bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Intracellular Angiotensin II in Vascular Smooth Muscle Cells
Authors: Haller Hermann , Lindschau Carsten , Erdmann Bettina , Quass Petra , Luft Friedrich C.
Published: June 2012

Angiotensin (Ang) II is present inside vascular smooth muscle cells (VSMCs); however, its intracellular functions, if any, are unknown. We tested the hypothesis that intracellular Ang II exerts effects on cytosolic Ca
2+
([Ca
2+
]
i
) in VSMCs. Ang II was administered via microinjection. Intracellular Ang II localization was demonstrated by fluorescein-labeled Ang II and electron microscopy. [Ca
2+
]
i
was monitored by confocal microscopy with fluo 3. Ang II was identified in endosomes and in the nucleus by both localizing techniques. Microinjection of Ang II (10
−10
mol/L) led to a rapid increase in [Ca
2+
]
i
in the cytosol and in the nucleus. The [Ca
2+
]
i
increase was due to the influx of extracellular Ca
2+
ions. The intracellular Ang II effect was totally inhibited by the concomitant injection of the Ang II antagonist CV-11947. Desensitization of extracellular Ang II receptors, on the other hand, did not influence the intracellular effects, nor did extracellular CV-11947. The increase in [Ca
2+
]
i
was observed not only in the microinjected cell but also in directly adjacent VSMCs. In contrast to the microinjected cells, the [Ca
2+
]
i
increase in the adjacent cells was mostly due to release from intracellular stores. Pretreatment with thapsigargin abolished the Ang II response in adjacent cells. Microinjection of inositol tris-phosphate induced a [Ca
2+
]
i
response in adjacent cells that was similar to the Ang II–induced effects. Preincubation of VSMCs with the uncoupling substances dimethyl sulfoxide and heptanol did not decrease the Ang II response but instead prevented a [Ca
2+
]
i
surge in adjacent cells. We conclude that intracellular Ang II binds to intracellular Ang II receptors and elicits an increased [Ca
2+
]
i
in the injected cell and, thereafter, cells in the immediate neighborhood. Cell-cell contact is necessary for the Ang II–mediated effects. The data suggest that intracellular Ang II may stimulate a cluster of VSMCs from a single cell via the release of second messengers.
Oxygen-Derived Free Radicals Contribute to Baroreceptor Dysfunction in Atherosclerotic Rabbits
Authors: Li Zhi , Mao Hui Z. , Abboud Francois M. , Chapleau Mark W.
Published: June 2012

The goal of the present study was to determine whether oxygen-derived free radicals contribute to baroreceptor dysfunction in atherosclerosis. Baroreceptor activity was measured from the carotid sinus nerve during pressure ramps in isolated carotid sinuses of anesthetized rabbits. Rabbits fed a 0.5% to 1.0% cholesterol diet for 7.9±0.4 months (mean±SE; range, 5.5 to 10) developed atherosclerotic lesions in the carotid sinuses. Maximum baroreceptor activity measured at 140 mm Hg and the slope of the pressure-activity curve were reduced in atherosclerotic (n=15) compared with normal (n=13) rabbits (425±34 versus 721±30 spikes per second and 6.2±0.6 versus 10.8±0.8 spikes per second per mm Hg, respectively,
P
<.05). The level of activity was inversely related to plasma cholesterol concentration (
r
=.86,
P
<.001) and total cholesterol load (plasma concentration×duration of diet,
r
=.92). Mean arterial pressure was normal in both groups. Exposure of the carotid sinus to the free-radical scavengers superoxide dismutase (SOD) and catalase significantly increased maximum baroreceptor activity by 25±4% in atherosclerotic rabbits (n=6) but caused only small and irreversible changes in activity in normal rabbits (n=8). Catalase alone but not SOD also increased baroreceptor activity in atherosclerotic rabbits (n=7). Exposure of the carotid sinus of normal rabbits to exogenous free radicals generated from the reaction between xanthine and xanthine oxidase inhibited baroreceptor activity in a dose-dependent and reversible manner (n=8,
P
<.05). The inhibition of activity was attenuated by SOD and catalase but was not attenuated by the inhibitor of hydroxyl radical formation, deferoxamine. Neither restoration of baroreceptor activity in atherosclerotic rabbits by catalase nor inhibition of activity by xanthine/xanthine oxidase could be explained by changes in the carotid pressure-diameter relation or prostacyclin formation. These results indicate that oxidant stress inhibits baroreceptor activity and that endogenous oxyradicals produced in atherosclerotic carotid sinuses contribute to baroreceptor dysfunction.
Function and Production of Nitric Oxide in the Coronary Circulation of the Conscious Dog During Exercise
Authors: Bernstein Robert D. , Ochoa Francisca Y. , Xu Xaobin , Forfia Paul , Shen Weiqun , Thompson Carl I. , Hintze Thomas H.
Published: June 2012

This study determined the changes in NO production from the coronary circulation of the conscious dog during exercise. The role of endogenous NO as it relates to coronary flow, myocardial work, and metabolism was also studied. Mongrel dogs were chronically instrumented for measurements of coronary blood flow (CBF), ventricular and aortic pressures, and ventricular diameter, with catheters in the aorta and coronary sinus. Acute exercise (5 minutes at 3.6, 5.9, and 9.1 mph) was performed, and hemodynamic measurements and blood samples were taken at each exercise level. Nitro-
l
-arginine (NLA, 35 mg/kg IV) was given to block NO synthesis, and the exercise was repeated. Blood samples were analyzed for oxygen, plasma nitrate/nitrite (an index of NO), lactate, glucose, and free fatty acid (FFA) levels. Acute exercise caused significant elevations in NO production by the coronary circulation (46±23, 129±44, and 63±32 nmol/min at each speed respectively,
P
<.05). After NLA, there was no measurable NO production at rest or during exercise. Blockade of NO synthesis resulted in elevations in myocardial oxygen consumption and reductions in myocardial FFA consumption for comparable levels of CBF and cardiac work. The metabolic changes after NLA occurred in the absence of alterations in myocardial lactate or glucose consumptions. NO production by the coronary circulation is increased with exercise and blocked by NLA. The absence of NO in the coronary circulation during exercise does not affect levels of CBF, because it shifts the relationship between cardiac work and myocardial oxygen consumption, suggesting that endogenous NO modulates myocardial metabolism.
Ca
2+
Waves in Lung Capillary Endothelium
Authors: Ying Xiaoyou , Minamiya Yoshihiro , Fu Chenzhong , Bhattacharya Jahar
Published: June 2012

