bluuebunny/crossref_metadata_2025 huggingface dataset:
Endoplasmic Reticulum Ca
2+
Depletion Unmasks a Caffeine-Induced Ca
2+
Influx in Human Aortic Endothelial Cells
Authors: Corda Stefano , Spurgeon Harold A. , Lakatta Edward G. , Capogrossi Maurizio C. , Ziegelstein Roy C.
Published: June 2012

Abstract
Intracellular Ca
2+
pools contribute to changes in cytosolic [Ca
2+
] ([Ca
2+
]
i
), which play an important role in endothelial cell signaling. Recently, endothelial ryanodine-sensitive Ca
2+
stores were shown to regulate agonist-sensitive intracellular Ca
2+
pools. Since caffeine binds the ryanodine Ca
2+
release channel on the endoplasmic reticulum in a variety of cell types, we examined the effect of caffeine on [Ca
2+
]
i
in human aortic endothelial cell monolayers loaded with the fluorescent probe indo 1. Under baseline conditions, 10 mmol/L caffeine induced a small increase in [Ca
2+
]
i
from 86±10 to 115±17 nmol/L (mean±SEM); this effect was similar to that of 5 μmol/L ryanodine and was unaffected by buffer Ca
2+
removal. After depletion of an intracellular Ca
2+
store by the irreversible endoplasmic reticulum Ca
2+
-ATPase inhibitor thapsigargin (1 μmol/L), ryanodine did not affect [Ca
2+
]
i
. In contrast, caffeine induced a large rapid increase in [Ca
2+
]
i
(176±19 to 338±35 nmol/L,
P
<.001) after thapsigargin exposure; this effect of caffeine was only observed when extracellular Ca
2+
was present. A similar increase in [Ca
2+
]
i
was induced by caffeine after depletion of ryanodine- and histamine-sensitive Ca
2+
stores or after pretreatment with the endoplasmic reticulum Ca
2+
-ATPase inhibitor cyclopiazonic acid (10 μmol/L). Thus, under baseline conditions the effect of caffeine on [Ca
2+
]
i
is similar to that of ryanodine and appears to be due to the release of an intracellular store. However, after depletion of an endoplasmic reticulum Ca
2+
store, caffeine, but not ryanodine, stimulates Ca
2+
influx, resulting in a large increase in [Ca
2+
]
i
. The data suggest that caffeine-induced Ca
2+
influx is controlled by the status of Ca
2+
loading of intracellular Ca
2+
stores in human aortic endothelial cells.
Identification of Receptor Binding and Activation Sites in Endothelin-1 by Use of Site-Directed Mutagenesis
Authors: Ergul Adviye , Tackett Randall L. , Puett David
Published: June 2012

Abstract
This study addresses the structural requirements for the intracellular processing and receptor binding properties of endothelin-1 (ET-1). Point mutants of preproendothelin-1 cDNA, with replacement of the codons for Lys
9
of ET-1 by ones for Ala and Glu and of Ile
20
and Trp
21
by ones encoding Ala, were expressed in COS-7 cells. Competitive binding experiments on rat vascular smooth muscle cells (A-10), which were shown to be an ET
A
receptor–rich cell line, between [
125
I]ET-1 and synthetic ET-1, wild-type recombinant ET-1, and recombinant [Ala
9
]ET-1, [Glu
9
]ET-1, [Ala
20
]ET-1, and [Ala
21
]ET-1 yielded
K
i
values of 0.2±0.02, 0.2±0.02, 0.04±0.01, 1.4±0.2, 1.6±0.2, and >50 nmol/L, respectively. In similar experiments with ET
B
receptor–rich human Girardi heart cells, the corresponding values were 0.2±0.03, 0.2±0.03, 0.2±0.04, 0.2±0.06, 1.4±0.4, and >50 nmol/L. The ET
A
receptor–mediated contractile responses to [Glu
9
]ET-1 and [Ala
20
]ET-1, measured by using canine coronary artery rings, were decreased approximately fourfold to fivefold compared with the response produced by synthetic or wild-type recombinant ET-1, whereas [Ala
9
]ET-1 was found to be more potent, and [Ala
21
]ET-1 did not produce any contraction. These results demonstrate that Ile
20
and Trp
21
are involved in binding to both receptor subtypes. Of considerable interest was the observation that [Glu
9
]ET-1 also blunts the ET
A
receptor subtype–mediated contractile response to ET-1 stimulus.
Cloned Human Inward Rectifier K
+
Channel as a Target for Class III Methanesulfonanilides
Authors: Kiehn J. , Wible B. , Ficker E. , Taglialatela M. , Brown A.M.
Published: June 2012

Abstract
Methanesulfonanilide derivatives such as dofetilide are members of the widely used Class III group of cardiac antiarrhythmic drugs. A methanesulfonanilide-sensitive cardiac current has been identified as I
Kr
, the rapidly activating component of the repolarizing outward cardiac K
+
current, I
K
. I
Kr
may be encoded by the human ether-related gene (hERG), which belongs to the family of voltage-dependent K
+
(K
v
) channels having six putative transmembrane segments. The hERG also expresses an inwardly rectifying, methanesulfonanilide-sensitive K
+
current. Here we show that hIRK, a member of the two-transmembrane-segment family of inward K
+
rectifiers that we have cloned from human heart, is a target for dofetilide. hIRK currents, expressed heterologously in
Xenopus
oocytes, are blocked by dofetilide at submicromolar concentrations (IC
50
=533 nmol/L at 40 mV and 20°C). The drug has no significant blocking effect on the human cardiac K
v
channels hK
v
1.2, hK
v
1.4, hK
v
1.5, or hK
v
2.1. The block is voltage dependent, use dependent, and shortens open times in a manner consistent with open-channel block. While steady state block is strongest at depolarized potentials, recovery from block is very slow even at hyperpolarized potentials (tau=1.17 seconds at −80 mV). Thus, block of hIRK may persist during diastole and might thereby affect cardiac excitability.
pH
i
Regulation in Myocardium of the Spontaneously Hypertensive Rat
Authors: Pérez Néstor G. , Alvarez Bernardo V. , Camilión de Hurtado María C. , Cingolani Horacio E.
Published: June 2012

