bluuebunny/crossref_metadata_2025 huggingface dataset:
[Ca
2+
]
i
Inhibition of K
+
Channels in Canine Pulmonary Artery
Authors: Post Joseph M. , Gelband Craig H. , Hume Joseph R.
Published: June 2012

Abstract
Experiments were performed on smooth muscle cells isolated from canine pulmonary artery to identify the type of K
+
channel modulated by hypoxia and examine the possible role of [Ca
2+
]
i
in hypoxic K
+
channel inhibition. Whole-cell patch-clamp experiments revealed that hypoxia (induced by the O
2
scavenger, sodium dithionite) reduced macroscopic K
+
currents, an effect that could be prevented by strong intracellular buffering of [Ca
2+
]
i
. The inhibitory effects of hypoxia were mimicked by acute exposure of cells to caffeine and could be prevented by caffeine pretreatment, suggesting an important obligatory role of [Ca
2+
]
i
in hypoxic inhibition of K
+
currents. Exposure of cells to low concentrations of 4-aminopyridine (4-AP, 1 mmol/L) prevented hypoxic inhibition of macroscopic K
+
currents, whereas low concentrations of tetraethylammonium were without effect, suggesting that the target K
+
channel inhibited by hypoxia is a voltage-dependent delayed rectifier K
+
channel, which is inhibited by [Ca
2+
]
i
. Hypoxia failed to consistently modify the activity of large-conductance (118 picosiemens [pS] in physiological K
+
) Ca
2+
-activated K
+
channels in inside-out membrane patches but reduced open probability of smaller-conductance (25-pS) delayed rectifier K
+
channels in cell-attached membrane patches. In inside-out membrane patches, 1 μmol/L Ca
2+
added to the cytoplasmic surface significantly reduced open probability of small-conductance (25-pS) 4-AP–sensitive delayed rectifier K
+
channels. Whole-cell current measurements using symmetrical K
+
to increase driving force for small currents active near the cell’s resting membrane potential revealed the presence of a 4-AP–sensitive K
+
current that activated near −65 mV and was inhibited by hypoxia. Simultaneous measurements of changes in [Ca
2+
]
i
, using the Ca
2+
indicator indo 1, and membrane potential revealed that hypoxia causes an initial rise of [Ca
2+
]
i
, which precedes hypoxia-induced membrane depolarization. It is concluded that in canine pulmonary arterial cells an early key event in hypoxic pulmonary vasoconstriction is release of Ca
2+
from caffeine-sensitive intracellular Ca
2+
stores, which causes inhibition of delayed rectifier K
+
channels and membrane depolarization, possibly leading to subsequent activation of Ca
2+
entry through voltage-dependent Ca
2+
channels.
Differential Activation of Mitogen-Activated Protein Kinases by H
2
O
2
and O
2
−
in Vascular Smooth Muscle Cells
Authors: Baas Arnold S. , Berk Bradford C.
Published: June 2012

Abstract
Increased generation of active oxygen species such as H
2
O
2
and O
2
−
may be important in vascular smooth muscle cell growth associated with atherosclerosis and restenosis. In previous work, we showed that H
2
O
2
stimulated vascular smooth muscle cell growth and proto-oncogene expression. In the present study, we compared the effects of H
2
O
2
and O
2
−
on cultured rat aortic vascular smooth muscle cell growth and signal transduction. O
2
−
was generated in a concentration-dependent manner by the naphthoquinolinedione LY83583. Vascular smooth muscle cell growth, as measured by [
3
H]thymidine incorporation, was stimulated by 200 μmol/L H
2
O
2
(110% increase versus 0.1% serum) and 1 μmol/L LY83583 (175% increase) to levels comparable to 10 ng/mL platelet-derived growth factor (210% increase). Since activation of mitogen-activated protein kinase (MAP kinase) is one of the earliest growth factor signal events, the activity of MAP kinase was measured by changes in mobility on Western blot and by phosphorylation of myelin basic protein. There was a concentration-dependent increase in MAP kinase activity by LY83583 (maximum, 10 μmol/L) but not by H
2
O
2
. The time course for activation of MAP kinase by LY83583 showed a maximum at 5 to 10 minutes with return to baseline by 20 minutes. Activation of MAP kinase by LY83583 was protein kinase C dependent. Expression of MAP kinase phosphatase-1 (MKP-1), a transcriptionally regulated redox-sensitive protein tyrosine/threonine phosphatase, was also measured. Although H
2
O
2
induced MKP-1 mRNA to a greater extent than did LY83583, the increased MKP-1 expression could not explain the inability of H
2
O
2
to stimulate MAP kinase, because mRNA levels were not detected until 60 minutes. The findings that both O
2
−
and H
2
O
2
stimulate vascular smooth muscle cell growth but only O
2
−
rapidly activates MAP kinase suggest that additional signal events are required for the mitogenic effects of H
2
O
2
.
Adenovirus-Mediated Gene Transfer In Vivo to Cerebral Blood Vessels and Perivascular Tissue
Authors: Ooboshi Hiroaki , Welsh Michael J. , Rios C. David , Davidson Beverly L. , Heistad Donald D.
Published: June 2012