Although cytosolic Ca
2+
importantly regulates organ function, lung microvascular [Ca
2+
]
i
regulation remains poorly understood because of the lack of direct in situ quantification. In the present study, we report the first endothelial [Ca
2+
]
i
quantification by the fura 2 method in microscopically imaged venular capillaries of the isolated blood-perfused rat lung. Sequential images indicated the presence of intercellular Ca
2+
waves that spontaneously originated from pacemaker endothelial cells and then spread for short distances along the capillary wall, inducing synchronous endothelial [Ca
2+
]
i
oscillations. Fast Fourier analyses of the oscillations revealed a dominant wave component with an amplitude of 37 nmol/L, frequency of 0.4 min
−1
, and velocity of 5 μm/s. The intracellular Ca
2+
wave was unaffected by blood flow stoppage or by infusions of Ca
2+
-containing or Ca
2+
-free dextran. Inhibition of the wave by thapsigargin in Ca
2+
-free dextran and by the gap junction uncoupler, heptanol, indicated that it was generated by endosomal Ca
2+
release in the pacemaker cell and was propagated by gap junctional communication. In the presence of histamine, enhancement of the wave accounted for a significant component of the coordinated [Ca
2+
]
i
increase in the capillary segment. No intercellular Ca
2+
waves were evident in adjoining alveolar epithelial cells. Our findings indicate a novel mechanism of [Ca
2+
]
i
regulation in the lung capillary under both resting and stimulated conditions. Pacemaker-induced Ca
2+
waves, generated intracellularly by unknown initiating mechanisms, communicated to adjoining cells to determine [Ca
2+
]
i
profiles in short interbranch segments of capillary walls.
Molecular Cloning and Characterization of Human Cardiac Homeobox Gene
CSX1
Authors: Shiojima Ichiro , Komuro Issei , Mizuno Takehiko , Aikawa Ryuichi , Akazawa Hiroshi , Oka Toru , Yamazaki Tsutomu , Yazaki Yoshio
Published: June 2012

Accumulating evidence has suggested that homeodomain-containing proteins play critical roles in regulating the tissue-specific gene expression essential for tissue differentiation and in determining the temporal and spatial patterns of development. In order to elucidate the mechanisms of human heart development, we have isolated a human homologue of the murine cardiac homeobox gene
Csx
(also called
Nkx-2.5)
and denoted it as
CSX1.
The amino acid sequence of the
CSX1
homeodomain is 100% and 67% identical to that of murine
Csx/Nkx-2.5
and
Drosophila tinman,
respectively.
CSX1
has at least three isoforms generated by an alternative splicing mechanism. One of these isoforms (
CSX1a
) encodes a protein of ≈35 kD that possesses the homeodomain, whereas the other two (
CSX1b
and
CSX1c
) encode a truncated protein of ≈12 kD that is identical to the
CSX1a
protein at the amino-terminal 112 amino acids but lacks the homeodomain. Northern blot analysis showed that
CSX1
transcripts are abundantly expressed in both fetal and adult hearts, but no signal was detected in other human tissues examined. Amplification of each isoform by reverse transcriptase–polymerase chain reaction revealed that all of the three isoforms are expressed in fetal and adult hearts and that the homeobox-containing isoform
CSX1a
is most abundant. The homeodomain-containing protein encoded by
CSX1a
binds to
Csx/Nkx-2.5
binding sequences and transactivates the sequence-containing luciferase reporter gene. Unexpectedly, the homeodomain-lacking protein encoded by
CSX1b
also transactivates the reporter gene, although
CSX1b
does not bind to the
Csx
/
Nkx-2.5
binding sequences. The highly conserved homeodomain sequence in evolution and the restricted expression in the heart suggest that
CSX1
plays an important role in the development and differentiation of the human heart and that there may be two different mechanisms in transcriptional regulation by the
CSX1
protein, homeodomain-dependent and -independent mechanisms.
Insulin Signaling and Its Regulation of System A Amino Acid Uptake in Cultured Rat Vascular Smooth Muscle Cells
Authors: Obata Toshiyuki , Kashiwagi Atsunori , Maegawa Hiroshi , Nishio Yoshihiko , Ugi Satoshi , Hidaka Hideki , Kikkawa Ryuichi
Published: June 2012

