bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Delayed Rectifier Current Blockade by E-4031 on Impulse Generation in Single Sinoatrial Nodal Myocytes of the Rabbit
Authors: Verheijck E. Etienne , van Ginneken Antoni C.G. , Bourier Jan , Bouman Lennart N.
Published: June 2012

Abstract
The role of the delayed rectifier current (I
K
) in impulse generation was studied in single sinoatrial nodal myocytes of the rabbit. We used the class III antiarrhythmic drug E-4031, which blocks I
K
in rabbit ventricular myocytes. In single sinoatrial nodal cells, E-4031 (0.1 μmol/L) significantly prolonged cycle length and action potential duration, depolarized maximum diastolic potential, and reduced both the upstroke velocity of the action potential and the diastolic depolarization rate. Half of the cells were arrested completely. At higher concentrations (1 and 10 μmol/L), spontaneous activity ceased in all cells. Three ionic currents fundamental for pacemaking, ie, I
K
, the long-lasting inward calcium current (I
Ca,L
), and the hyperpolarization-activated current (I
f
), were studied by using the whole-cell and amphotericin–perforated patch technique. E-4031 blocked part of the outward current during depolarizing steps as well as the tail current upon subsequent repolarization (I
TD
) in a dose-dependent manner. E-4031 (10 μmol/L) depressed I
TD
(88±4%) (n=6), reduced peak I
Ca,L
at 0 mV (29±15%) (n=4), but did not affect I
f
. Lower concentrations did not affect I
Ca,L
. Additional use of 5 μmol/L nifedipine demonstrated that I
TD
is carried in part by a calcium-sensitive current. Interestingly, complete blockade of I
K
and I
Ca,L
unmasked the presence of a background current component with a reversal potential of −32±5.4 mV (n=8) and a conductance of 39.5±5.6 pS/pF, which therefore can contribute both to the initial part of repolarization and to full diastolic depolarization. In conclusion, I
K
in conjunction with an inward background current plays an essential role in maintaining normal automaticity.
Expression, Genomic Organization, and Transcription of the Mouse Angiotensin II Type 2 Receptor Gene
Authors: Ichiki Toshihiro , Inagami Tadashi
Published: June 2012

Abstract
Although the rat angiotensin II type 2 receptor (AT
2
) was cloned and shown to be a member of the seven transmembrane domain–type receptor family, its signaling mechanism and biological roles have not been established. To acquire additional information on the structure and functions of AT
2
genomic DNA, we cloned the mouse AT
2
gene and examined its expression, transcription, and genomic organization. The amino acid sequence of the mouse AT
2
cDNA showed a 98.5% sequence identity with the rat AT
2
. In mouse fetus, mRNA of the AT
2
was highly expressed in the eviscerated carcass and brain. This expression decreased rapidly after birth. In 10-week-old mice, mRNA of the AT
2
could be detected in the brain by Northern blot analysis. However, reverse transcription–polymerase chain reaction showed that mRNA of the AT
2
was expressed in all organs examined, indicating that the AT
2
is expressed at a low level in other organs. Southern blot analysis of the genomic DNA of the mouse liver digested with
Bam
HI,
Eco
RI, and
Hin
dIII resulted in single bands, indicating that the AT
2
gene probably exists at a single locus in the mouse genome. The nucleotide sequence of the AT
2
gene (4.5 kb of the
Eco
RI fragment) revealed the presence of three exons. An entire coding sequence was included in the third exon. Primer extension experiments showed the presence of two transcription initiation sites in the mouse AT
2
gene. A DNA segment of about 1.5 kb of the promoter region (−1497 to +56 bp) of the mouse AT
2
gene was fused to a luciferase reporter gene. This promoter-luciferase construct was functional as a promoter when transfected into R3T3 cells. The promoter region contains several transcription
cis
elements, such as AP1 and C/EBP, in the −1497- to −874-bp segment of the promoter region. Deletion analysis showed that this segment of the DNA accounted for 70% of the promoter activity. The shortest deletion segment (−47 to +56 bp), which contains the TATA box, contributed about 15% of the promoter activity. These results clarified several functional features of the mouse AT
2
gene at a molecular level.
Myosin Heavy Chain Expression in Contracting Myocytes Isolated During Embryonic Stem Cell Cardiogenesis
Authors: Metzger Joseph M. , Lin Wan-In , Johnston Ross A. , Westfall Margaret V. , Samuelson Linda C.
Published: June 2012