Abstract
Gene transfer to blood vessels in vivo generally requires interruption of blood flow. Thus, gene transduction to cerebral blood vessels in vivo has not yet been achieved. In this study, we injected replication-deficient adenovirus into cerebrospinal fluid in an attempt to transduce genes to cerebral blood vessels. Recombinant adenovirus (1×10
9
infectious units) expressing nuclear-targeted bacterial β-galactosidase driven by the cytomegalovirus promoter was injected into the cisterna magna of Sprague-Dawley rats. The brains were examined histochemically after staining with 5-bromo-4-chloro-3-indolyl-β-
d
-galactopyranoside 1 to 7 days after injection of adenovirus. Leptomeningeal cells overlying the major arteries were efficiently transduced, and adventitial cells of large vessels and smooth muscle cells of small vessels were occasionally stained. β-Galactosidase was expressed on days 1 and 3 after injection but was undetectable by day 7. Expression of the gene was ‘targeted’ by altering the position of the head. When viral suspension was injected while the rat was in a nose-down position, the reporter gene was expressed extensively on the ventral surface of the brain, especially along the circle of Willis. When the position was changed to the nose-up or lateral position, the inferior or lateral region of the brain was stained primarily. Administration of the virus into the lateral ventricle provided extensive expression in ependymal cells and leptomeninges with some transduction to cerebral blood vessels. Thus, adenovirus injected into cerebrospinal fluid provides gene transfer in vivo to cerebral blood vessels and, with greater efficiency, to perivascular tissue. Furthermore, cisternal delivery may target specific brain regions by positioning of the head. This approach may be useful for studies of cerebral vascular biology and cerebral vascular gene therapy.
Identification of a
cis
-Acting Glucocorticoid Responsive Element in the Rat Angiotensin II Type 1A Promoter
Authors: Guo Deng-Fu , Uno Shusei , Ishihata Akira , Nakamura Norifumi , Inagami Tadashi
Published: June 2012

Abstract
Enhanced vascular responsiveness to angiotensin II at the AT
1
receptor has been considered one of the major contributing factors to vascular hypertrophy and high blood pressure. The transcription of the rat angiotensin II type 1A receptor gene is stimulated by glucocorticoids. To clarify the molecular mechanism for glucocorticoid action in rat vascular smooth muscle cells, we investigated the effects of dexamethasone on the promoter activity of the angiotensin II type 1A receptor by using promoter/luciferase reporter gene constructs and heterologous context constructs (containing the thymidine kinase promoter) in transfected vascular smooth muscle cells (<12 passages). There are three putative glucocorticoid responsive elements (GREs) in the promoter. However, only one GRE was found to respond to dexamethasone (1 μmol/L) and was located at positions −756 to −770 bp upstream from the transcription initiation site. When compared with the consensus sequence of GRE, 9 of 12 bases were identical. RU38486, a glucocorticoid antagonist, completely blocked the induction by dexamethasone, suggesting that the GRE was functional through a specific glucocorticoid receptor. The response to dexamethasone was lost in vascular smooth muscle cells at higher passage numbers (>8 passages) but was restored when the cells were transfected with a glucocorticoid-receptor expression construct. This finding provided additional support that the response to dexamethasone was mediated by the glucocorticoid receptor. The gel mobility supershift assay showed that the GRE binds in vitro–translated rat glucocorticoid receptors in a specific manner. Compared with the angiotensin II type 1A receptor promoter, no effect by dexamethasone was observed in vascular smooth muscle cells transfected with the angiotensin II type 1B receptor promoter/luciferase reporter gene constructs. We conclude from these experiments that the dexamethasone-induced increase in the transcription of the angiotensin II type 1A receptor gene occurred through the binding of GRE to the glucocorticoid-specific receptor.
Formation of F
2
-Isoprostanes During Oxidation of Human Low-Density Lipoprotein and Plasma by Peroxynitrite
Authors: Moore Kevin P. , Darley-Usmar Victor , Morrow Jason , Roberts L. J.
Published: June 2012

Abstract
F
2
-Isoprostanes are novel bioactive prostaglandin F
2
–like compounds produced by nonenzymatic free radical–catalyzed peroxidation of arachidonic acid. F
2
-Isoprostanes are initially formed in situ on phospholipids and subsequently released. Quantification of the F
2
-isoprostanes has been found to represent a valuable and reliable marker of lipid peroxidation. Oxidative modification of low-density lipoprotein (LDL) is a key process for the recognition of LDL by the scavenger receptors on macrophages. The oxidative mechanism responsible for the modification of LDL in vivo remains unclear, but an attractive candidate is the powerful oxidant peroxynitrite, which can be formed by reaction of nitric oxide and superoxide in the vessel wall. To further explore the potential role of peroxynitrite in the oxidative modification of plasma lipids, we investigated whether incubation of LDL and plasma with peroxynitrite or SIN-1, which decomposes to form nitric oxide and superoxide, catalyzes the formation of F
2
-isoprostanes. Incubation of LDL with peroxynitrite (0.125 to 1 mmol/L) or SIN-1 (0.5 and 1 mmol/L) induced a concentration-dependent increase in the formation of F
2
-isoprostanes, reaching a maximum of 5.5±2.05-fold (SEM) and 18.2±4.0-fold above control values, respectively. The increase of F
2
-isoprostanes induced by SIN-1 was essentially completely inhibited by superoxide dismutase. Incubation of plasma with peroxynitrite or SIN-1 yielded similar results. These results indicate that peroxynitrite can induce the formation of F
2
-isoprostanes in lipoproteins. Since F
2
-isoprostanes can exert potent biological activity such as vasoconstriction, they may contribute to the vascular pathobiology associated with atherosclerosis.
Differential Expression of Voltage-Gated K
+
Channel Subunits in Adult Rat Heart
Authors: Barry Dianne M. , Trimmer James S. , Merlie John P. , Nerbonne Jeanne M.
Published: June 2012