Hyperinsulinemia has been recognized as an independent risk factor for atherosclerosis. However, its exact mechanisms are still unclear. In our previous work, we showed that 10 nmol/L insulin stimulated neither mitogen-activated protein kinase (MAP kinase) activity nor [
3
H]thymidine incorporation but did stimulate S6 kinase through the specific insulin receptors in cultured rat vascular smooth muscle cells (VSMCs). In this study, we observed that ≥1 nmol/L insulin stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and activated IRS-1–dependent phosphatidylinositol 3′-kinase (PI 3′-kinase) and p70 S6 kinase (p70
S6K
) but not MAP kinase (extracellular signal-regulated kinase 2) and p90 S6 kinase (p90
RSK
). However, 10 nmol/L insulin-like growth factor I stimulated all these pathways. Finally, 10 nmol/L insulin stimulated α-aminoisobutyric acid (AIB) uptake, and wortmannin (100 nmol/L) completely inhibited insulin-stimulated AIB uptake, whereas rapamycin (20 nmol/L) had no such effect. Furthermore, cycloheximide (10 μg/mL) completely inhibited insulin-stimulated AIB uptake, but actinomycin D (5 μg/mL) failed to inhibit this. Thus, we reached the following conclusions: (1) Insulin (1 nmol/L) induced phosphorylation of IRS-1 and activated the PI 3′-kinase and p70
S6K
pathways in VSMCs, even though 10 nmol/L insulin did not significantly stimulate MAP kinase or p90
RSK
. (2) Stimulation of AIB uptake by insulin was regulated at the translational level via wortmannin-sensitive pathways but not p70
S6K
pathways.
Enhancer Stimulation Unmasks Latent Gene Transfer After Adenovirus-Mediated Gene Delivery Into Human Vascular Smooth Muscle Cells
Authors: Clesham Gerald J. , Browne Helena , Efstathiou Stacey , Weissberg Peter L.
Published: June 2012

Recombinant adenoviral vectors are being used increasingly for gene transfer studies in mammalian cells and gene therapy protocols in humans. High adenoviral titers are often required for successful transduction of vascular smooth muscle cells (VSMCs), defined as uptake and detectable expression of the foreign gene, but the relative contributions of efficiency of viral uptake and control of transcription are poorly understood. To explore the extent to which a lack of detectable gene expression may be due to inefficient transcription of a successfully transferred gene, we have used a replication-deficient adenovirus expressing β-galactosidase (RAd35β-Gal), under the control of the human cytomegalovirus major immediate-early promoter (CMV-IEP), which contains cAMP and nuclear factor-κB response elements, to investigate constitutive and inducible gene expression after gene transfer into human VSMCs. Histochemical staining with 5-bromo-4-chloro-3-indolyl β-
d
-galactopyranoside (X-gal), a quantitative spectrophotometric assay, SDS-PAGE, Western blotting, and Northern analysis were used to evaluate β−galactosidase expression in infected cells. After infection with RAd35β-Gal at 30, 100, and 1000 plaque-forming units per cell (pfu/cell), expression of β-galactosidase was augmented up to 17-, 19-, and 23-fold, respectively, in human VSMCs treated with forskolin and phorbol ester compared with unstimulated cells. After infection, the proportion of detectably transduced cells was increased by enhancer stimulation from 58% to 100% at 100 pfu/cell and from 9% to 62% at 10 pfu/cell, indicating quiescent viral DNA in unstimulated cells. At high adenoviral titers (1000 pfu/cell), the recombinant gene became the most abundant protein in cell extracts. These findings demonstrate that in human VSMCs, limited constitutive expression from the CMV-IEP, rather than failure of translocation of adenoviral DNA, may be responsible for the apparent failure of transduction at a low multiplicity of infection.
Measurement of Flow in Single Blood Vessels Including Cardiac Output by Local Thermodilution
Authors: FRONĚ A. , GANZ V.
Published: June 2012

On the basis of local thermodilution a method for measuring blood flow in a single vessel was elaborated. The measurement was accomplished by a special catheter with an injection orifice and a thermistor. The accuracy of the method was studied both in model and in vivo experiments on dogs (in jugular vein, in carotid and pulmonary artery) and good correlation was found with direct methods.
Observations on the Circulation of Domestic Cattle
Authors: DOYLE JOSEPH T. , PATTERSON JOHN L. , WARREN JAMES V. , DETWEILER DAVID K.
Published: June 2012

Comparative physiology has much to contribute to our understanding of the circulation. For this purpose large animals until recently have been neglected as subjects of circulatory studies by modern technics, yet nature's means of solving the circulatory problems which these animals present are of great interest and, in case of domestic cattle (Bos taurus), of obvious practical importance. Intravascular and intracardiac pressures were recorded, and cardiac output and related functions were measured, or calculated, and related to body size. The effects of environmental stimuli and of phonation ("moo") were observed. Interesting differences between the findings in the cow and those in normal man and in the giraffe are made the subject of comment.
Frequency Analysis of the Electrocardiogram
Authors: SCHER ALLEN M. , YOUNG ALLAN C.
Published: June 2012

The frequency analysis of the electrocardiogram reveals that contributions by frequencies of 100 cycles, or higher, per second are less than 10 per cent of the amplitude of the fundamental of the QRS comples, and it thus appears that transistor-driven amplifier-pen recorders presently available are adequate to record all the information contained in the electrocardiogram, if the standard paper speed is doubled, or quadrupled.
Effect of Liver Damage on Experimental Renal Hypertension in the Rat
Authors: LOYKE H. F. , PLUCINSKY J. J. , CRAWFORD T. L.
Published: June 2012

Renal hypertension was produced in 26 test rats and fell to normal levels when they received CCl
4
. Our data indicate that this is due to liver damage and not to general inanition. These changes are reversible, since cessation of CCl
4
injections led to blood pressure rise. Morphologically, only a moderate degree of fatty metamorphosis is necessary to cause the blood pressure to fall from hypertensive levels to normotension. The biochemical change involved variable changes in albumin and globulin.
Influence of Coronary Artery Pressure Upon Myocardial Elasticity
Authors: SALISBURY PETER F. , CROSS CECIL E. , RIEBEN P. ANDRE
Published: June 2012

The marked intensification of experimental left ventricular failure by veno-arterial pumping which was seen in earlier experiments suggested changes of myocardial elasticity as a mechanism. Two experimental procedures were therefore applied here, in which the pressure in the coronary arteries and veins could be varied at will, where the left ventricle was distended by an air-filled balloon, and where the coronary tree did not communicate with the left ventricle. Changes of the coronary arterial or venous pressures were accompanied by homodirectional changes of the left ventricular diastolic pressure which were of large magnitude and which could not be explained by unobserved blood flow into the left ventricle or by other factors.