Abstract
Mouse embryonic stem (ES) cells are totipotent cells derived from the inner cell mass of the preimplantation blastocyst and are capable of differentiating in vitro into cardiac myocytes. Attached cultures of differentiating ES cells were established to document the timing of contractile development by microscopic observation and to permit the microdissection of cardiac myocytes from culture. The onset of spontaneous contraction varied markedly in differentiation culture, with contraction being maintained on average for 9 days (range, 1 to 75 days). Indirect immunofluorescence microscopy showed that myosin expression was localized to the contracting cardiac myocytes in culture. Myosin heavy chain (MHC) isoform expression in microdissected ES cell–derived cardiac myocytes was determined by means of sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The distribution of MHC isoform expression in isolated ES cell cardiac myocytes was as follows: 27% expressed the β-MHC isoform, 33% expressed both the α- and β-MHC isoforms, and 40% expressed the α-MHC isoform. MHC phenotype was correlated to the duration of continuous contractile activity of the myocytes. Myocytes that had just initiated spontaneous contractile activity predominantly expressed the β-MHC (average days of contraction before isolation, 2.5±0.7). The α-MHC isoform was detected after more prolonged contractile activity in vitro (1 to 5 weeks). A strong correlation was obtained between MHC phenotype and days of contraction of the cardiac myocyte preparations isolated from ES cell cultures (
r
=.93). The apparent transition in MHC isoform expression during ES cell differentiation parallels the β- to α-MHC isoform transition characteristic of murine cardiac development in vivo. These findings are evidence that ES cell cardiac myocyte differentiation follows the normal developmental program of murine cardiogenesis.
Pathways of Rb
+
Influx and Their Relation to Intracellular [Na
+
] in the Perfused Rat Heart
Authors: Kupriyanov Valerie V. , Stewart Laura C. , Xiang Bo , Kwak June , Deslauriers Roxanne
Published: June 2012