Abstract
Polyclonal antibodies against each of the K
+
channel subunits (Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2) shown previously to be expressed in adult rat heart at the mRNA level were used to examine the distributions of these K
+
channel subunits in adult rat atrial and ventricular membranes. Immunohistochemistry on isolated adult rat ventricular myocytes revealed strong labeling with the anti-Kv4.2 and anti-Kv1.2 antibodies. Although somewhat weaker (than with anti-Kv1.2 or anti-Kv4.2), positive staining was also observed with the anti-Kv1.5 and anti-Kv2.1 antibodies. Ventricular myocytes exposed to the anti-Kv1.4 antibody, in contrast, did not appear significantly different from background. Qualitatively similar results were obtained on isolated adult rat atrial myocytes. Western blots of atrial and ventricular membrane proteins confirmed the presence of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 and revealed differences in the relative abundances of these subunits in the two membrane preparations. Kv4.2, for example, is more abundant in ventricular than in atrial membranes, whereas Kv1.2 and Kv2.1 are higher in atrial membranes; Kv1.5 levels are comparable in the two preparations. In contrast to these results, nothing was detected in Western blots of atrial or ventricular membrane proteins with the anti-Kv1.4 antibody at concentrations that revealed intense labeling of a 97-kD protein in adult rat brain membranes. A very faint band was detected at 97 kD in the atrial and ventricular preparations when the anti-Kv1.4 antibody was used at a 5- to 10-fold higher concentration. The simplest interpretation of these results is that Kv1.4 is not an abundant protein in adult rat atrial or ventricular myocytes. Therefore, it seems unlikely that Kv1.4 plays an important role in the formation of functional depolarization-activated K
+
channels in these cells. The relation(s) between the (other four) K
+
channel subunits and the depolarization-activated K
+
channels identified electrophysiologically in adult rat atrial and ventricular myocytes is discussed in the present study.
Interaction Between Strong Electrical Stimulation and Reentrant Wavefronts in Canine Ventricular Fibrillation
Authors: Bonometti Claudio , Hwang Chun , Hough Dustan , Lee John J. , Fishbein Michael C. , Karagueuzian Hrayr S. , Chen Peng-Sheng
Published: June 2012

Abstract
This study was designed to test the hypothesis that the effects of a strong electrical stimulus on reentrant wavefronts in ventricular fibrillation (VF) are dependent on the timing of the stimulus. We studied six open-chest dogs by computerized mapping techniques. A plaque electrode array with up to 509 bipolar electrodes was placed on the right ventricular epicardium. The interelectrode distance was 1.6 mm, and the interpolar distance was 0.5 mm. After eight baseline pacing stimuli (S
1
) with cycle lengths of 300 ms, a strong premature stimulus (S
2
) (73±10 mA) was given to induce VF. In subsequent episodes, a second strong premature stimulus (S
3
) was given at progressively longer S
2
-S
3
intervals in 20-ms increments. The results showed that, at baseline, the S
2
consistently induced figure-eight reentry and VF. The VF cycle length immediately after the S
2
averaged 108±17 ms. The S
3
resulted in one of the following responses: (1) termination of reentry and VF; (2) induction of different reentrant wavefronts or a focal pattern of repetitive activation; or (3) persistence of the same figure-eight reentry. The intervals between the S
3
and the immediately preceding activation at the site of the S
3
(the recovery intervals) were 39±12 ms (range, 20 to 60 ms) and 61±20 ms (range, 30 to 90 ms) for response patterns 1 and 2, respectively. The recovery intervals associated with response pattern 3 were either ≤30 ms (22±8 ms) or ≥80 ms (94±15 ms). The differences among these four intervals were significant (
P
<.001). We conclude that the effects of strong electrical stimulation on the reentrant wavefronts in VF are dependent on the recovery interval since the previous local activation. A protective zone occurred between 20 and 60 ms, during which time a strong electrical stimulus could terminate reentry and abort VF. This zone was followed by a vulnerable period during which new activation wavefronts could be induced. If a strong electrical stimulus was given shortly after or sufficiently long after the previous local activation, the same figure-eight reentrant pattern continued.
Cardiac V
1
And V
3
Myosins Differ in Their Hydrolytic and Mechanical Activities In Vitro
Authors: VanBuren Peter , Harris David E. , Alpert Norman R. , Warshaw David M.
Published: June 2012

Abstract
The two mammalian cardiac myosin heavy chain isoforms, α and β, have 93% amino acid homology, but hearts expressing these myosins exhibit marked differences in their mechanical activities. To further understand the function of these cardiac myosins as molecular motors, we compared the ability of these myosins to hydrolyze ATP and to both translocate actin filaments and generate force in an in vitro motility assay. V
1
myosin has twice the actin-activated ATPase activity and three times the actin filament sliding velocity when compared with V
3
myosin. In contrast, the force-generating ability of these myosins is quite different when the total force produced by a small population of myosin molecules (>50) is examined. V
1
myosin produces only one half the average cross-bridge force of V
3
myosin. With discrete areas of primary structural heterogeneity known to exist between α and β heavy chains, the differences we report in the hydrolytic and mechanical activities of the motors are explored in the context of potential structural and kinetic differences between the V
1
and V
3
myosins.
Induction of Nitric Oxide Synthase Activity by Cytokines in Ventricular Myocytes Is Necessary but Not Sufficient to Decrease Contractile Responsiveness to β-Adrenergic Agonists
Authors: Ungureanu-Longrois Dan , Balligand Jean-Luc , Simmons William W. , Okada Ikutaro , Kobzik Lester , Lowenstein Charles J. , Kunkel Steven L. , Michel Thomas , Kelly Ralph A. , Smith Thomas W.
Published: June 2012