The inverse relationship between coronary vascular pressures and myocardial distensibility was probably caused by the increasing volume of blood which was retained in the coronary arteries and veins when the coronary arterial or venous pressures were increased. This passive increase in coronary blood volume (turgor) must have changed the resiliency of the coronary tree. The changed elastic properties of the coronary tree then resulted in a change of the elastic properties of the heart.
Hemodynamic Effects of a Pressor Amine (Methoxamine) with Predominant Vasoconstrictor Activity
Authors: BREWSTER WILLIAM R. , OSGOOD PATRICIA F. , ISAACS JAMES P. , GOLDBERG LEON I. , W Chock Raymond T.
Published: June 2012

A pressor amine (methoxamine) which produces peripheral vasoconstriction, while lacking a significant, direct, positive inotropic effect upon ventricular heart muscle, has been shown to produce hemodynamic effects that are in distinct contrast to those resulting from the injection or infusion of
l
-epinephrine and
l
-norepinephrine in "physiologic" concentrations or of mephentermine sulfate. The latter 3 amines, possessing a positive inotropic action in addition to varying vasoconstrictor effects, decrease right and left atrial mean pressures while increasing femoral and pulmonary arterial pulse and mean pressures, right and left ventricular stroke works and the cardiac index.

Methoxamine injection or infusion, in contrast, resulted in linear increases in the calculated total peripheral resistance and left and right atrial mean pressures. There was a simultaneous rise in right and left ventricular stroke works which were maintained until a maximum rise in the mean arterial pressure was reached. The average stroke volume indices were maintained at near control values in both blocked and unblocked animals. The cardiac index of the normal unblocked dogs decreased in proportion to the decrease in heart rate.

Following the peak pressor response to methoxamine, a progressive fall in mean femoral arterial pressure occurred resulting from ventricular failure rather than "tachyphylaxis" to the vasoconstrictor activity of methoxamine. This decrease in systemic arterial pressure was accompanied by: (1) a further rise in calculated total peripheral resistance and in right and left atrial mean pressures, and (2) a fall in right and left ventricular stroke work and stroke volume indices and in the cardiac indices.
Maturation of Small Pulmonary Arteries in Puppies
Authors: PHILLIPS CLAY E. , DEWEESE JAMES A. , MANNING JAMES A. , MAHONEY EARLE B.
Published: June 2012

The histologic pulmonary vascular changes and the pulmonary arterial pressure changes which occur during the first 13 weeks of life have been studied in mongrel puppies. The histologic changes were based on measurements of the average lumen to wall ratios of arterial vessels 50 to 200 µ in diameter. The pulmonary arterial pressure changes were recorded by direct needle puncture of the main pulmonary artery in anesthetized thoracotomized animals. The average L/W ratios of all neonatal mongrel puppies from separate litters were less than 4.0, the boundary chosen to distinguish neonatal and adult pulmonary arterial vasculature. All puppies younger than 1 month had average L/W ratios below 4.0. Although average mean pulmonary arterial pressures were found to be near adult levels by age 1 week, the ratio of pulmonary to systemic arterial pressure, expressed as a percentage, did not consistently approach adult levels until 1 month of age. The dimensional evolution of small pulmonary arteries and the gradual decline in the ratio of pulmonary to systemic pressure toward near adult levels at 4 weeks suggested a cause-and-effect relationship between these 2 phenomena. Major operative procedures have been performed on puppies 3 to 5 weeks of age with survival. It is proposed that experimental pulmonary hypertension produced in 4-week-old mongrel puppies would closely approximate the situation of a high pressure congenital cardiac shunt in humans without excessive operative mortality.
Simulated Ischemia Increases the Susceptibility of Rat Cardiomyocytes to Hypercontracture
Authors: Ladilov Y.V. , Siegmund B. , Balser C. , Piper H.M.
Published: June 2012

The hypothesis that rat cardiomyocytes become susceptible to hypercontracture after anoxia/reoxygenation was investigated. The cells were gradually overloaded with Ca
2+
after different periods of simulated ischemia (substrate-free anoxia, medium at pH 6.4) followed by 20 minutes of reoxygenation. The cytosolic Ca
2+
concentration (measured with fura 2) at which the cells developed maximal hypercontracture (Ca
max
) was used as an index for their susceptibility to hypercontracture (SH). SH was increased in cardiomyocytes after prolonged periods of simulated ischemia; ie, these cells developed hypercontracture at significantly lower cytosolic Ca
2+
levels than did normoxic cells (Ca
max
, 0.80±0.05 μmol/L versus 1.27±0.05 μmol/L;
P
<.01). To find the possible cause of increased SH, the influence of Ca
2+
overload, acidosis, and protein dephosphorylation were studied. Prevention of cytosolic Ca
2+
overload in anoxic cardiomyocytes or imitation of ischemic acidosis in normoxic cells did not influence Ca
max
. In contrast, use of 10 μmol/L cantharidin (inhibitor of protein phosphatases 1 and 2A) during anoxic superfusion prevented the reduction of Ca
max
. Furthermore, treatment of normoxic cardiomyocytes with 20 mmol/L of the chemical phosphatase 2,3-butanedione monoxime reduced Ca
max
. Therefore, prolonged simulated ischemia increases susceptibility of cardiomyocytes to hypercontracture. This seems to be due to protein dephosphorylation.
Hydroxyl Radical Inhibits Sarcoplasmic Reticulum Ca
2+
-ATPase Function by Direct Attack on the ATP Binding Site
Authors: Xu Kai Y. , Zweier Jay L. , Becker Lewis C.
Published: June 2012