Abstract
The aims of this study were to characterize the routes of influx of the K
+
congener, Rb
+
, into cardiac cells in the perfused rat heart and to evaluate their links to the intracellular Na
+
concentration ([Na
+
]
i
) using
87
Rb and
23
Na nuclear magnetic resonance (NMR) spectroscopy. The rate constant for Rb
+
equilibration in the extracellular space was 8.5 times higher than that for the intracellular space. The sensitivity of the rate of Rb
+
accumulation in the intracellular space of the perfused rat heart to the inhibitors of the K
+
and Na
+
transport systems has been analyzed. The Rb
+
influx rates were measured in both beating and arrested hearts: both procaine (5 mmol/L) and lidocaine (1 mmol/L) halved the Rb
+
influx rate. In procaine-arrested hearts, the Na
+
,K
+
-ATPase inhibitor ouabain (0.6 mmol/L) decreased Rb
+
influx by 76±24% relative to that observed in untreated but arrested hearts. Rb
+
uptake was insensitive to the K
+
channel blocker 4-aminopyridine (1 mmol/L). The inhibitor of Na
+
/K
+
/2 Cl
−
cotransport bumetanide (30 μmol/L) decreased Rb
+
uptake only slightly (by 9±8%). Rb
+
uptake was dependent on [Na
+
]
i
: it increased by 58±34% when [Na
+
]
i
was increased with the Na
+
ionophore monensin (1 μmol/L) and decreased by 48±9% when [Na
+
]
i
was decreased by the Na
+
channel blockers procaine and lidocaine. Dimethylamiloride (15 to 20 μmol/L), an inhibitor of the Na
+
/H
+
exchanger, slightly reduced [Na
+
]
i
and Rb
+
entry into the cardiomyocytes (by 15±5%).
31
P NMR spectroscopy was used to monitor the energetic state and intracellular pH (pH
i
) in a parallel series of hearts. Treatment of the hearts with lidocaine, 4-aminopyridine, dimethylamiloride, or bumetanide for 15 to 20 minutes at the same concentrations as used for the Rb
+
and Na
+
experiments did not markedly affect the levels of the phosphate metabolites or pH
i
. These data show that under normal physiological conditions, Rb
+
influx occurs mainly through Na
+
,K
+
-ATPase; the contribution of the Na
+
/K
+
/2 Cl
−
cotransporter and K
+
channels to Rb
+
influx is small. The correlation between Rb
+
influx and [Na
+
]
i
during infusion of drugs that affect [Na
+
]
i
indicates that, in rat hearts at 37°C, Rb
+
influx can serve as a measure of Na
+
influx. We estimate that, at normothermia, at least 50% of the Na
+
entry into beating cardiac cells is provided by the Na
+
channels, with only minor contributions (<15%) from the Na
+
/K
+
/2 Cl
−
cotransporter and the Na
+
/H
+
exchanger.
Regression of Left Ventricular Hypertrophy Prevents Ischemia-Induced Lethal Arrhythmias
Authors: Kohya Tetsuro , Yokoshiki Hisashi , Tohse Noritsugu , Kanno Morio , Nakaya Haruaki , Saito Hideya , Kitabatake Akira
Published: June 2012

Abstract
To evaluate the preventive effect of regression of left ventricular hypertrophy (LVH) on sudden cardiac death (SCD), the incidence of ventricular tachycardia or ventricular fibrillation (VT/Vf) after left coronary artery occlusion in Langendorff preparations was studied in the following five groups: (1) spontaneously hypertensive rats (SHR) without treatment (SHR-N), (2) SHR treated with captopril (SHR-C), (3) SHR treated with the angiotensin II receptor antagonist TCV-116 (SHR-A), (4) SHR treated with hydralazine (SHR-H), and (5) Wistar-Kyoto (WKY) rats. Although blood pressure was equally lowered in all treated groups, SHR-C and SHR-A but not SHR-H showed regression of LVH. The incidence of VT/Vf was 5% in WKY rats, 63% in SHR-N (
P
<.005 versus WKY rats), 0% in SHR-C, 10% in SHR-A, and 45% in SHR-H (
P
<.05 versus WKY rats). Further evaluation of the effect of TCV-116 revealed that SHR treated with a low dose of TCV-116 (1 mg/kg per day) showed a decrease in left ventricular mass with only a little decrease in blood pressure and that the incidence of VT/Vf was reduced in association with the degree of regression of LVH. Electrophysiological study using microelectrode techniques revealed that in the LVH groups (SHR-N and SHR-H), the action potential duration (APD) of the left ventricular papillary muscle was more prolonged than in WKY rats, whereas APD shortened to a greater extent during superfusion with a hypoxia/no-glucose solution. APD showed no difference in the regression groups (SHR-C and SHR-A) compared with the WKY group. Shortening of APD at 75% repolarization 30 minutes after exposure to the hypoxia/no-glucose solution was 34% in WKY rats, 53% in SHR-N (
P
<.05 versus WKY rats), 32% in SHR-C, 28% in SHR-A, and 47% in SHR-H (
P
<.05 versus WKY rats). These results suggest that LVH has a greater susceptibility to VT/Vf during acute myocardial ischemia because of greater APD dispersion between the normal and ischemic zones. The reduction of electrical inhomogeneity in regressed LVH may prevent SCD caused by ischemia-induced lethal arrhythmias. Effective regression of LVH by angiotensin II blockade may play a beneficial role in the prevention of SCD.
Postischemic Changes in Cardiac Sarcoplasmic Reticulum Ca
2+
Channels
Authors: Zucchi Riccardo , Ronca-Testoni Simonetta , Yu Gongyuan , Galbani Paola , Ronca Giovanni , Mariani Mario
Published: June 2012