Abstract
Recent evidence has documented that increased activity of an inducible nitric oxide synthase (iNOS; type 2 NO synthase) in primary isolates of adult rat ventricular myocytes after exposure to soluble mediators in medium conditioned by lipopolysaccharide-activated macrophages is associated with a decrease in their contractile responsiveness to β-adrenergic agonists. It remained unclear which specific inflammatory cytokines in this medium contribute to the induction of iNOS activity in myocytes and whether induction of iNOS would result in an obligatory decline in contractile function. Interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) were both present in the lipopolysaccharide-activated macrophage-conditioned medium. However, only IL-1 receptor antagonist and not an anti–rat TNF-α antiserum diminished the extent of iNOS induction in myocytes exposed to this medium and prevented a decline in contractile responsiveness to isoproterenol. When recombinant cytokines were used, IL-1β, TNF-α, and IFN-γ each induced iNOS activity in cardiac myocytes at 24 hours. However, only the combination of IL-1β and IFN-γ reproducibly caused contractile dysfunction in cardiac myocytes. Among the constituents of the defined medium routinely used for maintenance of adult rat ventricular myocytes in primary culture, it was noted that insulin (10
−7
mol/L) was required for NO production, as detected by nitrite release in cytokine-pretreated myocytes, although insulin had no effect on the extent of induction of iNOS mRNA or maximal enzyme activity in myocyte cell lysates. Insulin was also required for the decrease in contractile responsiveness to isoproterenol to be manifest. Thus, induction of iNOS is necessary but not sufficient to cause inflammatory cytokine-induced contractile dysfunction in cardiac myocytes.
Cl
−
-HCO
3
−
Exchange in Developing Neonatal Rat Cardiac Cells
Authors: Korichneva Irina , Pucéat Michel , Cassoly Robert , Vassort Guy
Published: June 2012

Abstract
The Cl
−
-HCO
3
−
exchanger is the main anionic exchanger (AE) that alleviates alkaline loads in cardiac cells. We recently identified in adult ventricular cells two membrane proteins (80 and 120 kD) immunologically related to the erythroid band 3 and likely to mediate the anion exchange. In the present study, we further investigated the Cl
−
-HCO
3
−
exchanger activity concomitantly with the expression and intracellular localization of the band 3–like proteins during the development of neonatal rat cardiac cells maintained in culture for 17 days. Microspectrofluorometric measurements of pH
i
in single cells show that neonatal rat cardiomyocytes display a fully functional DIDS-sensitive Cl
−
-HCO
3
−
exchanger at early stages of development. Neither basal pH
i
nor the anion exchange activity changes with different stages of the culture. In Western blotting with an anti–whole erythroid band 3 antibody, we found both the 80- and the 120-kD band 3–like proteins in whole heart and cultured neonatal cardiac cells. The 80-kD protein was also recognized by an anti-AE1 antiserum, whereas the 120-kD protein was specifically detected by an anti–cardiac AE3 antibody. Thus, we propose that the proteins are encoded by two different genes, AE1 and AE3, respectively. Subcellular fractionation of isolated and cultured cardiomyocytes revealed the presence of both proteins in the membrane, nuclear, and myofibril fractions. The results obtained in biochemical experiments corroborate the confocal images of immunostained neonatal cells, which demonstrate perinuclear location of band 3–like proteins at an early stage of development and their appearance within myofilaments after cell maturation. Colocalization of band 3–like proteins with specific markers of the Golgi apparatus, wheat germ agglutinin and CTR433 antibody, suggests the presence of these proteins in the Golgi area. The later decoration of repetitive striations between each sarcomere indicates that the band 3–like proteins are assigned in development within a compartment similar to costameres, areas of attachment of myofibrils to sarcolemma. These areas are likely to play a major role in signal transduction of neurohormonal stimuli.
On the Molecular Nature of the Lidocaine Receptor of Cardiac Na
+
Channels
Authors: Bennett Paul B. , Valenzuela Carmen , Chen Li-Qiong , Kallen Roland G.
Published: June 2012

Abstract
The mechanism of inhibition of Na
+
channels by lidocaine has been suggested to involve low-affinity binding to rested states and high-affinity binding to the inactivated state of the channel, implying either multiple receptor sites or allosteric modulation of receptor affinity. Alternatively, the lidocaine receptor may be guarded by the channel gates. To test these distinct hypotheses, inhibition of Na
+
channels by lidocaine was studied by voltage-clamp methods in both native and heterologous expression systems. Native Na
+
channels were studied in guinea pig ventricular myocytes, and recombinant human heart Na
+
channels were expressed in
Xenopus laevis
oocytes. Fast inactivation was eliminated by mutating three amino acids (isoleucine, phenylalanine, and methionine) in the III-IV interdomain to glutamines or by enzymatic digestion with α-chymotrypsin. In channels with intact fast inactivation, lidocaine block developed with a time constant of 589±42 ms (n=7) at membrane potentials between −50 and +20 mV, as measured by use of twin pulse protocols. The IC
50
was 36±1.8 μmol/L. Control channels inactivated within 20 ms, and slow inactivation developed much later (time constant of slow inactivation, 6.2±0.36 s). The major component of block developed long after activated and open channels were no longer available for drug binding. Control channels recovered fully from inactivation in <50 ms at −120 mV (time constant, 11±0.5 ms; n=50). In 30 μmol/L lidocaine, 49±3% of the current recovered with a second larger time constant of 398±46 ms (n=5). After removal of fast inactivation, either enzymatically or by mutagenesis, the channels were no longer blocked by 50 μmol/L lidocaine (n=8). Much higher concentrations of lidocaine produced a tonic block (IC
50
, 0.4±0.07 mmol/L; n=13) and time dependence suggestive of open-channel block. The results indicate the importance of inactivated state block by therapeutic concentrations of lidocaine and suggest that the molecular site of action is the structural region of the channel that is responsible for inactivation.
Adrenomedullin as a Novel Antimigration Factor of Vascular Smooth Muscle Cells
Authors: Horio Takeshi , Kohno Masakazu , Kano Hiroaki , Ikeda Miwako , Yasunari Kenichi , Yokokawa Koji , Minami Mieko , Takeda Tadanao
Published: June 2012