Abstract
To elucidate the mechanisms controlling pH
i
in myocardium of the spontaneously hypertensive rat (SHR), experiments were performed in papillary muscles (isometrically contracting at 0.2 Hz) from SHR and age-matched normotensive Wistar-Kyoto (WKY) rats loaded with the pH-sensitive fluorescent probe BCECF-AM. An enhanced activity of the Na
+
-H
+
exchanger was detected in the hypertrophic myocardium of SHR. This conclusion was based on the following: (1) The myocardial pH
i
was more alkaline in SHR (7.23±0.03) than in WKY rats (7.10±0.03) (
P
<.05) in HEPES buffer. (2) SITS (0.1 mmol/L in HEPES buffer) did not alter pH
i
in the SHR (pH
i
7.26±0.03 and 7.28±0.03 before and after SITS, respectively). (3) The fall in pH
i
observed after 20 minutes of Na
+
-H
+
exchanger inhibition [5 μmol/L 5-(
N
-ethyl-
N
-isopropyl)amiloride (EIPA)] was greater in SHR (−0.16±0.01) than in WKY rats (−0.09±0.02,
P
<.05). (4) The velocity of pH
i
recovery from an intracellular acid load was faster in SHR than in WKY rats (0.068±0.02 versus 0.014±0.002 pH units/min at pH
i
6.99,
P
<.05). (5) After EIPA inhibition, the rate of pH
i
recovery from the same acid load decreased to a similar value in both rat strains (0.0032±0.002 pH units/min in SHR and 0.0032±0.002 pH units/min in WKY rats). Under the more physiological HCO
3
−
-CO
2
buffer, no significant difference in steady state myocardial pH
i
was detected between rat strains (7.15±0.03 in SHR and 7.11±0.05 in WKY rats). This finding suggested that an acidifying bicarbonate-dependent mechanism was fully compensating for the hyperactivity of the Na
+
-H
+
exchanger in SHR. The following pieces of evidence support an enhanced activity of the Na
+
-independent Cl
−
-HCO
3
−
exchanger as the mechanism accounting for the compensation: (1) SITS (0.1 mmol/L) increased steady state pH
i
in the presence of HCO
3
−
-CO
2
buffer in SHR (+0.08±0.02,
P
<.05) but not in WKY rats (+0.04±0.04). (2) The rate of pH
i
recovery from an alkaline load was faster in SHR than in WKY rats (0.075±0.028 versus 0.027±0.016 pH units per minute, respectively;
P
<.05). (3) The enhanced recovery from an alkaline load in the SHR was Na
+
independent. (4) No difference in the rate of pH
i
recovery was detected between SHR and WKY rats when the alkaline load was performed after SITS blockade. Comparison of net HCO
3
−
efflux at a given pH
i
suggests that an increased pH
i
is not the cause of the hyperactivity of the anion exchanger. Since this anion exchanger is not driving Na
+
, the offset of the increase in pH
i
induced by the antiport would not prevent an increase in intracellular Na
+
mediated by the Na
+
-H
+
exchanger.
Transmembrane Voltage Changes During Unipolar Stimulation of Rabbit Ventricle
Authors: Knisley Stephen B.
Published: June 2012

Abstract
This study tested the prediction of bidomain models that unipolar stimulation of anisotropic myocardium produces transmembrane voltage changes (ΔV
m
s) of opposite signs away from the electrode on perpendicular axes. Stimulation with a strength of 0.1 to 40 mA was applied from a point electrode on the left or right ventricle of isolated perfused rabbit hearts at 37°C to 38°C stained with the potentiometric dye di-4-ANEPPS. A laser scanner system recorded V
m
-sensitive fluorescence at 63 spots in an 8×8-mm region around the electrode. Cathodal stimulation in the refractory period produced regions of −ΔV
m
1 to 5 mm away from the electrode on an axis oriented parallel to the fast propagation axis to within 1.8±11° (
P
≥.7 for difference versus zero, n=7). Recording spots in these regions underwent +ΔV
m
when anodal stimulation was used. At recording spots on the slow propagation axis, cathodal stimulation produced +ΔV
m
and anodal stimulation produced −ΔV
m
. During diastolic stimulation, early excitation occurred near the electrode for cathodal stimulation or on the fast propagation axis as far as 2.8±1 mm away from the electrode for anodal stimulation. A “dog-bone” region of +ΔV
m
that included tissue near and away from the electrode on the slow propagation axis occurred when cathodal stimulation was given in diastole. Regions of +ΔV
m
occurred away from the electrode on the fast propagation axis when anodal stimulation was given in diastole. Thus, ΔV
m
differs in regions along and across myocardial fibers, indicating that ΔV
m
depends on anisotropic bidomain properties. Sites of early excitation are those where +ΔV
m
occurs, indicating that membrane channel excitation depends on the distribution of ΔV
m
.
Preconditioning of Bovine Endothelial Cells
Authors: Zhou Xiaobo , Zhai Xiaolin , Ashraf Muhammad
Published: June 2012

Abstract
We tested the hypothesis that anoxic preconditioning could protect coronary endothelial cells against anoxic and reoxygenation injury and that this preconditioning effect could be mediated by an adenosine A
2
receptor via the protein kinase C (PKC) pathway. Cells were preconditioned with 10-minute anoxia and 10-minute reoxygenation and were then subjected to anoxia for 60 minutes, followed by 120 minutes of reoxygenation. In some groups, the preconditioning effect was prevented by 8-sulfophenyltheophylline (SPT [50 μmol/L], a nonselective adenosine receptor antagonist) or calphostin C (100 nmol/L, a PKC inhibitor). In other groups, 2-
p
-(2-carboxyethyl)phenethylamino-5′-
N
-ethylcarboxamidoadenosine (CGS-21680 [20 nmol/L], an adenosine A
2
receptor agonist),
R
-(−)-
N
6
-(2-phenylisopropyl)-adenosine (R-PIA [50 nmol/L], an adenosine A
1
receptor agonist), or 4β-phorbol 12-myristate 13-acetate (PMA [100 nmol/L], a PKC activator) was given as a pretreatment to mimic the preconditioning effect. Endothelial cells were also pretreated with 100 nmol/L calphostin C to confirm whether inhibition of PKC can block the effects of adenosine A
2
receptor activation by CGS-21680 on anoxia and reoxygenation injury. Preconditioning reduced LDH release, increased adenosine release, promoted translocation of PKC from cytosol to membrane, increased cell viability, and preserved ATP content and cell morphology. Pretreatment with either CGS-21680 or PMA resulted in protection similar to that seen with anoxic preconditioning. The protection was totally abolished by SPT or calphostin C. The results suggest that (1) preconditioning protects coronary endothelial cells against anoxia and reoxygenation injury, (2) the protection is probably mediated by activation of adenosine A
2
receptors through the PKC pathway, and (3) the preservation of endothelial cells may be one of the mechanisms of myocardial preconditioning.
Chronic Inhalation of Nitric Oxide Inhibits Neointimal Formation After Balloon-Induced Arterial Injury
Authors: Lee Joon S. , Adrie Christophe , Jacob Howard J. , Roberts Jesse D. , Zapol Warren M. , Bloch Kenneth D.
Published: June 2012

