bluuebunny/crossref_metadata_2025 huggingface dataset:
Reactive carbonyls from tobacco smoke increase arterial endothelial layer injury
Authors: Mullick Adam E. , McDonald James M. , Melkonian Goar , Talbot Prudence , Pinkerton Kent E. , Rutledge John C.
Published: March 2015

We hypothesized that reactive carbonyls generated from smoke exposure cause increased arterial low-density lipoprotein (LDL) accumulation and endothelial layer permeability. In addition, we hypothesized that estrogen supplementation was protective against chronic environmental tobacco smoke (ETS) exposure to the artery wall. Quantitative fluorescence microscopy was used to determine artery injury after exposure. For our chronic studies, ovariectomized rats treated with subcutaneous placebo or 17β-estradiol pellets were exposed to ETS or filtered air for 6 wk. ETS exposure increased carotid artery LDL accumulation more than fourfold compared with filtered air exposure, an effect largely mediated by increased permeability. No protective effect of estradiol was observed. Acute ETS exposure of a buffer solution containing LDL resulted in a more than sixfold increase in the highly reactive carbonyl glyoxal. Perfusion of this solution through carotid arteries resulted in a 105% increase in permeability. Moreover, perfusion of glyoxal alone caused a 50% increase in carotid artery permeability. This endothelial damage and changes in lipid accumulation may serve as an initiating event in atheroma formation in individuals exposed to ETS.
NAD(P)H oxidase-generated superoxide anion accounts for reduced control of myocardial O2 consumption by NO in old Fischer 344 rats
Authors: Adler Alexandra , Messina Eric , Sherman Ben , Wang Zipping , Huang Harer , Linke Axel , Hintze Thomas H.
Published: March 2015

We investigated the role of nitric oxide (NO) in the control of myocardial O2 consumption in Fischer 344 rats. In Fischer rats at 4, 14, and 23 mo of age, we examined cardiac function using echocardiography, the regulation of cardiac O2 consumption in vitro, endothelial NO synthase (eNOS) protein levels, and potential mechanisms that regulate superoxide. Aging was associated with a reduced ejection fraction [from 75 ± 2%at4moto66 ± 3% ( P < 0.05) at 23 mo] and an increased cardiac diastolic volume [from 0.60 ± 0.04 to 1.00 ± 0.10 ml ( P < 0.01)] and heart weight (from 0.70 ± 0.02 to 0.90 ± 0.02 g). The NO-mediated control of cardiac O2 consumption by bradykinin or enalaprilat was not different between 4 mo (36 ± 2 or 34 ± 3%) and 14 mo (29 ± 1 or 25 ± 3%) but markedly ( P < 0.05) reduced in 23-mo-old Fischer rats (15 ± 3 or 7 ± 2%). The response to the NO donor S-nitroso- N-acetyl penicillamine was not different across groups (35%, 35%, and 44%). Interestingly, the eNOS protein level was not different at 4, 14, and 23 mo. The addition of tempol (1 mmol/l) to the tissue bath eliminated the depression in the control of cardiac O2 consumption by bradykinin (25 ± 3%) or enalaprilat (28 ± 3%) in 23-mo-old Fischer rats. We next examined the levels of enzymes involved in the production and breakdown of superoxide. The expression of Mn SOD, Cu/Zn SOD, extracellular SOD, and p67phox, however, did not differ between 4- and 23-mo-old rats. Importantly, there was a marked increase in gp91phox, and apocynin restored the defect in NO-dependent control of cardiac O2 consumption at 23 mo to that seen in 4-mo-old rats, identifying the role of NADPH oxidase. Thus increased biological activity of superoxide and not decreases in the enzyme that produces NO are responsible for the altered control of cardiac O2 consumption by NO in 23-mo-old Fischer rats. Increased oxidant stress in aging, by decreasing NO bioavailability, may contribute not only to changes in myocardial function but also to altered regulation of vascular tone and the progression of cardiac or vascular disease.
Doppler echocardiographic estimation of left ventricular end-diastolic pressure after MI in rats
Authors: Prunier Fabrice , Gaertner Roger , Louedec Liliane , Michel Jean-Baptiste , Mercadier Jean-Jacques , Escoubet Brigitte
Published: March 2015

The spectral Doppler mitral flow pattern, alone or combined with tissue Doppler mitral annulus velocity, can be used to predict left ventricular (LV) filling pressure in humans, whereas invasive hemodynamic measurements are still required in the rat. This study was undertaken to assess whether LV end-diastolic pressure (LVEDP) can be estimated using Doppler echocardiography in the rat after myocardial infarction (MI). Thirty-seven rats (23 rats with MI after left coronary artery ligation and 14 sham-operated rats) were evaluated 3 mo after surgery with echo-Doppler and invasive hemodynamic measurements. Pulse wave spectral Doppler at the mitral valve tip was used to measure the E wave, the E wave deceleration time (DT), and the A wave; spectral Doppler tissue imaging was used to measure the early diastolic lateral mitral annulus velocity ( E a). We found weak correlations between LVEDP and the peak velocity of the early mitral inflow ( E), E/peak velocity of the late mitral inflow, and DT, and strong correlations with E a and especially with E/ E a [ R 2= 0.89, LVEDP (in mmHg) = 0.987 E/ E a − 4.229]. Longitudinal followup of a subgroup of rats with MI revealed a marked rise of E/ E a between days 7 and 21 in rats with heart failure only. We conclude that Doppler echocardiography can be used for serial assessment of LV diastolic function in rats with MI.
ATP-sensitive K+channel activation by nitric oxide and protein kinase G in rabbit ventricular myocytes
Authors: Han Jin , Kim Nari , Joo Hyun , Kim Euiyong , Earm Yung E.
Published: March 2015