Abstract
The present study investigated the effect of adrenomedullin, a novel vasorelaxant peptide, on the migration of cultured rat vascular smooth muscle cells (SMCs) by using the Boyden-chamber method. Fetal calf serum (FCS) and platelet-derived growth factor (PDGF)–BB strongly stimulated SMC migration. Adrenomedullin clearly inhibited SMC migration stimulated with 5% and 10% FCS in a concentration-dependent manner. The migration induced by 10 and 25 ng/mL PDGF-BB was also inhibited by adrenomedullin in a concentration-dependent manner. Inhibition by adrenomedullin of FCS- and PDGF-induced SMC migration was paralleled by an increase in the cellular level of cAMP. In fact, the percent increase in cAMP level was strongly correlated with the percent decrease in migration activity of SMCs after treatment with adrenomedullin. 8-Bromo cAMP, a cAMP analogue, reproduced the inhibition by adrenomedullin of FCS- and PDGF-induced SMC migration. An activator of adenylate cyclase, forskolin, also reduced FCS- and PDGF-induced SMC migration. These data indicate that adrenomedullin inhibits the migration of SMCs stimulated with FCS and PDGF, probably through a cAMP-dependent process. On the basis of these results and the finding that adrenomedullin is synthesized in and secreted from vascular endothelial cells, adrenomedullin may play a role as a local antimigration factor in some pathophysiological states.
An Isolated Cardiac Conduction Disease Maps to Chromosome 19q
Authors: de Meeus Anne , Stephan Edouard , Debrus Sophie , Jean Marie-Kamala , Loiselet Jacques , Weissenbach Jean , Demaille Jacques , Bouvagnet Patrice
Published: June 2012

Abstract
Isolated cardiac conduction disease is an autosomal dominant defect that includes various combinations of bundle branch or fascicular blocks. These defects can cause sudden death due to a complete heart block. We used a genome-wide screening approach with polymorphic (CA)n repeat markers to determine the chromosomal position of the gene defect implicated in this disorder. The analyses were carried out on a large Lebanese kindred, which included individuals with either a complete or incomplete right bundle branch block (RBBB) with a vertical-axis deviation (≤−30 or ≥+100). Linkage to the disease locus was detected with the polymorphic marker D19S604 on the q arm of chromosome 19 (19q13.3) with a multipoint lod score of 7.18. Additionally, we were able to exclude the flanking loci D19S606 and D19S571, which are 13 cM apart because of recombination events in three affected individuals. The histidine-rich calcium-binding protein gene is found in this region and is an attractive candidate gene on the basis of its physiological properties and a tight linkage. There is no expansion in two exon 1 regions known for a variable number of triplet repeats.
Unique Conductance, Gating, and Selective Permeability Properties of Gap Junction Channels Formed by Connexin40
Authors: Beblo Dolores A. , Wang Hong-Zhan , Beyer Eric C. , Westphale Eileen M. , Veenstra Richard D.
Published: June 2012

Abstract
Connexin40 is selectively expressed in specialized cardiac conduction (nodal and His-Purkinje) tissues and the atrium, yet the channel properties formed by this gap junction protein have not been investigated. The conductance, gating, and selective permeability of rat connexin40 (Cx40) gap junction channels between pairs of Cx40-transfected mouse neuroblastoma (N2A) cells in culture were studied by using dual whole-cell voltage-clamp techniques. The macroscopic steady state junctional conductance gating was dependent on transjunctional voltage with a Boltzmann half-inactivation voltage of ±50 mV, a residual voltage-insensitive normalized junctional conductance of 35% of maximum, and a gating charge valence of 3. In the presence of 120 mmol/L potassium glutamate, the slope conductance of single rat Cx40 gap junction channels measured 158±2 pS (n=4). Lower conductance states equal to 21% to 48% of the main open-state conductance were also occasionally observed in two of the four cell pairs. Multichannel open probabilities were found to be heterogeneous. Ion substitution and dye transfer experiments were performed to determine the relative chloride/potassium conductance and dye permeability of anionic fluorescein derivatives in rat Cx40 channels. The rat Cx40 channel had a maximum conductance of 180±18 pS (n=3) in 120 mmol/L KCl and a detectable chloride permeability of 0.29 relative to potassium, indicating some selectivity for cations over anions. Cx40 gap junctions were permeable to 2′,7′-dichlorofluorescein (diCl-F) and also to the more polar 6-carboxyfluorescein dye; however, diCl-F dye transfer was not observed to increase with increasing junctional conductance.
Direct Effects of Smooth Muscle Relaxation and Contraction on In Vivo Human Brachial Artery Elastic Properties
Authors: Bank Alan J. , Wilson Robert F. , Kubo Spencer H. , Holte James E. , Dresing Thomas J. , Wang Hongyu
Published: June 2012