Oxygen-derived free radicals have been reported to damage the sarcoplasmic reticulum (SR) Ca
2+
-ATPase, potentially contributing to cellular Ca
2+
overload and myocardial damage after ischemia and reperfusion. To determine whether the ATP binding site on Ca
2+
-ATPase is involved in oxygen radical injury, SR vesicles containing bound Ca
2+
-ATPase were isolated from rabbit cardiac and skeletal muscle and exposed to a hydroxyl radical (·OH)–generating system consisting of H
2
O
2
and Fe
3+
-nitrilotriacetic acid in amounts that generate a magnitude of ·OH similar to that which occurs in the reperfused heart. ·OH exposure completely inhibited Ca
2+
-ATPase activity and SR
45
Ca uptake for both cardiac and skeletal muscle. In contrast, when the purified vesicles were premixed with l mmol/L ATP before exposure to ·OH, complete protection was observed: there was no loss of ATPase activity or
45
Ca transport. No significant protection occurred with adenosine, sucrose, AMP, or ADP (l mmol/L each). SDS–gel electrophoresis indicated that ·OH did not damage the primary structure of the enzyme. Electron paramagnetic resonance spin-trapping experiments demonstrated that ATP did not scavenge ·OH. These results suggest that ·OH denatures the SR Ca
2+
-ATPase by directly attacking the ATP binding site, and occupation of the active site by ATP protects against ·OH-induced loss of enzymatic activity and SR Ca
2+
transport. The depletion of ATP that occurs during ischemia may enhance the toxic effect of ·OH at the time of reperfusion.
Intracellular Ca
2+
Increases the Mitochondrial NADH Concentration During Elevated Work in Intact Cardiac Muscle
Authors: Brandes Rolf , Bers Donald M.
Published: June 2012

It is not clear how mitochondrial energy production is regulated in intact tissue when energy consumption suddenly changes. Whereas mitochondrial [NADH] ([NADH]
m
) may regulate cellular respiration rate and energetic state, it is not clear how [NADH]
m
itself is controlled during increased work in vivo. We have varied work and [Ca
2+
] in intact cardiac muscle while assessing [NADH]
m
using fluorescence spectroscopy. When increased work was accompanied by increasing average [Ca
2+
]
c
(by increasing [Ca
2+
]
o
or pacing frequency), [NADH]
m
initially fell and subsequently recovered to a new steady state level. Upon reduction of work, [NADH]
m
overshot and then returned to control levels. In contrast, when work was increased without increasing average [Ca
2+
]
c
(by increasing sarcomere length), [NADH]
m
fell similarly, but no recovery or overshoot was observed. This Ca
2+
-dependent recovery and overshoot may be attributed to Ca
2+
-dependent stimulation of mitochondrial dehydrogenases. We conclude that the immediate initial increase in respiration rate upon elevation of work is not activated by increased [NADH]
m
(since [NADH]
m
rapidly fell) or by [Ca
2+
]
c
(since work could also be increased at constant [Ca
2+
]
c
). However, during sustained high work, a Ca
2+
-dependent mechanism causes slow recovery of [NADH]
m
toward control values. This demonstrates a Ca
2+
-dependent feed-forward control mechanism of cellular energetics in cardiac muscle during increased work.
Electrophysiological Consequences of Purinergic Receptor Stimulation in Isolated Rat Pulmonary Arterial Myocytes
Authors: Hartley S. Anthony , Kozlowski Roland Z.
Published: June 2012

Neither the electrophysiological effects of purinergic receptor stimulation nor the role of ATP in regulating the tone of pulmonary arterial smooth muscle has been determined. Therefore, we investigated the effects of purine nucleotides on acutely dissociated smooth muscle cells from rat small pulmonary arteries using the patch-clamp recording technique. Extracellular application of ATP activated a fast transient inward current (which decayed in the continued presence of the nucleotide) and produced sustained periodic oscillations of predominantly inward current. Pharmacological and anion substitution experiments revealed that the transient inward current was carried by the movement of cations. In contrast, the periodic oscillations of current were due primarily to a Ca
2+
-activated Cl
−
current (I
Cl,Ca
) dependent on the release of Ca
2+
from intracellular stores. Experiments using ATP analogues revealed the following order of potency for activation of the fast transient inward current: 2-methylthio ATP (2-meSATP)>ATP>α,β-methylene ATP (α,β-meATP)>>ADP>UTP=adenosine. Cross desensitization was seen between applications of ATP, α,β-meATP, and 2-meSATP, suggesting that these agonists act via a common site. The order of potency for activation of I
Cl,Ca
was UTP=ATP>>ADP≥2-meSATP>α,β-meATP=adenosine. Both the fast transient inward current and I
Cl,Ca
evoked by ATP and its analogues were abolished by the nonselective P
2
purinoceptor antagonist suramin. These results show the existence of P
2X
and P
2U
purinoceptor subtypes in pulmonary arterial smooth muscle cells. Stimulation of these receptors results in activation of a fast transient inward cation current and I
Cl,Ca
, respectively. It is likely that ATP acts via these receptor subtypes to regulate pulmonary arterial tone under physiological or pathological conditions.
Suppression of Angiotensin-Converting Enzyme Expression and Activity by Shear Stress
Authors: Rieder M.J. , Carmona R. , Krieger J.E. , Pritchard K.A. , Greene A.S.
Published: June 2012