Abstract
We investigated the modifications of cardiac ryanodine receptors/sarcoplasmic reticulum Ca
2+
release channels occurring in ischemic preconditioning. In an isolated rat heart model, the injury produced by 30 minutes of global ischemia was reduced by preexposure to three 3-minute periods of global ischemia (preconditioning ischemia). The protection was still present 120 minutes after preconditioning ischemia but disappeared after 240 minutes. Three 1-minute periods of global ischemia did not provide any protection. In the crude homogenate obtained from ventricular myocardium, the density of [
3
H]ryanodine binding sites averaged 372±18 fmol/mg of protein in the control condition, decreased 5 minutes after preconditioning ischemia (290±15 fmol/mg,
P
<.01), was still significantly reduced after 120 minutes (298±17 fmol/mg,
P
<.05), and recovered after 240 minutes (341±21 fmol/mg). Three 1-minute periods of ischemia did not produce any change in ryanodine binding. The
K
d
for ryanodine (1.5±0.3 nmol/L) was unchanged in all cases. In parallel experiments, the crude homogenate or a microsomal fraction was passively loaded with
45
Ca, and Ca
2+
-induced Ca
2+
release was studied by the quick filtration technique. In both preparations, the rate constant of Ca
2+
-induced Ca
2+
release decreased 5 and 120 minutes after preconditioning ischemia (homogenate values: 19.7±1.4 and 18.9±0.9 s
−1
vs a control value of 25.4±1.7 s
−1
,
P
<.05 in both cases) and recovered after 240 minutes (23.0±1.9 s
−1
). The Ca
2+
dependence of Ca
2+
-induced Ca
2+
release was not affected by preconditioning ischemia. In conclusion, changes in sarcoplasmic reticulum Ca
2+
-release channels occur after brief ischemia and reperfusion, are closely correlated with the development of myocardial protection versus sustained ischemia, and might play a role in the pathogenesis of ischemic preconditioning.
Phosphotyrosine-Dependent Targeting of Mitogen-Activated Protein Kinase in Differentiated Contractile Vascular Cells
Authors: Khalil Raouf A. , Menice Constance B. , Wang C.-L. Albert , Morgan Kathleen G.
Published: June 2012

Abstract
Tyrosine phosphorylation has been linked to plasmalemmal targeting of
src
homology-2–containing proteins, activation of mitogen-activated protein (MAP) kinase, nuclear signaling, and proliferation of cultured cells. Significant tyrosine phosphorylation and MAP kinase activities have also been reported in differentiated cells, but the signaling role of tyrosine-phosphorylated MAP kinase in these cells is unclear. The spatial and temporal relation between phosphotyrosine and MAP kinase immunoreactivity was quantified in differentiated contractile vascular smooth muscle cells by using digital imaging microscopy. An initial association of MAP kinase with the plasmalemma required upstream protein kinase C activity but occurred in a tyrosine phosphorylation–independent manner. Subsequent to membrane association, a delayed redistribution of MAP kinase, colocalizing with the actin-binding protein caldesmon, occurred in a tyrosine phosphorylation–dependent manner. The apparent association of MAP kinase with the contractile proteins coincided with contractile activation. Thus, tyrosine phosphorylation appears to target MAP kinase to cytoskeletal proteins in contractile vascular cells. This targeting mechanism may determine the specific destination and thereby the specialized function of MAP kinase in other phenotypes.
α
1
-Adrenergic Receptor Stimulation Decreases Maximum Shortening Velocity of Skinned Single Ventricular Myocytes From Rats
Authors: Strang Kevin T. , Moss Richard L.
Published: June 2012