Abstract
The direct effect of smooth muscle relaxation on arterial elastic properties is controversial. Studies in animals show both a decrease and an increase in elastic modulus. In human subjects, the contribution of smooth muscle to arterial elastic mechanics has been limited by difficulty in separating the direct effects of a vasodilator drug on the arterial wall from the indirect effects due to reduced blood pressure. The purpose of the present study was to assess the direct contribution of vascular smooth muscle to brachial artery elastic mechanics in normal human subjects in vivo. We measured brachial artery compliance and incremental elastic modulus (E
inc
) in eight normal subjects (age, 22 to 51 years) by using intravascular ultrasound. A 3.5F 30-MHz intravascular ultrasound catheter was placed through a sheath into the brachial artery, and intra-arterial pressure, cross-sectional area, and wall thickness were measured simultaneously under baseline conditions and after the administration of intra-arterial nitroglycerin (100 μg) and norepinephrine (1.2 μg). A pressurized cuff surrounding the brachial artery was inflated to reduce transmural brachial artery pressure. Using this technique, we were able to measure the following arterial characteristics for the first time in human subjects in vivo: (1) the effective unstressed arterial radius and (2) the pressure-area, stress-strain, and pressure-E
inc
relations over a wide pressure range (0 to 100 mm Hg). Intra-arterial nitroglycerin increased brachial artery area by 22% and intra-arterial norepinephrine decreased brachial artery area by 17% at 100 mm Hg transmural pressure (
P
<.001 versus baseline). Nitroglycerin produced a significant (
P
<.01) nonparallel upward shift of the compliance pressure curve compared with baseline, and norepinephrine produced a smaller downward shift of the compliance-pressure curve. Pulse-wave velocity was decreased from 15.1±1.1 m/s at baseline to 13.2±0.7 m/s at 100 mm Hg after the administration of nitroglycerin (
P
<.05). Nitroglycerin and norepinephrine also significantly shifted the brachial artery stress-strain and E
inc
-strain curves in opposite directions. However, nitroglycerin did not significantly change E
inc
under isobaric conditions. This study describes and validates a new technique for determining brachial artery elastic properties in vivo over a wide pressure range. At constant pressure, nitroglycerin-induced smooth muscle relaxation increased brachial artery compliance and decreased pulse-wave velocity without significantly altering E
inc
.
Estrogen Relaxes Coronary Arteries by Opening BK
Ca
Channels Through a cGMP-Dependent Mechanism
Authors: White Richard E. , Darkow David J. , Falvo Lang Jessica L.
Published: June 2012

Abstract
Women rarely suffer cardiovascular dysfunction before menopause, but by the age of 65 a woman becomes as vulnerable to cardiovascular mortality as a man. It has been proposed that estrogens protect against cardiovascular disease; however, the physiological basis of estrogen protection is unknown. In the present study the mechanism of estrogen-induced relaxation of coronary arteries was investigated at the tissue, cellular, and molecular levels. Tissue studies demonstrated that 17β-estradiol relaxes porcine coronary arteries by an endothelium-independent mechanism involving K
+
efflux, and subsequent studies employing the patch-clamp technique confirmed that estrogen stimulates K
+
channel gating in coronary smooth muscle. Perforated-patch recordings from metabolically intact coronary myocytes revealed that 17β-estradiol more than doubles steady state outward currents in these cells at positive voltages. Studies of on-cell patches demonstrated a potent stimulatory effect of 17β-estradiol on the gating of the large-conductance, Ca
2+
- and voltage-activated K
+
(BK
Ca
) channels, while 17α-estradiol had no effect. Furthermore, blocking BK
Ca
channels in intact arteries inhibited estrogen-induced relaxation. The effect of 17β-estradiol on BK
Ca
channels was blocked by inhibiting cGMP-dependent protein kinase (PKG) activity and was mimicked by exogenous cGMP or by stimulating PKG activity. Therefore, we propose that 17β-estradiol relaxes coronary arteries by opening BK
Ca
channels via cGMP-dependent phosphorylation. This novel mechanism could account for the hypotensive effect of estrogens and help explain, at least in part, why postmenopausal estrogen therapy lowers the risk of cardiovascular disease.
Description of a Nonselective Cation Current in Human Atrium
Authors: Crumb William J. , Pigott John D. , Clarkson Craig W.
Published: June 2012

Abstract
Ion currents were examined in isolated human atrial myocytes by using the whole-cell patch-clamp technique. When currents were recorded with a K
+
-containing pipette solution, depolarizing voltage pulses elicited a rapidly activating outward current that decayed to an apparent steady state. Exposure of cells to 10 mmol/L 4-aminopyridine markedly reduced current amplitude; however, a rapidly activating current that was ≈30% of the steady state current amplitude remained. When pipette K
+
was replaced with Cs
+
, a similar rapidly activating current that reversed polarity at ≈0 mV was recorded. This current was seen in 100% of the cells tested from 17 different hearts (n=142), and its amplitude was ≈40% of the amplitude of the steady state current recorded in the presence of pipette K
+
. The current amplitude was not significantly different in cells isolated from adult (6.31±1.35 pA/pF, n=8) and pediatric (5.54±1.04 pA/pF, n=9) hearts. Studies designed to determine the charge-carrying species indicated that changes in bath Cl
−
concentration had no effect on either the amplitude or the reversal potential of this current, whereas removal of pipette Cs
+
and bath Na
+
dramatically reduced this current. In addition, this current was not modulated by either isoproterenol (1 μmol/L, 22°C) or cell swelling. This study provides the first description of a nonselective cation current in human atrial myocytes, which may play an important role in repolarization in human atria.
Relative Densities of Muscarinic Cholinergic and β-Adrenergic Receptors in the Canine Sinoatrial Node and Their Relation to Sites of Pacemaker Activity
Authors: Beau Scott L. , Hand Dwight E. , Schuessler Richard B. , Bromberg Burt I. , Kwon Brian , Boineau John P. , Saffitz Jeffrey E.
Published: June 2012