Abstract
Systemic and local intravascular NO administration inhibits neointimal formation after vascular injury in animal models. NO appears to attenuate smooth muscle proliferation both directly and indirectly by preventing the release of growth factors. Inhalation of low concentrations of NO dilates pulmonary vascular smooth muscle but does not cause systemic vasodilatation. Recently, NO inhalation was found to inhibit platelet function in vivo. We studied the effects of NO inhalation on neointimal formation after balloon-induced injury of the adult rat carotid artery. Beginning 60 minutes before carotid injury, rats breathed either air with 0 or 80 ppm NO for 14 days. Rats were killed, carotid arteries were fixed and paraffin-embedded, and neointimal formation was measured by analyzing the ratio of intimal to medial areas (I/M ratio) in carotid artery cross sections. Intimal hyperplasia was evident in both groups of animals, but I/M ratios were 43% less in animals breathing 80 ppm NO for 2 weeks than in animals breathing air alone (0.78±0.12 and 1.37±0.11 [mean±SE], respectively;
P
<.02). Similarly, 1 week after carotid injury, neointimal formation was less in rats breathing 80 ppm NO than in rats breathing air alone (I/M ratio, 0.39±0.11 versus 0.76±0.06;
P
<.02). Breathing 20 ppm NO for 2 weeks or 80 ppm NO for 1 week followed by air alone for 1 week did not attenuate neointimal formation measured at 14 days. In anesthetized rats breathing 80 ppm NO or air alone for 1 hour, neither systemic blood pressure nor bleeding time differed. These observations demonstrate that inhaling 80 ppm NO inhibits neointimal formation after balloon-induced carotid artery injury in rats. NO inhalation may represent a safe and novel method of preventing restenosis after percutaneous angioplasty.
Intrinsic Myofilament Alterations Underlying the Decreased Contractility of Stunned Myocardium
Authors: Dong Gao Wei , Liu Yongge , Mellgren Ronald , Marban Eduardo
Published: June 2012

Abstract
We investigated the mechanism of the decreased myofilament Ca
2+
responsiveness in stunned myocardium. The steady state force-[Ca
2+
] relationship was measured before and after skinning in thin ventricular trabeculae from control or stunned (20 minutes of ischemia, 20 minutes of reperfusion) rat hearts. [Ca
2+
]
i
was determined using microinjected fura 2 salt in intact muscles, whereas the myofilaments of chemically skinned trabeculae were activated directly with solutions of varied [Ca
2+
]. Maximal Ca
2+
-activated force (F
max
) before and after skinning was identical within either the control or stunned groups but was markedly depressed in both groups of stunned trabeculae (
P
<.001). After ischemia and reperfusion, the [Ca
2+
] required for 50% of maximal activation (Ca
50
) was increased in both intact (control, 0.60±0.09 μmol/L; stunned, 0.85±0.09 μmol/L;
P
<.001) and skinned (control, 1.13±0.24 μmol/L; stunned, 1.39±0.21 μmol/L;
P
=.0025) trabeculae. These data indicate that the decreased Ca
2+
responsiveness of stunned myocardium is due to intrinsic alterations of the myofilaments. Therefore, we tested the hypothesis that activation of proteases by reperfusion-induced Ca
2+
overload decreases the Ca
2+
responsiveness of the cardiac myofilaments. Force-[Ca
2+
] relations were compared before and 5 to 30 minutes after direct exposure of skinned trabeculae to calpain I (18 μg/mL, 20 minutes at [Ca
2+
]=10.8 μmol/L), a Ca
2+
-activated protease that is present in myocardium. Calpain I reduced F
max
from 94.3±8.3 to 56±8.5 mN/mm
2
while increasing Ca
50
from 0.94±0.11 to 1.36±0.21 μmol/L (
P
<.01). Calpastatin, a specific calpain inhibitor, prevented the effects of calpain I on skinned trabeculae. The results show that the reduced Ca
2+
responsiveness of stunned myocardium reflects alteration of the myofilaments themselves, not of soluble cytosolic factors, which can be faithfully reproduced by exposure to Ca
2+
-dependent protease.
Specific Accumulation of Lipoprotein(a) in Balloon-Injured Rabbit Aorta In Vivo
Authors: Nielsen Lars B. , Stender Steen , Kjeldsen Knud , Nordestgaard Børge G.
Published: June 2012