Abstract
α
1
-Adrenergic agonists have negative inotropic effects on mammalian myocardium under some conditions, and biochemical experiments measuring the Ca
2+
-activated actomyosin ATPase activity of myofibrillar preparations suggest that this may result from a decrease in cross-bridge cycling rate caused by phosphorylation of myofilament proteins. Experiments with intact ventricular preparations, however, have failed to demonstrate a mechanical manifestation of a decrease in cycling rate. The present study examined the effect of α
1
-adrenergic receptor stimulation on maximum shortening velocity in skinned single ventricular myocytes from rats. Enzymatically isolated myocytes were incubated with the β-receptor antagonist propranolol in the presence or absence of the α
1
-adrenergic receptor agonist phenylephrine and were then rapidly skinned to preserve the phosphorylation state of myofilament proteins. The velocity of unloaded shortening (V
o
) was determined by use of the slack-test method and compared between skinned control and phenylephrine-treated cells. The relationship between isometric tension and [Ca
2+
] was also assessed for each myocyte. V
o
was significantly lower in the α
1
-adrenergic receptor agonist–treated cells than in the control cells, but there was no effect on Ca
2+
sensitivity of isometric tension. In addition, the myosin heavy chain isoform composition accounted for a significant amount of the variation in V
o
within the treatment groups. On the basis of these and previous results we propose that α
1
-adrenergic receptor stimulation inhibits cross-bridge cycling rate at the level of myofilament proteins by a mechanism that may involve phosphorylation of troponin I by protein kinase C.
Ribozyme-Mediated Inhibition of Expression of Leukocyte-type 12-Lipoxygenase in Porcine Aortic Vascular Smooth Muscle Cells
Authors: Gu Jia-Li , Veerapanane Dange , Rossi John , Natarajan Rama , Thomas Lisa , Nadler Jerry
Published: June 2012

Abstract
Activation of a leukocyte-type 12-lipoxygenase (12-LO) has been proposed to be an important mechanism for angiotensin II– and glucose-induced vascular smooth muscle cell growth. Currently, no specific pharmacological inhibitors for the leukocyte-type 12-LO are available to test this hypothesis. We have therefore designed a chimeric DNA-RNA hammerhead ribozyme to produce cleavage at the first GUC sequence at nucleotide 7 of porcine leukocyte 12-LO mRNA. The ribozyme was tested in vitro with a 206-base 12-LO mRNA as substrate. We observed that the ribozyme specifically and dose-dependently cleaved porcine leukocyte 12-LO mRNA at the predicted site under physiological temperature. Furthermore, we also efficiently delivered the ribozyme into porcine aortic vascular smooth muscle cells by transfection with cationic liposomes. The ribozyme caused a dose-dependent decrease in levels of porcine leukocyte-type 12-LO mRNA in these cells and was more potent than an antisense oligonucleotide directed against porcine leukocyte 12-LO. The 12-LO ribozyme also attenuated 12-LO protein levels in the cells. The action of the ribozyme was primarily a result of its catalytic activity, since a modified ribozyme that lacks catalytic activity showed reduced effects. This represents the first ribozyme directed against a mammalian LO pathway. These results demonstrate the potential utility of new ribozyme technology to generate novel agents for gene modulation experiments to modify the development or progression of vascular disease in humans.
Mechanical Strain Induces Monocyte Chemotactic Protein-1 Gene Expression in Endothelial Cells
Authors: Wang Danny Ling , Wung Being-Sun , Shyy Yeun-Jund , Lin Cheng-Fu , Chao Yuh-Jen , Usami Shunichi , Chien Shu
Published: June 2012