Shear stress caused by the frictional forces of a fluid moving over a cell monolayer is an important regulator of gene expression. In this study, we investigated the effect of shear stress on angiotensin-converting enzyme (ACE) expression and promoter activity in vitro and on local vascular ACE activity in vivo. ACE activity measured in bovine pulmonary artery endothelial (BPAE) cells was reduced by 49.5% after exposure to a shear stress of 20 dyne/cm
2
for 18 hours. Short-term shearing (2 hours) elevated ACE activity in BPAE cells, whereas long-term shearing produced a time-dependent reduction in ACE activity by 23.3%, 33.5%, and 48.9% at 8, 12, and 18 hours, respectively. Northern blot analysis revealed that shear stress (20 dyne/cm sup 2 for 18 hours) significantly reduced ACE mRNA expression by 82%. To determine the mechanism of ACE activity and message reduction, the effect of shear on transcriptionally related events was determined in a rabbit aortic endothelial cell line (W3LUC) stably transfected with 1.3 kb of a rat ACE promoter/luciferase construct. Different shear stress magnitudes (5 to 20 dyne/cm
2
) caused suppression of luciferase activity by an average of 40.7%. ACE promoter activity was suppressed by 2 hours of shear stress (24.7%) and was further inhibited at time periods >8 hours. In vivo elevations in shear stress were created by placing a stainless steel clip over a 12-mm region of the rat abdominal aorta. Restriction of vessel diameter increased blood flow velocity and caused reduction in vascular ACE activity by 40%. These studies suggest that elevations in the level of shear stress alter endothelial cell function by suppressing ACE gene and protein expression in vitro and in vivo. (Circ Res. 1997;80:312-319.)
Hypoxia/Reoxygenation Stimulates Jun Kinase Activity Through Redox Signaling in Cardiac Myocytes
Authors: Laderoute Keith R. , Webster Keith A.
Published: June 2012

Hypoxia and reoxygenation are principal components of myocardial ischemia and reperfusion and have distinctive effects on the tissue. Both conditions have been associated with inflammation, necrosis, apoptosis, and myocardial infarction. Using a cell culture model of ischemia and reperfusion in which cardiac myocytes were exposed to cycles of hypoxia and reoxygenation, we report here that reoxygenation, but not hypoxia alone, caused sustained [nearly =] 10-fold increases in phosphorylation of the amino-terminal domain of the c-jun transcription factor. The activation was similar to treatments with anisomycin or okadaic acid and correlated with the hypoxia-mediated depression of intracellular glutathione. Reoxygenation-induced c-Jun kinase activity was reduced by preincubating myocytes during the hypoxia phase with the spin-trap agent alpha-phenyl N-tert-butylnitrone or with N-acetylcysteine. The kinase activation was also inhibited by the tyrosine kinase inhibitor genistein but not by other protein kinase inhibitors. These results implicate unquenched reactive oxygen intermediates as the stimulus that initiates a kinase pathway involving the stress-activated protein kinases (JNKs/SAPKs) in reoxygenated cardiac myocytes.
Different Compartments of Sarcoplasmic Reticulum Participate in the Excitation-Contraction Coupling Process in Human Atrial Myocytes
Authors: Hatem Stephane N. , Benardeau Agnes , Rucker-Martin Catherine , Marty Isabelle , de Chamisso Patricia , Villaz Michel , Mercadier Jean-Jacques
Published: June 2012

The excitation-contraction coupling process of human atrial myocytes was studied in voltage-clamped myocytes isolated from right atrial appendages obtained during cardiac surgery. Intracellular Ca
2
+ transients (Ca
i
transients) were monitored with 0.1 mmol/L indo 1 added to the internal dialyzing solution. Ryanodine receptors (RyRs) and sarcomeric alpha-actinin were stained with specific antibodies and visualized using plane and confocal microscopy. L-Type Ca
2
+ current (I
Ca
) elicited a prolonged Ca
i
transient, with an initial rapidly activating phase (slope 1, 23.6 +/- 1.2 s sup -1) followed by a slowly activating phase (slope 2, 5.8 +/- 0.4 s sup -1; P<.001 versus slope 1), resulting in a dome-shaped Ca
i
transient. Ryanodine (100 micro mol/L) inhibited 79 +/- 6% of the Ca
i
transient, indicating that it was due essentially to sarcoplasmic reticulum Ca
2
+ release. During step depolarizations, maximal activation of the Ca
i
transient or tail current (I sub tail) (in cells dialyzed with Ca
2
+ buffer-free internal solution) preceded that of I
Ca
and did not follow its voltage dependence (n=12). Test pulses lasting from 5 to 150 milliseconds elicited a similar time course of both Ca
i
transient and I
tail
(n=5). In a given cell, the two components of the Ca
i
transient could be dissociated by altering the intracellular Ca
2
+ load, by increasing the stimulation rate from 0.1 to 1 Hz, or by varying the amplitude of I
Ca
. Immunostaining of atrial sections and isolated myocytes showed that a large number of RyRs were located not only in a subsarcolemmal position but also deeper inside the cell, in a regularly spaced transverse band pattern at the level of Z lines. Together, our results indicate that, in human atrial myocytes, I
Ca
only partially controls the activation of RyRs, with the prolonged and dome-shaped Ca
i
transient of these cells probably reflecting the activation of RyRs not coupled to L-type Ca
2
+ channels. (Circ Res. 1997;80:345-353.)
Expression of Nonmuscle Myosin Heavy Chain-B Isoform in the Vessel Wall of Porcine Coronary Arteries After Balloon Angioplasty
Authors: De Leon H. , Scott N. A. , Martin F. , Simonet L. , Bernstein K. E. , Wilcox J. N.
Published: June 2012