The present investigation tested the hypothesis that nitric oxide (NO) potentiates ATP-sensitive K+(KATP) channels by protein kinase G (PKG)-dependent phosphorylation in rabbit ventricular myocytes with the use of patch-clamp techniques. Sodium nitroprusside (SNP; 1 mM) potentiated KATPchannel activity in cell-attached patches but failed to enhance the channel activity in either inside-out or outside-out patches. The 8-(4-chlorophenylthio)-cGMP Rp isomer (Rp-CPT-cGMP, 100 μM) suppressed the potentiating effect of SNP. 8-(4-Chlorophenylthio)-cGMP (8-pCPT-cGMP, 100 μM) increased KATPchannel activity in cell-attached patches. PKG (5 U/μl) added together with ATP and cGMP (100 μM each) directly to the intracellular surface increased the channel activity. Activation of KATPchannels was abolished by the replacement of ATP with ATPγS. Rp-pCPT-cGMP (100 μM) inhibited the effect of PKG. The heat-inactivated PKG had little effect on the KATPchannels. Protein phosphatase 2A (PP2A, 1 U/ml) reversed the PKG-mediated KATPchannel activation. With the use of 5 nM okadaic acid (a PP2A inhibitor), PP2A had no effect on the channel activity. These results suggest that the NO-cGMP-PKG pathway contributes to phosphorylation of KATPchannels in rabbit ventricular myocytes.
Altered NADH and improved function by anesthetic and ischemic preconditioning in guinea pig intact hearts
Authors: Riess Matthias L. , Camara Amadou K. S. , Chen Qun , Novalija Enis , Rhodes Samhita S. , Stowe David F.
Published: March 2015

NADH increases during ischemia because O2 shortage limits NADH oxidation at the electron transport chain. Ischemic (IPC) and anesthetic preconditioning (APC) attenuate cardiac reperfusion injury. We examined whether IPC and APC similarly alter NADH, i.e., mitochondrial metabolism. NADH fluorescence was measured at the left ventricular wall of 40 Langendorff-prepared guinea pig hearts. IPC was achieved by two 5-min periods of ischemia and APC by exposure to 0.5 or 1.3 mM sevoflurane for 15 min, each ending 30 min before 30 min of global ischemia. During ischemia, NADH initially increased in nonpreconditioned control hearts and then gradually declined below baseline levels. This increase in NADH was lower after APC but not after IPC. The subsequent decline was slower after IPC and APC. On reperfusion, NADH was less decreased after IPC or APC, mechanical and metabolic functions were improved, and infarct size was lower compared with controls. Our results indicate that IPC and APC cause distinctive changes in mitochondrial metabolism during ischemia and thus lead to improved function and tissue viability on reperfusion.
Overexpression of Na+/Ca2+ exchanger gene attenuates postinfarction myocardial dysfunction
Authors: Min Jiang-Yong , Sullivan Matthew F. , Yan Xinhua , Feng Xin , Chu Victor , Wang Ju-Feng , Amende Ivo , Morgan James P. , Philipson Kenneth D. , Hampton Thomas G.
Published: March 2015

We monitored myocardial function in postinfarcted wild-type (WT) and transgenic (TG) mouse hearts with overexpression of the cardiac Na+/Ca2+ exchanger. Five weeks after infarction, cardiac function was better maintained in TG than WT mice [left ventricular (LV) systolic pressure: WT, 41 ± 2; TG, 58 ± 3 mmHg; P < 0.05; maximum rising rate of LV pressure (+dP/d t max): WT, 3,750 ± 346; TG, 5,075 ± 334 mmHg/s; P < 0.05]. The isometric contractile response to β-adrenergic stimulation was greater in papillary muscles from TG than WT mice (WT, 13.2 ± 0.9; TG, 16.3 ± 1.0 mN/mm2 at 10−4 M isoproterenol). The sarcoplasmic reticulum (SR) Ca2+content investigated by rapid cooling contractures in papillary muscles was greater in TG than WT mouse hearts. We conclude that myocardial function is better preserved in TG mice 5 wk after infarction, which results from enhanced SR Ca2+ content via overexpression of the Na+/Ca2+ exchanger.
Ketamine abolishes ischemic preconditioning through inhibition of KATP channels in rabbit hearts
Authors: Han Jin , Kim Nari , Joo Hyun , Kim Euiyong
Published: March 2015

Although ketamine inhibits ATP-sensitive K (KATP) channels in rat ventricular myocytes and abolishes the cardioprotective effect of ischemic preconditioning in isolated rat hearts and in rabbits in in vivo, no studies to date specifically address the precise mechanism of this prevention of ischemic preconditioning by ketamine. This study investigated the mechanism of the blockade of ischemic preconditioning by ketamine in rabbit ventricular myocytes using patch-clamp techniques and in rabbit heart slices model for simulated ischemia and preconditioning. In cell-attached and inside-out patches, ketamine inhibited sarcolemmal KATP channel activities in a concentration-dependent manner. Ketamine decreased the burst duration and increased the interburst duration without a change in the single-channel conductance. In the heart slice model of preconditioning, heart slices preconditioned with a single 5-min anoxia, pinacidil, or diazoxide, followed by 15-min reoxygenation, were protected against subsequent 30-min anoxia and 1-h reoxygenation, and the cardioprotection was blocked by the concomitant presence of ketamine. These data are consistent with the notion that inhibition of sarcolemmal or mitochondrial KATP channels may contribute, at least in part, to the mechanism of the blockade of ischemic preconditioning by ketamine.
The ERK pathway regulates Na+-HCO 3 − cotransport activity in adult rat cardiomyocytes
Authors: Baetz Delphine , Haworth Robert S. , Avkiran Metin , Feuvray Danielle
Published: March 2015