Abstract
Monocyte chemotactic protein-1 (MCP-1), a potent monocyte chemoattractant secreted by endothelial cells (ECs), is believed to play a key role in the early events of atherogenesis. Since vascular ECs are constantly subjected to mechanical stresses, we examined how cyclic strain affects the expression of the MCP-1 gene in human ECs grown on a flexible membrane base deformed by sinusoidal negative pressure (peak level, −16 kPa at 60 cycles per minute). Northern blot analysis demonstrated that the MCP-1 mRNA levels in ECs subjected to strain for 1, 5, or 24 hours were double those in control ECs (
P
<.05). This strain-induced increase was mainly serum independent, and MCP-1 mRNA level returned to its control basal level 3 hours after release of strain. Culture media from strained ECs contained approximately twice the MCP-1 concentration and more than twice the monocyte chemotactic activity of media from control ECs (
P
<.05). Pretreatment of collected media with anti–MCP-1 antibody suppressed such activity. Monocyte adhesion to ECs subjected to strain for 12 hours was 1.8-fold greater than adhesion to unstrained control ECs (
P
<.05). A protein kinase C inhibitor, calphostin C, abolished the strain-induced MCP-1 gene expression. In addition, cAMP- or cGMP-dependent protein kinase inhibitors (KT5720 and KT5823, respectively) partially inhibited such expression. Pretreatment with EGTA or the intracellular Ca
2+
chelator BAPTA/AM strongly suppressed the strain-induced MCP-1 mRNA. Verapamil, a Ca
2+
channel blocker, greatly reduced MCP-1 mRNA levels in both strained and unstrained ECs. These results indicate that mechanical strain can stimulate monocyte chemotaxis and adhesion by increasing MCP-1 gene expression in ECs. This increased gene expression is predominantly mediated via the protein kinase C pathway and requires Ca
2+
influx. Such strain-induced MCP-1 expression might contribute to the trapping of monocytes in the subendothelial space. Strain-induced gene expression might provide a molecular mechanism for the role of hypertension in atherogenesis.
Voltage-Gated K
+
Currents Regulate Resting Membrane Potential and [Ca
2+
]
i
in Pulmonary Arterial Myocytes
Authors: Yuan Xiao-Jian
Published: June 2012

Abstract
The membrane potential (E
m
) of pulmonary arterial smooth muscle cells (PASMCs) regulates pulmonary arterial tone by controlling voltage-gated Ca
2+
channel activity, which is a major contributor to [Ca
2+
]
i
. The resting membrane is mainly permeable to K
+
; thus, the resting E
m
is controlled by K
+
permeability through sarcolemmal K
+
channels. At least three K
+
currents, voltage-gated K
+
(K
V
) currents, Ca
2+
-activated K
+
(K
Ca
) currents, and ATP-sensitive (K
ATP
) currents, have been identified in PASMCs. In this study, both patch-clamp and quantitative fluorescent microscopy techniques were used to determine which kind(s) of K
+
channels (K
V
, K
Ca
, and/or K
ATP
) is responsible for controlling E
m
and [Ca
2+
]
i
under resting conditions in rat PASMCs. When the bath solution contained 1.8 mmol/L Ca
2+
and the pipette solution included 0.1 mmol/L EGTA, depolarizations (−40 to +80 mV) elicited both K
Ca
and K
V
currents. Removal of extracellular Ca
2+
and increase of intracellular EGTA concentration (to 10 mmol/L) eliminated the Ca
2+
influx–dependent K
Ca
current. 4-Aminopyridine (4-AP, 5 to 10 mmol/L) but not charybdotoxin (ChTX, 10 to 20 nmol/L) significantly reduced K
V
current under these conditions. In current-clamp experiments, 4-AP decreased E
m
(depolarization) and induced Ca
2+
-dependent action potentials; this depolarization increased [Ca
2+
]
i
in intact PASMCs. Neither ChTX nor the specific blocker of K
ATP
channels, glibenclamide (2 to 10 μmol/L), caused membrane depolarization and the increase in [Ca
2+
]
i
. However, pretreatment of PASMCs with ChTX enhanced the 4-AP–induced increase in [Ca
2+
]
i
. These results suggest that the 4-AP–sensitive K
V
currents that are active in the resting state are the major contributors to regulation of E
m
and thus [Ca
2+
]
i
in rat PASMCs.
23
Na and
31
P Nuclear Magnetic Resonance Studies of Ischemia-Induced Ventricular Fibrillation
Authors: Pike M. M. , Luo C. S. , Yanagida S. , Hageman G. R. , Anderson P. G.
Published: June 2012