Abstract
To explore whether lipoprotein(a), Lp(a), may accumulate preferentially to LDL in the arterial wall at sites of injury, cholesterol-fed rabbits were injected intravenously with radiolabeled Lp(a) and/or LDL 3.1±0.1 days (mean±SEM, n=30) after a balloon injury of the thoracic aorta. After 5 to 10 minutes’ exposure to labeled lipoproteins, more labeled LDL than labeled Lp(a) was recovered in the intima–inner media of the balloon-injured segment (n=9; paired
t
test,
P
<.0001); however, the amount of tightly bound labeled lipoprotein was similar for the two lipoprotein fractions. In the second set of experiments,
131
I-Lp(a) (or
131
I-LDL) was injected 26 hours before and
125
I-Lp(a) (or
125
I-LDL) 3 hours before the aorta was removed. Permeability and fractional loss of labeled Lp(a) (n=8) versus LDL (n=7) in the balloon-injured aortic intima–inner media were: permeability, 0.46±0.10 μL/cm
2
per hour versus 1.41±0.32 μL/cm
2
per hour (nonpaired
t
test,
P
<.0001); and fractional loss, 0.12±0.02 h
−1
versus 0.44±0.05 h
−1
(nonpaired
t
test,
P
=.0001), respectively. Finally, after 23 hours’ exposure to labeled lipoproteins, the total accumulation and the amount of tightly bound labeled Lp(a) in the balloon-injured intima–inner media were, respectively, 174% (n=6; ANOVA,
P
=.03) and 256% (ANOVA,
P
=.005) of the values for labeled LDL. For labeled Lp(a) in the balloon-injured compared with the normal aortic intima–inner media, the recovery after 5 to 10 minutes, the permeability, and the accumulation after 23 hours were all increased, whereas the fractional loss was unchanged. These data suggest that the accumulation of Lp(a) is much larger in injured vessels than in normal vessels. Moreover, the data support the idea of a specific accumulation of Lp(a) compared with LDL in injured vessels.
Inhibitory Effects of TA-993, a New 1,5-Benzothiazepine Derivative, on Platelet Aggregation
Authors: Odawara Akio , Kikkawa Kohei , Katoh Makoto , Toryu Hiromi , Shimazaki Tamotu , Sasaki Yasuhiko
Published: June 2012

Abstract
TA-993, an
l
-
cis
4′,8-dimethyl derivative of the Ca
2+
antagonist diltiazem, and some of its metabolites inhibited platelet aggregation induced by collagen, ADP, epinephrine, platelet activating factor, arachidonic acid, and U-46619 in human platelets in vitro. Among the metabolites, MB3 was the most potent (IC
50
, <1 μmol/L; several hundred times more potent than the parent compound). The
d
isomer of MB3 was >100 times less potent than the
l
isomer. Unlike acetylsalicylic acid (ASA), TA-993 inhibited both primary and secondary phases of ADP-induced platelet aggregation and also exhibited a disaggregating effect on human platelet aggregates. The inhibitory effect of TA-993 was enhanced when used in combination with ASA. In ex vivo studies involving rats, TA-993 (≈0.3 to 100 mg/kg PO) dose-dependently inhibited collagen-induced platelet aggregation (ED
50
, 3 mg/kg PO). In the whole-blood platelet aggregation system in rats, orally administered TA-993 was also inhibitory in single (3 to 30 mg/kg) or repeated daily (10 mg/kg per day for 10 days) dosage. Orally administered TA-993 dose-dependently inhibited ADP-induced platelet aggregation ex vivo in dogs (0.3 to 10 mg/kg), significantly protected mice against collagen+epinephrine–induced thromboembolic death (10 mg/kg), and inhibited thrombus formation in an arteriovenous shunt in rats (30 mg/kg). The Ca
2+
-antagonistic action of TA-993 was very weak in depolarized canine basilar arteries: the potency was ≈1/10 that of diltiazem (
d
-
cis
) and
d
-TA-993. These results suggest that antiplatelet action is more characteristic of the
l
-
cis
than the
d
-
cis
1,5-benzothiazepine structure and that TA-993 may become a clinically useful antiplatelet agent of this structure series.
Vasopressin Stimulates Ca
2+
Spiking Activity in A7r5 Vascular Smooth Muscle Cells via Activation of Phospholipase A
2
Authors: Byron Kenneth L.
Published: June 2012

Abstract
[Arg
8
]-vasopressin (AVP) is both a potent vasoconstrictor and a mitogen for vascular smooth muscle cells. AVP binds to a single class of receptors (V
1a
) in the A7r5 rat aortic smooth muscle cell line (
K
d
≈2 nmol/L). Stimulation of these cells with AVP results in an increase in cytoplasmic free Ca
2+
concentration ([Ca
2+
]
i
) by releasing intracellular Ca
2+
stores and increasing Ca
2+
influx; the EC
50
for these effects is ≈5 nmol/L. AVP has recently been reported to stimulate arachidonic acid release in primary cultures of rat aortic smooth muscle over a much lower concentration range (EC
50
≈0.05 nmol/L). The present study examined the effects of varying concentrations of AVP on spontaneous Ca
2+
spiking activity in fura 2–loaded A7r5 cells. Frequency of Ca
2+
spiking increased with increasing [AVP] in the range of 10 to 500 pmol/L. Higher concentrations of AVP inhibited spiking but elicited the characteristic [Ca
2+
]
i
changes ascribed to the release of Ca
2+
stores and increased Ca
2+
entry. The effects of both low and high concentrations of AVP were inhibited by [1-(β-mercapto-β,β,-pentamethylenepropionic acid),2-
O
-methyltyrosine]arginine vasopressin, a selective V
1a
vasopressin antagonist. Nimodipine (50 nmol/L), a blocker of L-type voltage-sensitive Ca
2+
channels, abolished the Ca
2+
-spiking activity without inhibiting a maximal [Ca
2+
]
i
response to AVP (1 μmol/L). AVP-stimulated Ca
2+
spiking, but not release of intracellular Ca
2+
stores, was also abolished by ONO-RS-082 (1 μmol/L), an inhibitor of phospholipase A
2
. These results suggest that occupation of a small fraction of V
1a
vasopressin receptors by AVP results in stimulation of phospholipase A
2
and leads to increased Ca
2+
-spiking activity. This effect may be important for fine tuning of vascular tone, whereas maximal stimulation by AVP (full receptor occupancy) may be required for more vigorous or sustained vasoconstriction or mitogenesis.
Binding of Cytosolic Proteins to Myofibrils in Ischemic Rat Hearts
Authors: Barbato Roberta , Menabò Roberta , Dainese Paola , Carafoli Ernesto , Schiaffino Stefano , Di Lisa Fabio
Published: June 2012