The sarcolemmal Na+-HCO[Formula: see text] cotransporter (NBC) is stimulated by intracellular acidification and acts as an acid extruder. We examined the role of the ERK pathway of the MAPK cascade as a potential mediator of NBC activation by intracellular acidification in the presence and absence of angiotensin II (ANG II) in adult rat ventricular myocytes. Intracellular pH (pHi) was recorded with the use of seminaphthorhodafluor-1. The NH[Formula: see text]method was used to induce an intracellular acid load. NBC activation was significantly decreased with the ERK inhibitors PD-98059 and U-0126. NBC activity after acidification was increased in the presence of ANG II (pHi range of 6.75–7.00). ANG II plus PD-123319 (AT2 antagonist) still increased NBC activity, whereas ANG II plus losartan (AT1 antagonist) did not affect it. ERK phosphorylation (measured by immunoblot analysis) during intracellular acidification was increased by ANG II, an effect that was abolished by losartan and U-0126. In conclusion, the MAPK(ERK)-dependent pathway facilitates the rate of pHirecovery from acid load through NBC activity and is involved in the AT1 receptor-mediated stimulation of such activity by ANG II.
Stanniocalcin-1 is a naturally occurring L-channel inhibitor in cardiomyocytes: relevance to human heart failure
Authors: Sheikh-Hamad David , Bick Roger , Wu Gang-Yi , Christensen Birgitte Mønster , Razeghi Peter , Poindexter Brian , Taegtmeyer Heinrich , Wamsley Ann , Padda Ranjit , Entman Mark , Nielsen Søren , Youker Keith
Published: March 2015

Cardiomyocytes of the failing heart undergo profound phenotypic and structural changes that are accompanied by variations in the genetic program and profile of calcium homeostatic proteins. The underlying mechanisms for these changes remain unclear. Because the mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) is expressed in the heart, we reasoned that STC1 might play a role in the adaptive-maladaptive processes that lead to the heart failure phenotype. We examined the expression and localization of STC1 in cardiac tissue of patients with advanced heart failure before and after mechanical unloading using a left ventricular assist device (LVAD), and we compared the results with those of normal heart tissue. STC1 protein is markedly upregulated in cardiomyocytes and arterial walls of failing hearts pre-LVAD and is strikingly reduced after LVAD treatment. STC1 is diffusely expressed in cardiomyocytes, although nuclear predominance is apparent. Addition of recombinant STC1 to the medium of cultured rat cardiomyocytes slows their endogenous beating rate and diminishes the rise in intracellular calcium with each contraction. Furthermore, using whole cell patch-clamp studies in cultured rat cardiomyocytes, we find that addition of STC1 to the bath causes reversible inhibition of transmembrane calcium currents through L-channels. Our data suggest differential regulation of myocardial STC1 protein expression in heart failure. In addition, STC1 may regulate calcium currents in cardiomyocytes and may contribute to the alterations in calcium homeostasis of the failing heart.
Differential effects of pulsatile versus steady flow on coronary endothelial membrane potential
Authors: Qiu Wei-Ping , Hu Qinghua , Paolocci Nazareno , Ziegelstein Roy C. , Kass David A.
Published: March 2015

Steady shear stress stimulates transient hyperpolarization coupled to calcium-sensitive potassium (KCa) channels and sustained depolarization linked to chloride-selective channels. Physiological flow is pulsatile not static, and whereas in vivo data suggest phasic shear stress may preferentially activate KCa channels, its differential effects on both currents remain largely unknown. To determine this interaction, coronary endothelial cells were cultured in glass capillary flow tubes, loaded with the voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol, and exposed to constant or pulsatile shear stress. The latter was generated by a custom servoperfusion system employing physiological pressure and flow waveforms. Steady shear induced a sustained depolarization inhibited by the Cl- channel blocker DIDS. Even after exposure to steady flow, subsequent transition to pulsatile shear stress further stimulated DIDS-sensitive depolarization. DIDS pretreatment “unmasked” a pulsatile flow-induced hyperpolarization of which magnitude was further enhanced by nifedipine, which augments epoxygenase synthesis. Pulse-shear hyperpolarization was fully blocked by KCa channel inhibition (charybdotoxin + apamin), although these agents had no influence on membrane potential altered by steady flow. Thus KCa-dependent hyperpolarization is preferentially stimulated by pulsatile over steady flow, whereas both can stimulate Cl--dependent depolarization. This supports studies showing greater potency of pulsatile flow for triggering KCa-dependent vasorelaxation.
Erythrocyte consumption of nitric oxide in presence and absence of plasma-based hemoglobin
Authors: Tsoukias Nikolaos M. , Popel Aleksander S.
Published: March 2015

Experimental measurements have suggested a consumption rate of nitric oxide (NO) by red blood cells (RBCs) that is orders of magnitude smaller than that of an equivalent concentration of free hemoglobin in solution. This difference has been attributed to external diffusion limitations in the transport of NO from the plasma to the surface of the RBC or to resistance in the transport through the erythrocytic membrane. A detailed mathematical model is developed to quantify the resistance to NO transport around a single RBC and to predict the consumption rate in the presence and absence of extracellular hemoglobin. We provide a description for the NO consumption rate as a function of hematocrit, RBC radius, membrane permeability, and extracellular hemoglobin concentration. We predict a first-order rate constant for NO consumption in blood between 7.5 × 102and 6.5 × 103s−1at a hematocrit of 45% for membrane permeability values between 0.1 and 40 cm/s. Our results suggest that the difference in NO uptake by RBCs and free hemoglobin is smaller than previously reported and it is hematocrit dependent.
Estrogen and testosterone have opposing effects on chronic cardiac remodeling and function in mice with myocardial infarction
Authors: Cavasin Maria A. , Sankey Steadman S. , Yu Ai-Li , Menon Shreevidya , Yang Xiao-Ping
Published: March 2015