Abstract
Nonmuscle myosin heavy chain-B isoform (NMMHC-B) is expressed by proliferating vascular smooth muscle cells (SMCs), and its expression in primary lesions has been proposed to be predictive of restenosis after atherectomy. The present study was designed to study the time-course expression of NMMHC-B after angioplasty of porcine coronary arteries by in situ hybridization and immunohistochemistry. Domestic juvenile swine underwent percutaneous transluminal coronary angioplasty (PTCA) of the left anterior descending and circumflex coronary arteries with standard clinical angioplasty catheters. To identify proliferating cells, 5′-bromo-2′-deoxyuridine (BrdU) was administered and detected by immunohistochemistry on serial sections. Vessels were examined at 3, 7, and 14 days after balloon angioplasty, and uninjured coronary vessels were used as controls. Normal arteries showed hybridization to
35
S-labeled NMMHC-B riboprobes localized mainly in the medial layer. NMMHC-B expression in the adventitia was markedly increased 3 days after balloon angioplasty. Seven and 14 days after injury, NMMHC-B mRNA–containing cells were localized in the adventitia and neointima at the arterial injury site. Cell proliferation, as indicated by BrdU staining, colocalized with NMMHC-B mRNA expression 3 and 7 days after angioplasty. These data indicate that cells proliferating in the adventitia and neointima express NMMHC-B; however, its expression is not limited to the proliferative state, since NMMHC-B mRNA was also found in quiescent SMCs of normal coronary arteries and in nonproliferating adventitial and neointimal cells 14 days after angioplasty.
Increased Hepatocyte Permeability Surface Area Product for
86
Rb With Increase in Blood Flow
Authors: Goresky Carl A. , Simard André , Schwab Andreas J.
Published: June 2012

Abstract
Liver cell recruitment (the equivalent of capillary recruitment in other organs) was explored by carrying out multiple indicator dilution experiments with labeled rubidium across the liver of the anesthetized dog under basal conditions and after bleeding with saline replacement infusion, which increases liver blood flow. A mixture of
51
Cr-labeled red blood cells (a vascular reference),
22
Na (which immediately equilibrates in the extracellular space, the sum of the sinusoidal plasma and Disse or interstitial spaces, the expected distribution space for labeled rubidium in the absence of cellular entry), and
86
Rb was injected into the portal vein, and normalized outflow patterns, expressed as outflowing fractions of each injected tracer per milliliter versus time, were obtained. In relation to the labeled red blood cell curve, the labeled sodium curve is displaced by flow-limited distribution into the Disse or interstitial space; it is lower on the upslope, reaches a lower and delayed peak, and decays more slowly. The early part of the labeled rubidium curve lies within the labeled sodium curve; it reaches a much reduced peak, and the later return of tracer entering cells is so slow that it is obscured by recirculation. Modeling of the concentrative cellular uptake of rubidium from the Disse space provided an influx permeability surface area product for labeled rubidium. This increases with flow over the observed flow range, demonstrating that sinusoidal recruitment occurs with increase in hepatic blood flow.
C2 Region–Derived Peptides of β-Protein Kinase C Regulate Cardiac Ca
2+
Channels
Authors: Zhang Zhi-Hao , Johnson John A. , Chen Long , El-Sherif Nabil , Mochly-Rosen Daria , Boutjdir Mohamed
Published: June 2012

Abstract
We have previously shown that α
1
-adrenergic activation inhibited β-adrenergic–stimulated L-type Ca
2+
current (
I
Ca
). To determine the role of protein kinase C (PKC) in this regulation, the inositol trisphosphate pathway was bypassed by direct activation of PKC with 4β-phorbol 12-myristate 13-acetate (PMA). To minimize Ca
2+
-induced Ca
2+
inactivation, Ba
2+
current (
I
Ba
) was recorded through Ca
2+
channels in adult rat ventricular myocytes. We found that PMA (0.1 μmol/L) consistently inhibited basal
I
Ba
by 40.5±7.4% and isoproterenol (ISO, 0.1 μmol/L)–stimulated
I
Ba
by 48.9±7.8%. These inhibitory effects were not observed with the inactive phorbol ester analogue α-phorbol 12,13-didecanoate (0.1 μmol/L). To identify the PKC isozymes that mediate these PMA effects, we intracellularly applied peptide inhibitors of a subclass of PKC isozymes, the C2-containing cPKCs. These peptides (βC2-2 and βC2-4) specifically inhibit the translocation and function of C2-containing isozymes (α-PKC, β
I
-PKC, and β
II
-PKC), but not the C2-less isozymes (δ-PKC and ε-PKC). We first used the pseudosubstrate peptide (0.1 μmol/L in the pipette), which inhibits the catalytic activity of all the PKC isozymes, and found that PMA-induced inhibition of ISO-stimulated
I
Ba
was reduced to 16.8±7.4% but was not affected by the scrambled pseudosubstrate peptide. The effects of PMA on basal and ISO-stimulated
I
Ba
were then determined in the presence of C2-derived peptides or control peptides. When the pipette contained 0.1 μmol/L of βC2-2 or βC2-4, PMA-induced inhibition of basal
I
Ba
was 26.1±4.5% and 23.6±2.2%, respectively. Similarly, ISO-stimulated
I
Ba
was inhibited by 29.9±6.6% and 29.3±7.8% in the presence of βC2-2 and βC2-4, respectively. In contrast, there was no significant change in the effect of PMA in the presence of control peptides, scrambled βC2-4, or pentalysine. Finally, PMA-induced inhibition of basal and ISO-stimulated
I
Ba
was almost completely abolished in cells dialyzed with both βC2-2 and βC2-4. Together, these data suggest a role for C2-containing isozymes in mediating PMA-induced inhibition of L-type Ca
2+
channel activity.
Inhibitory Effects of Glibenclamide on Cystic Fibrosis Transmembrane Regulator, Swelling-Activated, and Ca
2+
-Activated Cl
−
Channels in Mammalian Cardiac Myocytes
Authors: Yamazaki Jun , Hume Joseph R.
Published: June 2012

