosine phosphatase 1B activation
Authors: Hassid Aviv , Yao Jian , Huang Shile
Published: December 2017

Cell motility is an important determinant of vascular disease. We examined mechanisms underlying the effect of nitric oxide (NO) on motility in cultured primary aortic smooth muscle cells from newborn rats. The NO donor S-nitroso- N-acetyl-penicillamine (SNAP) increased the activity of protein tyrosine phosphatase 1B (PTP-1B). This effect was mimicked by a cGMP analog and blocked by the guanyl cyclase antagonist 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, indicating the involvement of cGMP. Treatment of cells with antisense, but not control oligodeoxynucleotide (ODN), against PTP-1B attenuated the inhibitory effect of NO on cell motility. Cell shape and adhesion are important determinants of cell motility. We report that SNAP induced cell rounding and reduced adhesion and caused dissociation of actin stress fibers. Moreover, SNAP reduced phosphotyrosine levels in focal adhesion proteins, paxillin, and focal adhesion kinase. The PTP inhibitor phenylarsine oxide or decrease of PTP-1B protein levels via the use of antisense ODN prevented NO-induced cell-shape change, altered adhesion, and migration. These results indicate that NO regulates cell shape, adhesion, and migration by dephosphorylation of focal adhesion proteins via a mechanism that requires PTP-1B activity.
Independent effects of preload, afterload, and contractility on left ventricular torsion
Authors: Dong Sheng-Jing , Hees Paul S. , Huang Wen-Mei , Buffer Sam A. , Weiss James L. , Shapiro Edward P.
Published: December 2017

Shortening of oblique left ventricular (LV) fibers results in torsion. A unique relationship between volume and torsion is therefore expected, and the effects of load and contractility on torsion should be predictable. However, volume-independent behavior of torsion has been observed, and the effects of load on this deformation remain controversial. We used magnetic resonance imaging (MRI) with tagging to study the relationships between load and contractility, and torsion. In ten isolated, blood-perfused canine hearts, ejection was controlled by a servopump: end-diastolic volume (EDV) was controlled by manipulating preload parameters and end-systolic volume (ESV) by manipulating afterload using a three-element windkessel model. MRI was obtained at baseline, two levels of preload alteration, two levels of afterload alteration, and dobutamine infusion. An increase in EDV resulted in an increase in torsion at constant ESV (preload effect), whereas an increase in ESV resulted in a decrease in torsion at constant EDV (afterload effect). Dobutamine infusion increased torsion in association with an increase in LV peak-systolic pressure (PSP), even at identical EDV and ESV. Multiple regression showed correlation of torsion with preload (EDV), afterload (ESV), and contractility (PSP; r = 0.67). Furthermore, there was a close linear relationship between torsion and stroke volume (SV) and ejection fraction (EF) during load alteration, but torsion during dobutamine infusion was greater than expected for the extent of ejection. Preload and afterload influence torsion through their effects on SV and EF, and there is an additional direct inotropic effect on torsion that is independent of changes in volume but rather is force dependent. There is therefore potential for the torsion-volume relation to provide a load-independent measure of contractility that could be measured noninvasively.
Minimal model of arterial chaos generated by coupled intracellular and membrane Ca2+oscillators
Authors: Parthimos D. , Edwards D. H. , Griffith T. M.
Published: December 2017

We have developed a mathematical model of arterial vasomotion in which irregular rhythmic activity is generated by the nonlinear interaction of intracellular and membrane oscillators that depend on cyclic release of Ca2+from internal stores and cyclic influx of extracellular Ca2+, respectively. Four key control variables were selected on the basis of the pharmacological characteristics of histamine-induced vasomotion in rabbit ear arteries: Ca2+concentration in the cytosol, Ca2+concentration in ryanodine-sensitive stores, cell membrane potential, and the open state probability of Ca2+-activated K+channels. Although not represented by independent dynamic variables, the model also incorporates Na+/Ca2+exchange, the Na+-K+-ATPase, Cl−fluxes, and Ca2+efflux via the extrusion ATPase. Simulations reproduce a wide spectrum of experimental observations, including 1) the effects of interventions that modulate the functionality of Ca2+stores and membrane ion channels, 2) paradoxes such as the apparently unpredictable dual action of Ca2+antagonists and low extracellular Na+concentration, which can abolish vasomotion or promote the appearance of large-amplitude oscillations, and 3) period-doubling, quasiperiodic, and intermittent routes to chaos. Nonlinearity is essential to explain these diverse patterns of experimental vascular response.