Premenopausal women are much less prone to develop cardiovascular disease than men of similar age, but this advantage no longer applies after menopause. We previously found that male mice have a significantly higher rate of cardiac rupture than females during the acute phase of myocardial infarction (MI); however, the effects of sexual hormones on chronic remodeling are unknown. We hypothesized that estrogen (E) may protect the heart from chronic remodeling and deterioration of function post-MI, whereas testosterone (T) may have adverse effects. Mice (4 wk old) of both genders were divided into four groups: female groups consisted of 1) sham ovariectomy (S-Ovx) + placebo (P) (S-Ovx + P), 2) S-Ovx + T, 3) Ovx + P, and 4) Ovx + T; and male groups consisted of 1) sham castration (S-Cas)+ P (S-Cas + P), 2) S-Cas + 17β-estradiol (E), 3) Cas + P, and 4) Cas + E. MI was induced 6 wk later. Echocardiography was performed to assess cardiac function and left ventricular dimensions (LVD). Myocyte cross-sectional area (MCSA) was measured at the end of the study. In females, both testosterone and ovariectomy decreased ejection fraction (EF) and increased LVD, and when combined they aggravated cardiac function and remodeling further. Testosterone significantly increased MCSA. In males, castration or estrogen increased EF and reduced LVD, whereas castration significantly reduced MCSA. Our data suggest that estrogen prevents deterioration of cardiac function and remodeling after MI, but testosterone worsens cardiac dysfunction and remodeling and has a pronounced effect when estrogen levels are reduced.
Cardiac modulations of ANG II receptor expression in rats with hypoxic pulmonary hypertension
Authors: Adamy Christophe , Oliviero Patricia , Eddahibi Saadia , Rappaport Lydie , Samuel Jane-Lise , Teiger Emmanuel , Chassagne Catherine
Published: March 2015

Right ventricular myocardial hypertrophy during hypoxic pulmonary hypertension is associated with local renin-angiotensin system activation. The expression of angiotensin II type 1 (AT1) and type 2 (AT2) receptors in this setting has never been investigated. We have therefore examined the chronic hypoxia pattern of AT1 and AT2expression in the right and left cardiac ventricles, using in situ binding and RT-PCR assays. Hypoxia produced right, but not left, ventricular hypertrophy after 7, 14, and 21 days, respectively. Hypoxia for 2 days was associated in each ventricle with a simultaneous and transient increase ( P < 0.05) in AT1 binding and AT1 mRNA levels in the absence of any significant change in AT2 expression level. Only after 14 days of hypoxia, AT2 binding increased ( P < 0.05) in the two ventricles, concomitantly with a right ventricular decrease ( P < 0.05) in AT2 mRNA. Along these data, AT1 and AT2 binding remained unchanged in both the left and hypertrophied right ventricles from rats treated with monocrotaline for 30 days. These results indicate that chronic hypoxia induces modulations of AT1 and AT2 receptors in both cardiac ventricles probably through direct and indirect mechanisms, respectively, which modulations may participate in myogenic (at the level of smooth or striated myocytes) rather than in the growth response of the heart to hypoxia.
Sphingosine 1-phosphate and control of vascular tone
Authors: Dantas Ana Paula V. , Igarashi Junsuke , Michel Thomas
Published: March 2015

Sphingosine-1-phosphate (S1P) is a platelet-derived lipid mediator that activates the endothelial isoform of nitric oxide synthase (eNOS) in endothelial cells. However, the role of S1P in endothelium-dependent vasodilation and the signaling pathways elicited by S1P in intact vessels are largely unknown. We found that S1P induces dose-dependent transient relaxation of isolated pressurized mesenteric arterioles (EC5010 ± 3 nM); maximal vasodilation (55 ± 8%) is seen ∼2 min after S1P addition and returns to baseline by 5 min. S1P promotes comparable responses in arterioles from wild-type but not eNOSnullmice. S1P-induced vasodilation is abrogated by removal of endothelium or by the addition of the NOS inhibitor Nω-monomethyl-l-arginine but is not affected by the cyclooxygenase inhibitor indomethacin, nor by the blockade of K+channels by using 4-aminopyridine. S1P-induced vasodilation is attenuated by pertussis toxin, by the phosphoinositide 3-kinase (PI3-kinase) inhibitor wortmannin, and by the calcium chelator BAPTA. With the use of high-sensitivity protein immunoblots in extracts from single pressurized vessels, we found that S1P, but not bradykinin, promotes the phosphorylation of eNOS at Ser1179. Maximum S1P-induced eNOS Ser1179phosphorylation was reached at the time of maximum vasorelaxation, but enzyme phosphorylation persisted for several minutes after vasodilation had resolved. Thus regulatory pathways distinct from eNOS Ser1179dephosphorylation serve to terminate agonist-promoted vasorelaxation. Taken together, our findings demonstrate that S1P, an important intercellular mediator of platelet-vessel wall interactions, is a effective arteriolar vasodilator that acts via G protein-dependent, calcium-sensitive, and PI3-kinase-modulated signaling pathways.
Left ventricular targeting of reporter gene expression in vivo by human BNP promoter in an adenoviral vector
Authors: LaPointe Margot C. , Yang Xiao-Ping , Carretero Oscar A. , He Quan
Published: March 2015