Abstract
To clarify the role of Na
+
i
, pH
i
, and high-energy phosphate (HEP) levels in the initiation and maintenance of ischemia-induced ventricular fibrillation (VF), interleaved
23
Na and
31
P nuclear magnetic resonance spectra were collected on perfused rat hearts during low-flow ischemia (51 minutes, 1.2 mL/g wet wt). When untreated, 50% of the hearts from normal (sham) rats and 89% of the hypertrophied hearts from aortic-banded (band) rats (
P
<.01 versus sham) exhibited VF. Phosphocreatine content was significantly higher in sham than band hearts during control perfusion (53.3±1.6 versus 39.8±2.0 μmol/g dry wt). Before VF at 20 minutes of ischemia, Na
+
i
accumulation was greater in hearts that eventually developed VF than in hearts that did not develop VF for both band and sham groups (144% versus 128% of control in sham;
P
<.005) and was the strongest metabolic predictor of VF; ATP depletion was also greater for VF hearts in the sham group. Infusion of the Na
+
-H
+
exchange inhibitor 5-(
N
,
N
-hexamethylene)-amiloride prevented VF in sham and band hearts; reduced Na
+
i
accumulation but similar HEP depletion were observed compared with VF hearts before the onset of VF. Rapid changes in Na
+
i
, pH
i
, and HEP began with VF, resulting in intracellular Na
+
i
overload (≈300% of control) and increased HEP depletion. A delayed postischemic functional recovery occurred in VF hearts, which correlated temporally with the recovery of Na
+
i
. In conclusion, alterations in Na
+
i
were associated with spontaneous VF transitions, consistent with involvement of excess Na
+
i
accumulation in VF initiation and maintenance and with previously reported alterations in Ca
2+
i
with VF. Hypertrophied band hearts exhibited enhanced susceptibility to ischemia-induced VF, possibly linked to a lower HEP reserve.
Elevated Levels of cAMP Inhibit Protein Kinase C– Independent Mechanisms of Endothelial Platelet-Derived Growth Factor–B Chain and Intercellular Adhesion Molecule-1 Gene Induction by Lysophosphatidylcholine
Authors: Ochi Hiroshi , Kume Noriaki , Nishi Eiichiro , Kita Toru
Published: June 2012

Abstract
Lysophosphatidylcholine (lyso-PC), a polar phospholipid product increased in atherogenic lipoproteins and atherosclerotic lesions, has been shown to differentially induce functional intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 and mRNA for platelet-derived growth factor (PDGF)–A and –B chains and heparin-binding epidermal growth factor–like growth factor in various cultured endothelial cells. In this study, we have demonstrated increased expression of cell- and matrix-associated forms of PDGF–B chain (PDGF-B) protein elicited by lyso-PC and further characterized potential signal transduction mechanisms responsible for lyso-PC–induced gene expression, focusing on PDGF-B and ICAM-1 genes in cultured human umbilical vein endothelial cell models. Cycloheximide almost completely inhibited PDGF-B but not ICAM-1 mRNA induction elicited by lyso-PC, suggesting that dependence on de novo protein synthesis for PDGF-B is different from that for ICAM-1. Prolonged exposure to phorbol myristate acetate (PMA), which depletes protein kinase C (PKC), or staurosporine, a PKC inhibitor, did not block lyso-PC–induced increases in PDGF-B or ICAM-1 mRNA. Forskolin and dibutyryl cAMP, which elevate intracellular cAMP levels, blocked both PDGF-B and ICAM-1 upregulation elicited by lyso-PC; however, these cAMP-elevating agents did not suppress ICAM-1 upregulation by PMA. Taken together, PDGF-B and ICAM-1 gene induction by lyso-PC may involve different signaling mechanisms; however, both appear to be independent of PMA-regulatable PKC activation but are suppressed by increased levels of intracellular cAMP.
Evidence That Expression of Inducible Nitric Oxide Synthase in Response to Endotoxin Is Augmented in Atherosclerotic Rabbits
Authors: Sobey Christopher G. , Brooks Robert M. , Heistad Donald D.
Published: June 2012