Abstract
Myofibrillar proteins (MPs) were extracted from isolated and perfused rat hearts subjected to different periods of ischemia to investigate the occurrence of protein degradation and/or the association of cytosolic proteins with the myofibrillar pellet. A 23-kD band was detected by SDS-PAGE of MPs after 5 minutes of ischemia, with its density gradually increasing to a plateau after 20 minutes. Longer periods of ischemia were associated with the appearance of a 39-kD band. Irrespective of the duration of ischemia, both these bands persisted during reperfusion. A partial proteolytic degradation of troponin T (TnT) and troponin I (TnI) has been claimed to be responsible for the generation of these peptides. However, the N-terminal sequence of the 39-kD band was identical to that of GAPDH, whereas Edman sequencing after pepsin digestion showed that the 23 kD is αB-crystallin. The binding of the two cytosolic proteins to myofibrils was confirmed by immunofluorescence analysis on cryosections of ischemic hearts. In vitro studies showed that acidosis was sufficient to induce the binding of αB-crystallin, whereas the inhibition of ATP depletion prevented the binding of GAPDH. Thiol oxidation is unlikely to promote GAPDH binding, since perfusion with iodoacetate under aerobic conditions or treatment of homogenates with
N
-ethylmaleimide or diamide failed to induce GAPDH association with the myofibrils. These changes of the myofibrillar proteins could be considered as intracellular markers of the evolution of the ischemic damage. In addition, the binding of the 23-kD peptide might be involved in alterations of contractility.
Creatine Kinase Is the Main Target of Reactive Oxygen Species in Cardiac Myofibrils
Authors: Mekhfi Hassane , Veksler Vladimir , Mateo Philippe , Maupoil Véronique , Rochette Luc , Ventura-Clapier Renée
Published: June 2012

Abstract
Reactive oxygen species (ROS) have been reported to alter cardiac myofibrillar function as well as myofibrillar enzymes such as myosin ATPase and creatine kinase (CK). To understand their precise mode and site of action in myofibrils, the effects of the xanthine/xanthine oxidase (X/XO) system or of hydrogen peroxide (H
2
O
2
) have been studied in the presence and in the absence of phosphocreatine (PCr) in Triton X-100–treated cardiac fibers. We found that xanthine oxidase (XO), with or without xanthine, induced a decrease in maximal Ca
2+
-activated tension. We attributed this effect to the high contaminating proteolytic activity in commercial XO preparations, since it could be prevented by a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), and it could be mimicked by trypsin. In further experiments, XO was pretreated with 1 mmol/L PMSF. Superoxide anion production by the X/XO system, characterized by electron paramagnetic resonance spin-trapping technique, was not altered by PMSF. A slight increase in maximal force was then observed either with X/XO (100 μmol/L per 30 mIU/mL) or H
2
O
2
. pMgATP–rigor tension relationships have been established in the presence and in the absence of PCr to separate the effects of ROS on myosin ATPase and myofibrillar-bound CK. In the presence of PCr, pMgATP
50
, the pMgATP necessary to induce half-maximal rigor tension, was reduced from 5.03±0.17 (n=21) to 4.22±0.22 (n=4) after 25 minutes of incubation in the presence of 30 mIU/mL XO and 100 μmol/L xanthine or to 4.04±0.1 (n=11) after incubation in the presence of 2.5 mmol/L H
2
O
2
. The ROS effects were partially prevented or antagonized by 1 mmol/L dithiothreitol. No effect was observed on pMgATP
50
when PCr was absent. pCa-tension relationships have been evaluated to assess the effects of ROS on active tension development. Incubations with H
2
O
2
induced an increase in Ca
2+
sensitivity and resting tension when MgATP was provided through myofibrillar CK (PCr and MgADP as substrates) but not when MgATP was added directly. These results suggest that myofibrillar CK was inhibited by ROS. Active stiffness and the time constant of tension changes after quick stretches applied to the fibers were dose-dependently increased by H
2
O
2
only in the presence of PCr. In addition, myofibrillar CK but not myosin ATPase enzymatic activity was depressed after incubation with either ROS. These results suggest that ROS mainly alters CK in myofibrils, probably by the oxidation of its essential sulfhydryl groups. Such CK inactivation results in a decrease in the intramyofibrillar ATP-to-ADP ratio. The effects of ROS on cytosolic and bound CKs may take part in the overall process of myocardial stunning after cardiac ischemia and reperfusion.
Differential Sensitivity of Venular and Arteriolar α-Adrenergic Receptor Constriction to Inhibition by Hypoxia
Authors: Leech Cindi Jo , Faber James E.
Published: June 2012

Abstract
Reflex adrenergic constriction of the venous circulation is considerably less sensitive than the arterial circulation to local metabolic inhibition, but the basis for this difference remains unclear. The purpose of the present study was to determine whether α-adrenergic receptor (AR) constriction of venular smooth muscle is in fact protected against inhibition by hypoxia, per se, and to examine possible mechanisms for this protection. An intermediate level of α
1
-AR (norepinephrine+rauwolscine) or α
2
-AR (UK 14,304+prazosin) tone was induced in rat cremaster skeletal muscle arterioles and venules (control lumen diameter, 134 and 194 μm, respectively), and tissue bath P
o
2
was lowered from the control value (30 mm Hg). Arteriolar α
2
-AR tone was inhibited by 29% at 5 mm Hg P
o
2
(
P
<.05), whereas arteriolar α
1
-, venular α
1
-, and venular α
2
-AR constrictions were unaffected. Like these findings obtained for in situ vessels with normal blood flow, α
1
-AR tone induced in vascularly “isolated” venules and basal diameter were again unaffected by hypoxia, whereas α
2
-AR tone was actually enhanced by 19% (
P
<.05). This constriction was prevented by indomethacin but not by endothelin or nitric oxide blockade; importantly, however, venular α
2
- and α
1
-AR tone still remained insensitive to inhibition by hypoxia. ATP-sensitive K
+
(K
ATP
) channels, which are known to participate in hypoxic inhibition of arteriolar smooth muscle, were examined for a role in this differential arteriolar versus venular sensitivity to hypoxia. Use of the K
ATP
antagonists glibenclamide and U-37883A and the K
ATP
channel opener cromakalim suggested that venular, unlike arteriolar, smooth muscle had no detectable basal or inducible K
ATP
activity. Also, unlike arteriolar α
2
-AR constriction, venular α
2
-AR tone did not depend on K
ATP
activity. Finally, venular α
2
-AR tone was unaffected by nifedipine (0.06 to 3 μmol/L), whereas venular α
1
-AR tone was inhibited by 50% (
P
<.05), findings opposite those found for arteriolar α
1
and α
2
tone. These data demonstrate that venular α
1
- and α
2
-AR constrictions are insensitive to inhibition by hypoxia and suggest that this may be due to a paucity of K
ATP
channels on venular smooth muscle. In addition, venular α
1
- but not α
2
-ARs appear to couple to dihydropyridine-sensitive voltage-operated Ca
2+
channels.
Differential Regulation of
l
-Arginine Transport and Nitric Oxide Production by Vascular Smooth Muscle and Endothelium
Authors: Durante William , Liao Lan , Iftikhar Irfan , O’Brien William E. , Schafer Andrew I.
Published: June 2012