To selectively introduce genes into the mouse myocardium, we used a recombinant adenovirus encoding a transgene composed of a cardiac-specific promoter [the proximal human brain natriuretic peptide (hBNP) promoter] coupled to a luciferase reporter gene (Ad.hBNPLuc). Activity in vitro and in vivo was compared with Ad.CMVLuc, which contained the cytomegalovirus (CMV) enhancer/promoter. We tested cell-specific and inducible regulation of Ad.hBNPLuc in vitro. Expression was higher in neonatal cardiac myocytes than in a fibroblast cell line and was induced by interleukin-1β, phenylephrine, and isoproterenol in myocytes. For in vivo experiments, Ad.hBNPLuc, Ad.CMVLuc, or vehicle was injected into the left ventricular (LV) free wall of the mouse heart. In Ad.hBNPLuc-injected mice, luciferase activity was only detected in the heart. In contrast, Ad.CMVLuc-injected mice had detectable luciferase activity in all tissues examined. Our studies indicate that 1) the cardiac-specific hBNP promoter and direct cardiac injection limit expression of the transgene to the LV free wall; and 2) transgene expression in vitro is inducible and cardiac myocyte specific. Thus the use of the proximal hBNP promoter in recombinant adenoviral vectors may allow cardiac-specific and inducible expression of therapeutic genes in vivo and prevent some of the side effects of systemic adenovirus administration.
Attenuation of chronic hypoxic pulmonary hypertension by simvastatin
Authors: Girgis Reda E. , Li Dechun , Zhan Xinhua , Garcia Joe G. N. , Tuder Rubin M. , Hassoun Paul M. , Johns Roger A.
Published: March 2015

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to improve multiple normal endothelial cell functions and inhibit vascular wall cell proliferation. We hypothesized that one such agent, simvastatin, would attenuate chronic hypoxic pulmonary hypertension. Male adult Sprague-Dawley rats were exposed (14 days) to normoxia (N), normoxia plus once-a-day administered simvastatin (20 mg/kg ip) (NS), hypoxia (10% inspired O2 fraction) (H), or hypoxia plus simvastatin (HS). Mean pulmonary artery pressure, measured in anesthetized, ventilated rats with an open-chest method, was reduced from 25 ± 2 mmHg in H to 18 ± 1 in HS ( P < 0.001) but did not reach normoxic values (12 ± 1 mmHg). Similarly, right ventricular/left ventricular plus interventricular septal weight was reduced from 0.53 ± 0.02 in the H group to 0.36 ± 0.02 in the HS group ( P < 0.001). The increased hematocrit in H (0.65 ± 0.02) was prevented by simvastatin treatment (0.51 ± 0.01, P < 0.001). Hematocrit was similar in N versus NS. Alveolar vessel muscularization and medial thickening of vessels 50–200 μM in diameter induced by hypoxia were also significantly attenuated in the HS animals. Lung endothelial nitric oxide synthase (eNOS) protein expression in the HS group was less than H ( P < 0.01) but was similar in N versus NS. We conclude that simvastatin treatment potently attenuates chronic hypoxic pulmonary hypertension and polycythemia in rats and inhibits vascular remodeling. Enhancement of lung eNOS expression does not appear to be involved in mediating this effect.
TLR4 inactivation and rBPI21 block burn-induced myocardial contractile dysfunction
Authors: Thomas James A. , Tsen May F. , White D. Jean , Horton Jureta W.
Published: March 2015

Both large burns and severe gram-negative sepsis are associated with acute myocardial contractile dysfunction. Because others have reported that burn injury may be followed by transient endotoxemia, we hypothesized that bacterial endotoxin induces contractile impairment after burn trauma. We tested this hypothesis in two rodent models. In each model, postburn myocardial contractility was assessed using Langendorff preparations of excised hearts. In the first model, mice expressing either a mutant form of or no Toll-like receptor 4 (TLR4), a critical element of the mammalian endotoxin receptor, were resistant to postburn myocardial contractile dysfunction. In the second model, starting 30 min or 4 h after burn injury, rats were infused with recombinant bactericidal/permeability-increasing protein (rBPI21), a protein that binds and neutralizes endotoxin. Hearts from rBPI21-treated animals were completely protected from postburn contractile impairment. Because burn-induced contractile dysfunction can be prevented either by blocking signaling through the endotoxin receptor or by neutralizing circulating LPS, bacterial endotoxin may contribute to impaired myocardial contractility after burn injury.
Neuroprotection in hypothermia linked to redistribution of oxygen in brain
Authors: Sakoh Masaharu , Gjedde Albert
Published: March 2015

Hypothermia improves the outcome of acute ischemic stroke, traumatic injury, and inflammation of brain tissue. We tested the hypothesis that hypothermia reduces the energy metabolism of brain tissue to a level that is commensurate with the prevailing blood flow and hence allows adequate distribution of oxygen to the entire tissue. To determine the effect of 32°C hypothermia on brain tissue, we measured the sequential changes of physiological variables by means of PET in pigs. Cerebral blood flow and oxygen consumption (cerebral metabolic rate of oxygen) declined to 50% of the baseline in 3 and 5 h, respectively, thus elevating the oxygen extraction fraction to 140% of the baseline at 3 h. The results are consistent with the claim that cooling of the brain to 32°C couples both energy metabolism and blood flow to a lower rate of work of the entire tissue.
Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism
Authors: Norman M. Ursula , Reeve Shane B. , Dive Vincent , Smith A. Ian , Lew Rebecca A.
Published: March 2015

The closely related metalloendopeptidases EC 3.4.24.15 (EP24.15; thimet oligopeptidase) and 24.16 (EP24.16; neurolysin) cleave a number of vasoactive peptides such as bradykinin and neurotensin in vitro. We have previously shown that hypotensive responses to bradykinin are potentiated by an inhibitor of EP24.15 and EP24.16 (26), suggesting a role for one or both enzymes in bradykinin metabolism in vivo. In this study, we have used selective inhibitors that can distinguish between EP24.15 and EP24.16 to determine their activity in cultured endothelial cells (the transformed human umbilical vein endothelial hybrid cell line EA.hy926 or ovine aortic endothelial cells). Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-d-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation.
Do angiotensin-converting enzyme inhibitors directly stimulate the kinin B1receptor?
Authors: Fortin Jean-Philippe , Gobeil Fernand , Adam Albert , Regoli Domenico , Marceau François
Published: March 2015