Abstract
The site of earliest extracellular electrical activation in the sinoatrial node (SAN) is known to shift in response to autonomic stimuli, but the mechanisms underlying this phenomenon and the determinants of the location of dominant pacemaker activity have not been elucidated. The present study was designed to characterize the spatial distribution of muscarinic cholinergic and β-adrenergic receptors in the canine SAN and to determine whether a consistent relationship exists between autonomic receptor densities and the site of dominant pacemaker activity. We used quantitative light-microscopic autoradiography of radioligand binding sites to characterize the spatial distribution of muscarinic cholinergic and β-adrenergic receptor subtypes in tissue sections containing the SAN and adjacent right atrial muscle from 18 canine hearts. Muscarinic receptor density was 5.4 times greater in SAN cells than in atrial myocytes (
P
<.01). Total β-adrenergic receptor density was more than 3 times greater in SAN cells than in atrial myocytes (
P
<.0001), due entirely to the significantly greater number of β
1
-adrenergic receptors in the SAN. The region of dominant pacemaker activity, localized in 4 hearts with in vitro mapping, consistently exhibited greater densities of muscarinic and β
1
-adrenergic receptors than other SAN regions. Muscarinic receptor density in the dominant pacemaker region was 18±2% and 29±7% higher than in adjacent superior and inferior regions, respectively. β
1
-Receptor density in the dominant site was 53±5% and 26±4% higher than in adjacent superior and inferior SAN regions, respectively. Thus, the SAN is richly endowed with both muscarinic cholinergic and β
1
-adrenergic receptors. Differential responsiveness to autonomic inputs, related perhaps to regional differences in receptor density, may help determine the spatial localization of pacemaker activity.
Rapamycin Selectively Inhibits Angiotensin II–Induced Increase in Protein Synthesis in Cardiac Myocytes In Vitro
Authors: Sadoshima Junichi , Izumo Seigo
Published: June 2012

Abstract
It has been suggested that phosphorylation of a 40S ribosomal protein, S6, regulates protein synthesis. Two distinct families of S6 kinase have been identified, the
rsk
-encoded 85- to 92-kD S6 kinase (RSK) and the 70- or 85-kD S6 kinase (p70
S6K
). We have previously shown that hypertrophic stimuli, such as angiotensin II (Ang II), rapidly activate RSK in cardiac myocytes. However, RSK and p70
S6K
are regulated by distinct mechanisms, and p70
S6K
, but not RSK, is the physiological S6 kinase in vivo in other cell types. Using cultured neonatal rat ventricular myocytes, we examined whether Ang II activates p70
S6K
and investigated the effect of rapamycin, a potent yet indirect inhibitor of p70
S6K
, on the Ang II–induced hypertrophic response. Immunoblot analyses indicate that cardiac myocytes express the 70- and 85-kD forms of p70
S6K
. Ang II caused a rapid and sustained activation of p70
S6K
through the type I Ang II receptor. Rapamycin inhibited Ang II–induced activation of p70
S6K
in a dose-dependent manner, with an IC
50
of 0.14 ng/mL (0.15 nmol/L). Rapamycin did not inhibit Ang II–induced activation of tyrosine kinase, mitogen-activated protein kinase, RSK, and protein kinase C. The effect of rapamycin is unlikely to be mediated by its effect on p34
cdc2
and p33
cdk2
because Ang II did not activate these cell cycle–dependent kinases in cardiac myocytes. In contrast, a dose-dependent inhibition of p70
S6K
by rapamycin is very closely correlated with its inhibition of the Ang II–induced increase in protein synthesis. Interestingly, rapamycin did not affect the Ang II–induced activation of specific gene expression, including the immediate-early gene c-
fos
and fetal type genes, such as atrial natriuretic factor and skeletal α-actin. Moreover, rapamycin did not suppress Ang II–induced phenotypic changes at the protein level, such as increased atrial natriuretic factor secretion, expression of β-myosin heavy chain, and organization of actin into sarcomeric units. These results indicate that p70
S6K
is activated by Ang II and that a rapamycin-sensitive signaling mechanism, most likely p70
S6K
, plays an essential role in the Ang II–induced increase in overall protein synthesis but not in Ang II–induced specific phenotypic changes in cardiac myocytes.
Decreased Elastin Synthesis in Normal Development and in Long-term Aortic Organ and Cell Cultures Is Related to Rapid and Selective Destabilization of mRNA for Elastin
Authors: Johnson D.J. , Robson P. , Hew Yin , Keeley F.W.
Published: June 2012

Abstract
We have previously shown that aortic organ cultures from 1- to 3-day-old chickens initially mimic the high levels of elastin production seen in vivo. However, more prolonged incubation of these tissues results in decreased synthesis of elastin. In the present study, we demonstrate that decreased production of elastin in these aortic organ cultures is selective for elastin compared with collagen and is correlated with decreased steady state levels of mRNA for elastin. These decreases in steady state levels of elastin mRNA are due at least in part to a rapid and selective destabilization of mRNA for elastin, the half-life of which falls from ≈25 hours in fresh aortic tissues to ≈15 hours after incubation for only 8 hours. Destabilization of elastin mRNA can be prevented by incubation in the presence of blockers of DNA transcription (5,6-dichlorobenzimidazole riboside and actinomycin D) and mRNA translation (cycloheximide). Furthermore, the half-life of aortic elastin mRNA decreases from ≈25 hours in the 1-day-old chicken to ≈7 hours in the 8-week-old chicken, demonstrating that destabilization of mRNA is an important contributing factor in the decline in production of aortic elastin taking place during normal postnatal growth.
Mononuclear Leukocytes Invade Rabbit Arterial Intima During Thickening Formation via CD18- and VLA-4–Dependent Mechanisms and Stimulate Smooth Muscle Migration
Authors: Kling Dorothee , Fingerle Jürgen , Harlan John M. , Lobb Roy R. , Lang Florian
Published: June 2012