Abstract
Since NO production is dependent on the availability of
l
-arginine, we examined whether
l
-arginine transport and NO synthesis are coregulated by vascular smooth muscle cells and endothelial cells cultured from the same vessel wall source.
l
-Arginine transport by both bovine aortic smooth muscle cells (BASMCs) and endothelial cells (BAECs) was primarily Na
+
independent (≈70%) and was mediated by both a high- and low-affinity transport system. Treatment of BASMCs with tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) resulted in a significant increase in
l
-arginine transport (≈20%) and in the induction of NO release. Exposure of BASMCs to interferon gamma (IFN-γ) or lipopolysaccharide (LPS) also stimulated NO release but did not affect
l
-arginine transport. In contrast, incubation of BAECs with TNF-α or LPS strikingly enhanced
l
-arginine uptake (2.5-fold), whereas IL-1β and IFN-γ had no effect. Treatment of BAECs with any of the inflammatory mediators did not stimulate NO production. These results demonstrate that
l
-arginine uptake and NO synthesis by these cells are differentially regulated. In BASMCs, the coinduction of
l
-arginine transport and NO formation may function to provide increased levels of substrate to the cell during activation of the NO synthase enzyme. In contrast, the selective stimulation of
l
-arginine uptake in BAECs indicates that
l
-arginine transport is dissociated from NO generation in these cells.
Structure and Characterization of the 5′-Flanking Region of the Mouse Smooth Muscle Myosin Heavy Chain (SM1/2) Gene
Authors: Watanabe Masafumi , Sakomura Yasunari , Kurabayashi Masahiko , Manabe Ichiro , Aikawa Masanori , Kuro-o Makoto , Suzuki Toru , Yazaki Yoshio , Nagai Ryozo
Published: June 2012

Abstract
We have previously shown that smooth muscle myosin heavy chain isoforms (SMs), including SM1, SM2, and SMemb, are differentially expressed during vascular development and in vascular lesions, such as atherosclerosis. The SM1/2 gene is expressed exclusively in smooth muscle cells and generates SM1 and SM2 mRNAs by alternative splicing. Whereas SM1 is constitutively expressed from early development, SM2 appears only after birth. In this study, we have isolated and characterized the 5′-flanking region of the mouse SM1/2 gene. Transient transfection assays using a series of promoter-luciferase chimeric constructs demonstrated that tandem elements of the CCTCCC sequence, located at −89 and −61 bp relative to the transcription start site, were essential for transcriptional activity of the SM1/2 gene in primary cultured rabbit aortic smooth muscle cells and smooth muscle cell lines derived from the rabbit aorta but not in non–smooth muscle cells. Gel mobility shift assays indicated that CCTCCC was a binding site for nuclear proteins prepared from smooth muscle cells. Double-stranded oligonucleotides containing either the CACC box or the Sp1 consensus sequence efficiently competed with the CCTCCC elements for binding the nuclear extracts. Site-specific mutations of CCTCCC elements resulted in a significant reduction of the promoter activity. Moreover, CCTCCC elements are evolutionary conserved between mouse and rabbit. In conclusion, the results of this study indicate an important role for the interaction of the CCTCCC sequence with Sp1 or related factors in activating transcription from the SM1/2 gene promoter.
Lidocaine Block of LQT-3 Mutant Human Na
+
Channels
Authors: An R.-H. , Bangalore R. , Rosero S.Z. , Kass R.S.
Published: June 2012

In transiently transfected mammalian cells we have identified pharmacological consequences of a naturally occurring deletion mutation, ΔKPQ, of the human heart Na
+
channel α subunit that previously has been linked to one form of the long QT syndrome, an inherited heart disease. Our results show that the Class I
B
antiarrhythmic agent lidocaine blocks maintained inward current through and slows recovery from inactivation of ΔKPQ-encoded Na
+
channels. Block is greater for maintained than for peak current. Because incomplete inactivation of mutant Na
+
channels is now thought to underlie the prolonged ventricular action potential, which is the phenotype of this disease, and we find that the ΔKPQ mutation speeds the recovery from inactivation of drug-free mutant channels, our results provide evidence, for the first time, that clinically relevant dysfunctional properties of an ion channel can be selectively targeted on the basis of the molecular properties conferred on the channel by an inherited genetic disorder.
Calcium Preconditioning Elicits Strong Protection Against Ischemic Injury via Protein Kinase C Signaling Pathway
Authors: Miyawaki Hiroshi , Zhou Xiaobo , Ashraf Muhammad
Published: June 2012

We tested the hypothesis that elevation of [Ca
2+
]
i
during Ca
2+
preconditioning (CPC) is a strong activator of protein kinase C (PKC) and confers unique protection against ischemic injury. CPC consisted of three cycles of Ca
2+
depletion (1 minute each) and Ca
2+
repletion (5 minutes each). Langendorff-perfused rat hearts were subjected to 40 minutes of global ischemia followed by 30 minutes of reperfusion. Significant functional recovery and decreased lactate dehydrogenase release were observed in CPC hearts compared with ischemic control hearts. In addition, ATP contents were significantly higher and cell structure was better preserved in CPC hearts than in ischemic control hearts. Administration of chelerythrine, a specific PKC inhibitor, completely abolished the CPC-induced cardioprotection. In other groups, in which Ca
2+
influx during CPC was inhibited with verapamil, amiloride, and low Na
+
perfusion, cardioprotection was significantly reduced. The prominent increase in the membrane PKC activity after CPC was in agreement with immunolocalization of PKC-α and PKC-δ in the cell membrane of CPC hearts. These results demonstrate that (1) a transient increase in [Ca
2+
]
i
is a prominent feature of CPC and is a strong stimulus for the activation of PKC, (2) the elevation of [Ca
2+
]
i
likely occurs via an L-type Ca
2+
channel and Na
+
-Ca
2+
exchanger, and (3) PKC plays a crucial role in the subcellular mechanisms of protection by CPC.
Activation of Purinergic Receptors Triggers Oscillatory Contractions in Adult Rat Ventricular Myocytes
Authors: Zhang Bin-Xian , Ma Xiuye , McConnell Bradley K. , Damron Derek S. , Bond Meredith
Published: June 2012