It has been recently claimed that the human B1receptors for kinins bind angiotensin-converting enzyme (ACE) inhibitors via a potential zinc-binding domain and are pharmacologically stimulated by these drugs. We verified whether ACE inhibitors stimulate B1receptors in vitro. The isolated rabbit aorta or mouse stomach responded by negligible contractions to the application of captopril, enalaprilat, or zofenoprilat. The human isolated umbilical vein also failed to respond to enalaprilat. All of these preparations were responsive to the B1receptor agonists des-Arg9-bradykinin (BK) or Lys-des-Arg9-BK. Furthermore, enalaprilat applied continuously had no significant interaction with the effects of Lys-des-Arg9-BK on the rabbit aorta. Enalaprilat failed to stimulate [3H]arachidonate release, translocate the receptors (confocal microscopy), or stimulate ERK1/2 phosphorylation (immunoblot) in HEK-293 cells stably expressing the rabbit B1receptor conjugated to yellow fluorescent protein. The phospho-ERK1/2 content of arterial smooth muscle cells of human or rabbit origin was increased by treatment with Lys-des-Arg9-BK but not with enalaprilat. ACE inhibitors do not act as bona fide agonists of the kinin B1receptors.
Dobutamine potentiates arterial chemoreflex sensitivity in healthy normal humans
Authors: Velez-Roa Sonia , Kojonazarov Baktybek , Ciarka Agnieszka , Godart Pascal , Naeije Robert , Somers Virend K. , van de Borne Philippe
Published: March 2015

β-Adrenergic agonists may increase chemosensitivity in humans. We tested the hypothesis that the β1-agonist dobutamine increases peripheral chemosensitivity in a double-blind placebo-controlled randomized and crossover study. In 15 healthy subjects, we examined the effects of dobutamine on breathing, hemodynamics, and sympathetic nerve activity (measured using microneurography) during normoxia, isocapnic hypoxia (10% O2), posthypoxic maximal voluntary end-expiratory apnea, hyperoxic hypercapnia, and cold pressor test (CPT). Dobutamine increased ventilation (7.5 ± 0.3 vs. 6.7 ± 0.2 l/min, P = 0.0004) during normoxia, markedly enhanced the ventilatory (16.1 ± 1.6 vs. 11.4 ± 0.7 l/min, P < 0.0001) and sympathetic (+403 ± 94 vs. +222 ± 5%, P < 0.03) responses at the fifth minute of isocapnic hypoxia, and enhanced the sympathetic response to the apnea performed after hypoxia (+501 ± 107% vs. +291 ± 38%, P < 0.05). No differences were observed between dobutamine and placebo on the responses to hyperoxic hypercapnia and CPT. Dobutamine increases ventilation during normoxia and potentiates the ventilatory and sympathetic responses to hypoxia in healthy subjects. Dobutamine does not affect the responses to hyperoxic hypercapnia and CPT. We conclude that dobutamine enhances peripheral chemosensitivity.
The mechanism of excitation-contraction coupling in phenylephrine-stimulated human saphenous vein
Authors: Crowley Christine M. , Lee Cheng-Han , Gin Stephanie A. , Keep Albert M. , Cook Richard C. , van Breemen Cornelis
Published: March 2015

The human saphenous vein (HSV) is the most widely used graft in coronary artery revascularization procedures and is susceptible to spasm perioperatively. The aim of this study is to elucidate the mechanism(s) of agonist-induced excitation-contraction coupling in this vessel. Isometric contraction experiments were combined with in situ smooth muscle intracellular Ca2+ concentration ([Ca2+]i) imaging by confocal microscopy of intact undistended HSV segments during activation with phenylephrine (PE; 50 μM). Stimulation with PE produced a sustained contraction. Preincubation with 5 μM nifedipine, a blocker of the L-type voltage-operated Ca2+ channel, or 50 μM SKF-96365, a blocker of both the voltage- and receptor-operated channels, reduced force generation by 25–30%. Ca2+ imaging revealed that PE elicited only a transient rise in [Ca2+]i, suggesting that Ca2+plays only a minor role. However, a requirement for basal Ca2+ levels was demonstrated when PE contractions could not be maintained in Ca2+-free medium. In light of the transient Ca2+ response, it appears that signals other than Ca2+ must maintain the tonic contraction elicited by PE, such as those that sensitize the myofilaments to Ca2+. Application of HA-1077 (a Rho kinase inhibitor) at the peak of the contraction completely abolished the plateau phase of the response, whereas application of genistein (a tyrosine kinase inhibitor) reduced this phase by ∼50%. The foregoing results suggest that, whereas the transient Ca2+ signal can contribute to the development of force, maintenance of the plateau phase of the PE contraction in the HSV is the result of myofilament Ca2+ sensitization by Rho kinase and tyrosine phosphorylation. The elucidation of the mechanisms of excitation-contraction coupling in the HSV may be useful for the development of therapeutic strategies for the alleviation of vein graft spasm.
Suppression of cortical functional hyperemia to vibrissal stimulation in the rat by epoxygenase inhibitors
Authors: Peng Xinqi , Carhuapoma Juan R. , Bhardwaj Anish , Alkayed Nabil J. , Falck John R. , Harder David R. , Traystman Richard J. , Koehler Raymond C.
Published: March 2015