Abstract
The role of mononuclear leukocytes for the migration of smooth muscle cells (SMCs) during intimal thickening was investigated in the rabbit model of electrically stimulated carotid artery. The approach was to inhibit leukocyte entry into the arterial intima with antibodies against the adhesion molecules very late activation antigen-4 (VLA-4) and CD11/CD18. In electrically stimulated control rabbits treated either with saline or a nonspecific antibody, all types of granulocytes, monocytes, and lymphocytes migrated across an intact endothelium into the acellular subendothelial space, followed by the movement of SMCs from the media into the intima within 36 hours of applying electrical current. Treatment of the rabbits with monoclonal antibody (mAb) HP1/2 directed toward the α
4
subunit (CD49d) of VLA-4 inhibited mononuclear leukocyte invasion (consisting of monocytes and lymphocytes) by ≈70% compared with the IgG-treated control rabbits and completely abolished the minimal influx of basophils and eosinophils after 36 hours. Neutrophil infiltration, however, remained unaffected by anti–VLA-α
4
treatment. Under these conditions, SMC migration across the internal elastic lamina was reduced by 50%. The use of mAb HP1/2 together with mAb 60.3 (directed to the β
2
chain of CD11/CD18) completely abolished the influx of monocytes, lymphocytes, and all types of granulocytes into the arterial intima. This complete blockade of leukocyte infiltration resulted in a 70% reduction of intimal SMC accumulation. Together with our previous findings excluding neutrophils as stimulators of SMC migration, the present results indicate that mononuclear leukocytes promote lesion development by stimulating SMC migration.
Binding and Phagocytosis of Apoptotic Vascular Smooth Muscle Cells Is Mediated in Part by Exposure of Phosphatidylserine
Authors: Bennett M.R. , Gibson D.F. , Schwartz S.M. , Tait J.F.
Published: June 2012

Abstract
Apoptosis of vascular smooth muscle cells has recently been demonstrated to occur in vitro and in vivo. Uptake of apoptotic cells into adjacent normal cells appears to be rapid and specific. We have investigated binding and phagocytosis of apoptotic vascular smooth muscle cells by normal smooth muscle cell monolayers. Vascular smooth muscle cells were infected with the proto-oncogene c-
myc
or the adenovirus E1A gene, induced to undergo apoptosis in low-serum conditions, and then incubated with normal smooth muscle cells. Apoptosis was accompanied by a marked increase in exposure of phosphatidylserine on the outer surface of the cell, which was recognized by binding to annexin V. Liposomes containing phosphatidylserine but not phosphatidylinositol inhibited uptake of apoptotic cells in a dose-dependent manner to a maximum of 50% inhibition; annexin V also inhibited the uptake of apoptotic cells in a dose-dependent and calcium-dependent manner. Binding of apoptotic bodies did not appear to be mediated by endogenous annexin V, as evidenced by the inability of an antibody to annexin V to inhibit uptake. Smooth muscle cells were also able to recognize exposed phosphatidylserine on other cell types, as judged by their ability to bind erythrocytes having a high degree of exposed phosphatidylserine. We conclude that smooth muscle cells express phosphatidylserine during apoptosis, and this exposure partly mediates binding and phagocytosis of dead cells. This mechanism may be important in promoting rapid cell removal in the vessel wall.
Effects of Pacing on Stationary Reentrant Activity
Authors: Davidenko Jorge M. , Salomonsz Remy , Pertsov Arkady M. , Baxter William T. , Jalife José
Published: June 2012

Abstract
It is well known that electrical pacing may either terminate or change the rate and/or ECG appearance of reentrant ventricular tachycardia. However, the dynamics of interaction of reentrant waves with waves initiated by external pacing are poorly understood. Prevailing concepts are based on simplistic models in which propagation occurs in one-dimensional rings of cardiac tissue. Since reentrant activation in the ventricles occurs in two or three dimensions, such concepts might be insufficient to explain the mechanisms of pacing-induced effects. We used numerical and biological models of cardiac excitation to explore the phenomena, which may take place as a result of electrical pacing during functionally determined reentry. Computer simulations of a two-dimensional array of electrically coupled FitzHugh-Nagumo cells were used to predict the response patterns expected from thin slices of sheep ventricular epicardial muscle, in which self-sustaining reentrant activity in the form of spiral waves was consistently initiated by premature stimulation and monitored by means of video mapping techniques. The results show that depending on their timing and shape, externally induced waves may collide with the self-sustaining spiral and result in one of three possible outcomes: (1) direct annihilation of the spiral, (2) multiplication of the spiral, or (3) shift of the spiral center (ie, core). Multiplication and shift of the spiral core were attended by changes in rate and morphology of the arrhythmia as seen by “pseudo-ECGs.” Furthermore, delayed termination (ie, termination of the activity one to three cycles after the stimulus) occurred after both multiplication and shift of the spiral center. Both numerical predictions and experimental results support the hypothesis that whether a pacing stimulus will terminate a reentrant arrhythmia or modify its ECG appearance depends on whether the interactions between the externally induced wave and the spiral wave result in the de novo formation of one or more “wavebreaks.” The final outcome depends on the stimulus parameters (ie, position and size of the electrodes and timing of the stimulus) as well as on the position of the newly formed wavebreak(s) in relation to that of the original wave.