Extracellular ATP is an important neurotransmitter that modulates cardiac function by activation of purinergic receptors. In this study, the effect of P
2
purinergic receptor activation on contractions and on [Ca
2+
]
i
was investigated in adult rat ventricular myocytes. Fura 2 was used to measure [Ca
2+
]
i
, and video edge detection was used to measure contraction. Superfusion of 2-methylthio-adenosine-5′-triphosphate (2-M-S-ATP) over quiescent myocytes induced oscillations in contraction and in [Ca
2+
]
i
. The frequency of the oscillatory contractions increased with increasing concentrations of 2-M-S-ATP, but the amplitude of contractions varied from cell to cell and was independent of the concentration of 2-M-S-ATP. During electrical stimulation, activation of purinergic receptors in myocytes potentiated the amplitude of contraction and induced arrhythmias. In populations of quiescent myocytes, the plateau phase of the [Ca
2+
]
i
signal evoked by 2-M-S-ATP could be shown to represent summed oscillations in [Ca
2+
]
i
in individual cells. Pretreatment of quiescent myocytes with thapsigargin or caffeine reduced or abolished the oscillations in contractions and in [Ca
2+
]
i
triggered by 2-M-S-ATP, indicating a dependence of the oscillations on uptake and release of Ca
2+
by the sarcoplasmic reticulum. These data demonstrate the novel phenomenon that activation of purinergic receptors in quiescent myocytes stimulates oscillations in [Ca
2+
]
i
and contraction. In electrically stimulated myocytes, activation of purinergic receptors triggers oscillatory contractions and potentiates the amplitude of electrically triggered contractions.
Distribution of cAMP-Activated Chloride Current and CFTR mRNA in the Guinea Pig Heart
Authors: James Andrew F. , Tominaga Tomoko , Okada Yasunobu , Tominaga Makoto
Published: June 2012

Guinea pig ventricular myocytes exhibit a Cl
−
-selective current regulated by the cAMP-dependent pathway. We have investigated the distribution of cAMP-activated Cl
−
channel current density and cystic fibrosis transmembrane-conductance regulator (CFTR) mRNA in three regions of the guinea pig heart: the atrium, and the epicardium and endocardium of the free wall of the left ventricle. The regional differences in the Cl
−
current density were investigated in enzymatically isolated myocytes using the whole-cell patch-clamp technique. Forskolin (1 μmol/L) activated Cl
−
-selective currents in all ventricular myocytes and 21% of atrial myocytes examined. The conductance density, estimated as the outward chord conductance normalized to cell capacitance, was greatest in epicardial myocytes (79.8±8.4 pS/pF, n=21) and significantly lower in endocardial (59.8±9.5 pS/pF, n=22) and atrial (10.9±5.0 pS/pF, n=38) myocytes. The regional differences in CFTR mRNA expression levels were investigated by competitive reverse-transcribed polymerase chain reaction. The regional distribution of the mRNA levels was similar to that of the Cl
−
conductance density, ie, highest in the epicardium (23 230±1840 molecules/μg total RNA, n=3), significantly lower in endocardium (10 610±780 molecules/μg total RNA, n=3), and lowest in atrium (1450±290 molecules/μg total RNA, n=3). The data indicate that regional differences in CFTR mRNA expression in the guinea pig heart are responsible, at least in part, for the regional differences in cAMP-activated Cl
−
current density.
Maximal Actomyosin ATPase Activity and In Vitro Myosin Motility Are Unaltered in Human Mitral Regurgitation Heart Failure
Authors: Nguyen The-Tin T. , Hayes Eric , Mulieri Louis A. , Leavitt Bruce J. , ter Keurs Henk E.D.J. , Alpert Norman R. , Warshaw David M.
Published: June 2012

Myofibrillar but not actomyosin ATPase is depressed in failing myocardium from patients with dilated cardiomyopathy. Since there is a similar depression of myofibrillar ATPase in mitral regurgitation myocardium, we investigated whether or not the hydrolytic and mechanical performances of myosin are altered by comparing the maximal actomyosin ATPase activity and the in vitro myosin motility of myocardial myosin from patients with mitral regurgitation heart failure with that of patients with normal ventricular function. The results show that there is no significant difference (
P
>.05) between nonfailing and failing values for either the maximal actomyosin ATPase activity (0.3 s
−1
·head
−1
) or the myosin motility (1 μm/s). These observations suggest that changes, other than in the myosin heavy chain, contribute to the altered myocardial performance in mitral regurgitation myocardium.
Heparin-Binding Epidermal Growth Factor-Like Growth Factor Regulates Fibroblast Growth Factor-2 Expression in Aortic Smooth Muscle Cells
Authors: Peifley Kimberly A. , Alberts Gregory F. , Hsu Debbie K.W. , Feng Sheau-Line Y. , Winkles Jeffrey A.
Published: June 2012

Heparin-binding epidermal growth factor–like growth factor (HB-EGF) is a vascular smooth muscle cell (SMC) mitogen and chemotactic factor that is expressed by endothelial cells, SMCs, monocytes/macrophages, and T lymphocytes. Both the membrane-anchored HB-EGF precursor and the secreted mature HB-EGF protein are biologically active; thus, HB-EGF may stimulate SMC growth via autocrine, paracrine, and juxtacrine mechanisms. In the present study, we report that HB-EGF treatment of serum-starved rat aortic SMCs can induce fibroblast growth factor (FGF)-2 (basic FGF) gene expression but not FGF-1 (acidic FGF) gene expression. Increased FGF-2 mRNA expression is first detectable at 1 hour after HB-EGF addition, and maximal FGF-2 mRNA levels, corresponding to an ≈46-fold level of induction, are present at 4 hours. The effect of HB-EGF on FGF-2 mRNA levels appears to be mediated primarily by a transcriptional mechanism and requires de novo synthesized proteins. HB-EGF induction of FGF-2 mRNA levels can be inhibited by treating cells with the anti-inflammatory glucocorticoid dexamethasone or the glycosaminoglycan heparin. Finally, Western blot analyses indicate that HB-EGF–treated SMCs also produce an increased amount of FGF-2 protein. These results indicate that HB-EGF expressed at sites of vascular injury or inflammation in vivo may upregulate FGF-2 production by SMCs.
Attenuated Glycogenolysis Reduces Glycolytic Catabolite Accumulation During Ischemia in Preconditioned Rat Hearts
Authors: Weiss Robert G. , de Albuquerque Cicero P. , Vandegaer Koenraad , Chacko V.P. , Gerstenblith Gary
Published: June 2012