Application of glutamate to glial cell cultures stimulates the formation and release of epoxyeicosatrienoic acids (EETs) from arachidonic acid by cytochome P-450 epoxygenases. Epoxygenase inhibitors reduce the cerebral vasodilator response to glutamate and N-methyl-d-aspartate. We tested the hypothesis that epoxygenase inhibitors reduce the somatosensory cortical blood flow response to whisker activation. In chloralose-anesthetized rats, percent changes in cortical perfusion over whisker barrel cortex were measured by laser-Doppler flowmetry during whisker stimulation. Two pharmacologically distinct inhibitors were superfused subdurally: 1) N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), an epoxygenase substrate inhibitor; and 2) miconazole, a reversible cytochrome P-450 inhibitor acting on the heme moiety. Superfusion with 5 μmol/l MS-PPOH decreased the hyperemic response to whisker stimulation by 28% (from 25 ± 9 to 18 ± 7%, means ± SD, n = 8). With 20 μmol/l MS-PPOH superfusion, the response was decreased by 69% (from 28 ± 9% to 9 ± 4%, n = 8). Superfusion with 20 μmol/l miconazole decreased the flow response by 67% (from 31 ± 6% to 10 ± 3%, n = 8). Subsequent superfusion with vehicle restored the response to 26 ± 11%. Indomethacin did not prevent MS-PPOH inhibition of the flow response, suggesting that EET-related vasodilation was not dependent solely on cyclooxygenase metabolism of 5,6-EET. Neither MS-PPOH nor miconazole changed baseline flow, reduced the blood flow response to an adenosine A2agonist, or decreased somatosensory evoked potentials. The marked reduction of the cortical flow response to whisker stimulation with two different types of epoxygenase inhibitors indicates that EETs play an important role in the physiological coupling of blood flow to neural activation.
Heme oxygenase-mediated vasodilation involves vascular smooth muscle cell hyperpolarization
Authors: Naik Jay S. , Walker Benjimen R.
Published: March 2015

Chronic hypoxia is associated with both blunted agonist-induced and myogenic vascular reactivity and is possibly due to an enhanced production of heme oxygenase (HO)-derived carbon monoxide (CO). However, the mechanism of endogenous CO-meditated vasodilation remains unclear. Isolated pressurized mesenteric arterioles from chronically hypoxic rats were administered the HO substrate heme-l-lysinate (HLL) in the presence or absence of iberiotoxin, 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), ryanodine, or free radical spin traps ( N- tert-butyl-α-phenylnitrone and 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt). The effects of HLL administration on vascular smooth muscle (VSM) membrane potential were assessed in superior mesenteric artery strips in the presence and absence of zinc protoporphyrin IX or iberiotoxin. The vasodilatory responses to exogenous CO were assessed in the presence and absence of ODQ or iberiotoxin. HLL administration produced a dose-dependent vasodilatory response that was nearly eliminated in the presence of iberiotoxin. Neither ODQ, spin traps, nor ryanodine altered the vasodilatory response to HLL, although ODQ abolished the vasodilatory response to S-nitroso- N-acetyl-penicillamine. HLL administration produced a zinc protoporphyrin IX- and iberiotoxin-sensitive VSM cell hyperpolarization. Iberiotoxin and ODQ inhibited the vasodilatory response to exogenous CO. Thus the vasodilatory response to endogenous CO involves cGMP-independent activation of VSM large-conductance Ca2+-activated K+ channels and does not likely involve the formation of Ca2+ sparks emanating from ryanodine-sensitive stores.
Effect of a high-salt diet on oxidant enzyme activity in skeletal muscle microcirculation
Authors: Lenda Deborah M. , Boegehold Matthew A.
Published: March 2015

Increased salt intake attenuates the endothelium-dependent dilation of skeletal muscle arterioles by abolishing local nitric oxide (NO) activity. There is evidence of oxidative stress in arteriolar and venular walls of rats fed a high-salt diet, and depressed arteriolar responses to acetylcholine (ACh) in these rats are reversed by scavengers of reactive oxygen species (ROS). In this study, we tested the hypothesis that this salt-dependent increase in microvascular ROS and the resulting attenuation of endothelium-dependent dilation are due to increased expression and/or activity of oxidant enzymes in the microvascular wall. Resting arteriolar and venular wall oxidant activity, as assessed by tetranitroblue tetrazolium reduction, was consistently higher in the spinotrapezius muscle of rats fed a high-salt diet (7% NaCl, HS) for 4–5 wk than in those fed a normal diet (0.45% NaCl, NS) for the same duration. Western analysis of protein from isolated microvessels showed no difference between HS and NS rats in the expression of NAD(P)H oxidase or xanthine oxidase. Inhibition of NAD(P)H oxidase and/or xanthine oxidase with diphenyleneiodonium chloride and oxypurinol, respectively, reduced resting arteriolar wall oxidant activity to normal levels in HS rats but had no effect in NS rats, suggesting that the basal activities of NAD(P)H oxidase and xanthine oxidase are increased in HS microvessels. However, inhibition of these enzymes in HS rats did not restore normal arteriolar responses to ACh, suggesting that this stimulus activates an alternate source of ROS that eliminates the role of NO in the subsequent dilation.
Morphological and functional changes in cardiac myocytes isolated from mice overexpressing TNF-α
Authors: Janczewski Andrzej M. , Kadokami Toshiaki , Lemster Bonnie , Frye Carole S. , McTiernan Charles F. , Feldman Arthur M.
Published: March 2015