Abstract
Recent studies have provided evidence that sulfonylureas, in addition to blocking ATP-sensitive K
+
(K
ATP
) channels, also inhibit cystic fibrosis transmembrane regulator (CFTR) Cl
−
channels in epithelial and cardiac cells. The purpose of this study was to test whether the sulfonylurea glibenclamide might also inhibit other types of cardiac Cl
−
channels. Whole-cell patch-clamp techniques were used to compare the effects of glibenclamide on CFTR Cl
−
currents in guinea pig ventricular myocytes, swelling-activated Cl
−
currents in guinea pig atrial myocytes, and Ca
2+
-activated Cl
−
currents in canine ventricular myocytes. Glibenclamide markedly inhibited CFTR Cl
−
currents in a voltage-independent manner at 22°C, with estimated IC
50
values of 12.5 and 11.0 μmol/L at +50 and −100 mV, respectively. The outwardly rectifying swelling-activated Cl
−
current in atrial cells was less sensitive to glibenclamide, and the block exhibited voltage dependence. At 22°C, the estimated IC
50
values were 193 and 470 μmol/L at +50 and −100 mV, respectively, and block was enhanced at 35°C. Macroscopic Cl
−
currents activated by a rise in intracellular Ca
2+
, induced by either Ca
2+
-induced Ca
2+
release or by external application of the Ca
2+
ionophore A23187, were also markedly inhibited at 22°C by glibenclamide in a voltage-independent manner. The estimated IC
50
values were 61.5 and 69.9 μmol/L at +50 and −100 mV, respectively. These results suggest that glibenclamide, an inhibitor of cardiac CFTR Cl
−
channels, also inhibits swelling-activated and Ca
2+
-activated Cl
−
channels at higher concentrations. The results also suggest that studies attributing the beneficial or deleterious effects of sulfonylurea compounds in the heart solely to blockade of K
ATP
channels should use submicromolar concentrations of these agents to minimize possible secondary interactions with cardiac Cl
−
channels.
Delayed Enhanced Nitric Oxide–Mediated Coronary Vasodilation Following Brief Ischemia and Prolonged Reperfusion in Conscious Dogs
Authors: Kim Song-Jung , Ghaleh Bijan , Kudej Raymond K. , Huang Cheng-Hsiung , Hintze Thomas H. , Vatner Stephen F.
Published: June 2012

Abstract
The goal of this study was to determine both the early and delayed effects of a brief (10-minute) coronary artery occlusion (CAO) and prolonged (5-day) reperfusion (CAR) on coronary endothelial function. Fourteen mongrel dogs were chronically instrumented to measure aortic and left ventricular pressures, wall thickness, and left circumflex coronary blood flow (CBF). Before CAO and during CAR, coronary vascular reactivity was investigated by 15-second CAO and subsequent reactive hyperemia (RH) and by the selective intracoronary infusion of acetylcholine (ACh, 10 μg/min) and bradykinin (BK, 2.5 μg/min), endothelium-dependent vasodilators, and sodium nitroprusside (SNP, 40 μg/min), an endothelium-independent vasodilator. CBF responses to ACh and BK began to increase after 6 hours of CAR, reached a peak after 1 to 2 days of CAR, and then subsided over the subsequent 4 days. After 1 day of CAR, compared with before CAO, enhanced CBF responses (
P
<.05), associated with increased coronary sinus oxygen content, were observed for ACh (+66±20%), BK (+74±24%), and RH (+24±5%) but not SNP (−2±10%). Production of NO metabolites (nitrate and nitrite), measured as their coronary arteriovenous difference×CBF, was significantly increased after 1 to 2 days of CAR, both at baseline (153±56%) and during BK infusion (220±76%) (
P
<.05). Holding CBF at pre-CAO levels during the initial CAR period did not attenuate the delayed enhanced endothelial vasodilation to ACh and BK. However, NO blockade with intracoronary
N
G
-nitro-
l
-arginine blocked the enhanced coronary vasodilation to ACh and BK. Thus, in contrast to previous studies, these data indicate that brief ischemic episodes induce delayed enhanced coronary endothelial function, which is delayed in onset and prolonged in duration. This can be explained by an upregulation of coronary vascular NO production, potentially involved in the mechanism of the delayed window of preconditioning.
Positive Inotropy Mediated by Diacylglycerol in Rat Ventricular Myocytes
Authors: Pi YeQing , Sreekumar R. , Huang XuPei , Walker Jeffery W.
Published: June 2012

Abstract
Many neurohormones stimulate phospholipid hydrolysis and elevate diacylglycerol in the mammalian heart, but the physiological consequences of these intracellular events are unclear. Regulation of myocardial contraction by diacylglycerol was investigated in the present study by releasing the diacylglycerol analogue dioctanoylglycerol (diC
8
) within adult rat ventricular myocytes by using a light-sensitive caged compound. This approach permitted us to avoid exposure of myocytes to extracellular diC
8
and yet to control the amount of diC
8
released into the cells. Photorelease of diC
8
produced a slowly developing (half-time, 1.9±0.1 minute; n=26) but robust (406±42%) enhancement of twitch amplitude in electrically paced myocytes (0.5 Hz, 1 mmol/L Ca
2+
Ringer’s solution [pH 7.4], 22°C). This positive inotropic effect was dose dependent, stereospecific for the S-enantiomer of diC
8
, synergistically enhanced by arachidonic acid, and blocked by the protein kinase C inhibitor chelerythrine. The data provide evidence that diacylglycerol can induce a strong positive inotropic effect in mammalian ventricular muscle, possibly by activating protein kinase C. By contrast, perfusion of diC
8
extracellularly onto myocytes caused a 42±2% decline in twitch amplitude, in accordance with previous reports. To account for this dependence on how diC
8
is applied, we postulate that diC
8
has distinct physiological actions at intracellular and extracellular sites. The peptide neurohormone endothelin-1, which elevates diacylglycerol in cardiac tissues, produced a positive inotropic effect that was similar to the response to photoreleased diC
8
. The diacylglycerol/protein kinase C pathway has now become a good candidate for mediator of at least a component of the positive inotropy associated with agents that stimulate phospholipid turnover in adult mammalian myocardium.