Prior transient episodes of ischemia (“ischemic preconditioning”) reduce lactate accumulation and attenuate acidosis during a subsequent prolonged ischemic insult. The mechanisms responsible for attenuated glycolytic catabolite accumulation have not been established but may include earlier exhaustion of glycogen stores, slowed glycogenolysis before complete glycogen depletion, and/or inhibition of glycolysis. Simultaneous repeated measures of myocardial glycogen and the rates of glycolysis, glycogenolysis, glucose utilization, and glycolytic ATP production were obtained during total ischemia by
13
C nuclear magnetic resonance spectroscopy in control and ischemia-preconditioned isolated rat hearts. Both [
13
C]glycolytic and [
13
C]glycogenolytic rates were significantly lower during total ischemia in preconditioned compared with control hearts (0.77±0.04 versus 1.06±0.06 μmol/min per gram wet weight [
P
<.01] for glycolysis and 0.15±0.07 versus 0.78±0.12 μmol/min per gram wet weight [
P
<.001] for glycogenolysis, respectively, at 2.5 minutes of ischemia). Slowed glycolysis was present even during the early minutes of ischemia, when significant amounts of available [
13
C]glycogen were still present. Importantly, the reduction in the rate of glycogenolysis was larger and out of proportion to the reduction in glycolysis and occurred despite an increase in glucose utilization in preconditioned hearts (2.23±0.15 versus 1.5±0.10 μmol/min per gram wet weight at 1.25 minutes,
P
<.01). During early ischemia, conversion of glycogen phosphorylase to the
a
or “active” form was less in preconditioned than in control hearts (29.1±2.6% versus 41.2±9.8%, respectively;
P
<.05). Taken together, these findings demonstrate that ischemic preconditioning significantly depresses glycolytic catabolite accumulation during sustained ischemia not by more severe glycolytic inhibition or exhaustion of glycogen stores but by depressed glycogenolysis from the onset of ischemia.
Increased Nitric Oxide Synthesis and Action Preclude Choroidal Vasoconstriction to Hyperoxia in Newborn Pigs
Authors: Hardy Pierre , Peri Krishna G. , Lahaie Isabelle , Varma Daya R. , Chemtob Sylvain
Published: June 2012

We tested the hypothesis that hyperoxia does not cause adequate constriction of choroidal vessels of the newborn (1 to 5 days old) pig, resulting in increased O
2
delivery to the retina, possibly due to excess production and/or effects of vasodilators such as nitric oxide (NO). Hyperoxia (100% O
2
, 45 minutes) led to a decrease in retinal blood flow (RBF) of both newborn and juvenile (5 to 6 weeks old) pigs and also reduced choroidal blood flow (ChBF) in juvenile but not in newborn pigs; the absence of hyperoxia-induced ChBF response in the newborn was associated with a rise in choroidal O
2
delivery. Ibuprofen (prostaglandin G/H synthase inhibitor) and 1,3-dimethyl-2-thiourea (a free radical scavenger) did not modify the choroidal hemodynamic responses to hyperoxia in newborn pigs. However, in newborn animals treated with the NO synthase (NOS) inhibitor
N
G
-nitro-
l
-arginine methyl ester (L-NAME), hyperoxia caused a decrease in blood flow and O
2
delivery to the choroid. Consistent with these effects of L-NAME, hyperoxia induced an increase in choroidal cGMP in newborn pigs ventilated with 100% O
2
and stimulated nitrite production in isolated choroids exposed to hyperoxia from newborn but not juvenile pigs; these effects were inhibited by NOS blockers. Also, both constitutive and inducible NOS activities were higher in choroidal tissues from newborn than from juvenile animals. In addition, the vasorelaxant effect of the NO donor sodium nitroprusside in vitro was also greater on choroids from newborn than from juvenile pigs. Finally, L-NAME prevented the hyperoxia-induced increase in peroxidation products in the choroid of newborns. It is concluded that hyperoxia does not lead to a decrease in blood flow and O
2
delivery to the choroid of the newborn because of increased NO synthesis and effects; since the choroid is the main source of O
2
supply to the retina, the present data contribute in providing an explanation for the increased susceptibility of the immature neonate to hyperoxia-induced retinopathy.
Vasoactive Substances Regulate Vascular Smooth Muscle Cell Apoptosis
Authors: Pollman Matthew J. , Yamada Takehiko , Horiuchi Masatsugu , Gibbons Gary H.
Published: June 2012

This study tests the hypothesis that the control of vascular smooth muscle cell (VSMC) apoptosis is regulated by the antagonistic balance between vasoactive substances such as NO and angiotensin II (Ang II). Moreover, it is postulated that the cellular signaling pathways involved in regulating vessel tone are also coupled to the regulation of programmed cell death. Using an in vitro model system, we documented that the addition of NO donor molecules
S
-nitroso-
N
-acetylpenicillamine or sodium nitroprusside to VSMC dose-dependently induced apoptosis as documented by DNA laddering and quantified by analysis of cellular chromatin morphology. The mediator role of the guanylate cyclase signaling pathway in NO-induced apoptosis was evidenced by (1) induction of apoptosis by the 8-bromo-cGMP analogue, (2) potentiation of NO-induced apoptosis by cGMP-specific phosphodiesterase inhibition, and (3) the prevention of NO-induced apoptosis by the inhibition of the cGMP-dependent protein kinase Iα. In contrast, Ang II directly antagonized NO donor– and cGMP analogue–induced apoptosis via activation of the type I Ang II receptor. These findings suggest that the countervailing balance between NO and Ang II may determine the overall cell population within the vessel wall by regulating genetic programs determining cell death as well as cell growth.