Transgenic (TG) TNF1.6 mice, which cardiac specifically overexpress tumor necrosis factor-α (TNF-α), exhibit heart failure (HF) and increased mortality, which is markedly higher in young (<20 wk) males (TG-M) than females (TG-F). HF in this model may be partly caused by remodeling of the extracellular matrix and/or structure/function alterations at the single myocyte level. We studied left ventricular (LV) structure and function using echocardiography and LV myocyte morphometry, contractile function, and intracellular Ca2+ (Ca[Formula: see text]) handling using cell edge detection and fura 2 fluorescence, respectively, in 12-wk-old TG-M and TG-F mice and their wild-type (WT) littermates. TG-F mice showed LV hypertrophy without dilatation and only a small reduction of basal fractional shortening (FS) and response to isoproterenol (Iso). TG-M mice showed a large LV dilatation, higher mRNA levels of β-myosin heavy chain and atrial natriuretic factor versus TG-F mice, reduced FS relative to both WT and TG-F mice, and minimal response to Iso. TG-F and TG-M myocytes were similarly elongated (by ≈20%). The amplitude of Ca[Formula: see text] transients and contractions and the response to Iso were comparable in WT and TG-F myocytes, whereas the time to 50% decline (TD50%) of the Ca[Formula: see text]transient, an index of the rate of sarcoplasmic reticulum Ca2+ uptake, was prolonged in TG-F myocytes. In TG-M myocytes, the amplitudes of Ca[Formula: see text] transients and contractions were reduced, TD50% of the Ca[Formula: see text] transient was prolonged, and the inotropic effect of Iso on Ca[Formula: see text] transients was reduced approximately twofold versus WT myocytes. Protein expression of sarco(endo)plasmic reticulum Ca2+-ATPase 2 and phospholamban was unaltered in TG versus WT hearts, suggesting functional origins of impaired Ca2+ handling in the former. These results indicate that cardiac-specific overexpression of TNF-α induces myocyte hypertrophy and gender-dependent alterations in Ca[Formula: see text] handling and contractile function, which may at least partly account for changes in LV geometry and in vivo cardiac function in this model.
Side population cells and Bcrp1 expression in lung
Authors: Summer Ross , Kotton Darrell N. , Sun Xi , Ma Bei , Fitzsimmons Kathleen , Fine Alan
Published: March 2015

Side population (SP) cells are a rare subset of cells found in various tissues that are highly enriched for stem cell activity. SP cells can be isolated by dual-wavelength flow cytometry because of their capacity to efflux Hoechst dye, a process mediated by the ATP-binding cassette transporter breast cancer resistance protein (Bcrp) 1. By performing flow cytometry of enzymedigested mouse lung stained with Hoechst dye, we found that SP cells comprise 0.03–0.07% of total lung cells and are evenly distributed in proximal and distal lung regions. By RT-PCR, we found that lung SP cells express hepatocyte nuclear factor-3β, but not thyroid transcription factor-1. Surface marker analysis revealed lung SP cells to be stem cell antigen 1 positive, Bcrp1 positive, lineage marker negative, and heterogeneous at the CD45 locus. As expected, we did not detect lung SP cells in Bcrp1-deficient animals. We, therefore, employed nonisotopic in situ hybridization and immunostaining for Bcrp1 as a strategy to localize these cells in vivo. Expression was observed in distinct lung cell types: bronchial and vascular smooth muscle cells and round cells within the distal air space. We confirmed the expression of Bcrp1 in primary bronchial smooth muscle cell cultures (BSMC) and in lavaged distal airway cells, but neither possessed the capacity to efflux Hoechst dye. In BSMC, Bcrp1 was localized to an intracellular compartment, suggesting that the molecular site of Bcrp1 expression regulates SP phenotype.
Oxidative stress and inflammation contribute to lung toxicity after a common breast cancer chemotherapy regimen
Authors: Abushamaa Amir M. , Sporn Thomas A. , Folz Rodney J.
Published: March 2015

Delayed pulmonary toxicity syndrome after high-dose chemotherapy (HDC) and autologous hematopoietic support occurs in up to 64% of women with advanced-stage breast cancer. Using a similar, but nonmyeloablative, HDC treatment regimen in mice, we found both immediate and persistent lung injury, coincident with marked decreases in lung tissue glutathione reductase activity and accompanied by increases in lung oxidized glutathione, bronchoalveolar lavage (BAL) lipid peroxidation, and BAL total cell counts. Most interestingly, at 6 wk, BAL total cell counts had increased fourfold, with lymphocyte cell counts increasing >11-fold. A single supplemental dose of glutathione prevented early lung injury at 48 h but showed no lung-protective effects at 6 wk, whereas single doses of other thiol-sparing agents (Ethyol and glutathione monoethyl ester) showed no benefit. These data suggest that this HDC regimen results in acute and persistent lung toxicity, induced in part by oxidative stress, that culminates with an acute lung cellular inflammatory response. Continuous glutathione supplementation and/or attenuation of the delayed pulmonary inflammatory response may prove beneficial in preventing lung toxicity after the use of these chemotherapeutic agents.
Prenatal hypoxia decreases lung extracellular superoxide dismutase expression and activity
Authors: Giles Brenda-Louise , Suliman Hagir , Mamo Lisa B. , Piantadosi Claude A. , Oury Tim D. , Nozik-Grayck Eva
Published: March 2015

Extracellular superoxide dismutase (EC-SOD), which scavenges extracellular superoxide (O[Formula: see text]·), is highly regulated in the developing lung. In the prenatal rabbit, EC-SOD is predominantly intracellular and inactive, and postnatally, active EC-SOD is secreted. We hypothesized that prenatal hypoxia would delay the normal postnatal secretion of active EC-SOD in the lung. Pregnant New Zealand White rabbits were exposed to hypobaric hypoxia (15,000 ft × 36 h) to alter fetal O2tension or were maintained in room air. Lungs were harvested from preterm (28 days), term (30 ± 1 day), and 1-wk-old kits. After prenatal hypobaric hypoxia, EC-SOD mRNA expression was significantly decreased in lungs of full-term kits, whereas EC-SOD protein decreased at all ages. Immunohistochemical staining for EC-SOD showed that hypoxia delayed secretion of the isoenzyme in the airways and pulmonary vasculature. Furthermore, pulmonary EC-SOD enzyme activity was significantly decreased in the 1-wk-old kits exposed to prenatal hypoxia. We conclude that prenatal hypoxia downregulates EC-SOD expression at both the transcriptional and posttranslational levels. Furthermore, prenatal hypoxia delays secretion of active EC-SOD enzyme. These findings have important implications for the effects of prenatal asphyxia on postnatal response to oxidant stress.