Abstract
Atherosclerotic lesions contain monocytes/macrophages and vascular smooth muscle cells and thus may have an increased capacity for generation of nitric oxide by inducible nitric oxide synthase (NOS). We used three approaches (contractile responses, generation of
l
-citrulline from
l
-arginine, and staining with NADPH-diaphorase) to test the hypothesis that after administration of lipopolysaccharide (LPS) in vivo, generation of nitric oxide by inducible NOS is augmented in atherosclerotic arteries. New Zealand White (normal, n=18) and Watanabe heritable hyperlipidemic (atherosclerotic, n=21) rabbits were anesthetized and injected intravenously with vehicle or LPS. Contractile responsiveness of aortic segments was examined in vitro 4 hours after injection of LPS in vivo. There was a substantial (approximately fivefold) decrease in contractile sensitivity of aortas from LPS-treated atherosclerotic rabbits and a small (approximately twofold) decrease in normal rabbits. Incubation of aortic segments with aminoguanidine, which inhibits inducible NOS, restored contractile responsiveness after LPS treatment. In vitro assay of conversion of [
14
C]
l
-arginine to [
14
C]
l
-citrulline by aortic segments demonstrated marked (approximately fivefold) increase in calcium-independent conversion of [
14
C]
l
-arginine by LPS-treated atherosclerotic, but not normal, aortas. NADPH-diaphorase staining demonstrated positive cells only in the endothelium of normal rabbits and in the lesions and media of the atherosclerotic aortas in both vehicle- and LPS-treated rabbits. The general distribution of these NADPH-diaphorase–positive cells resembled that of smooth muscle cells and not macrophages. Thus, impairment of contractile responses, generation of
l
-citrulline, and staining with NADPH-diaphorase suggest that atherosclerotic arteries have increased capacity for generation of nitric oxide by inducible NOS.
Optical Transmembrane Potential Measurements During Defibrillation-Strength Shocks in Perfused Rabbit Hearts
Authors: Zhou Xiaohong , Ideker Raymond E. , Blitchington Timothy F. , Smith William M. , Knisley Stephen B.
Published: June 2012

Abstract
To study the optical transmembrane potential change (ΔF) induced during shocks, optical recordings were obtained in 15 isolated perfused rabbit hearts treated with the potentiometric dye di-4-ANEPPS and diacetyl monoxime. Shock electrodes were sutured on the right and left ventricles. A laser beam 30 μm in diameter was used to optically excite di-4-ANEPPS. Fluorescence from a region 150 μm in diameter was recorded during a shock. In the macroscopic study (six animals), there were nine recording spots that were 3 mm apart between the two shock electrodes. In the microscopic study, there were three recording regions that were 3 mm away from either shock electrode and midway between them, with nine recording spots that were 30 μm (three animals), 100 μm (three animals), and 300 μm (three animals) apart in each region. After 20 S
1
stimuli, a 10-ms truncated exponential S
2
shock of defibrillation-threshold strength was given during the plateau of the last S
1
action potential. In the microscopic study, shocks were also given during diastole, with ΔF recordings at the three recording regions. Shocks of both polarities were tested. ΔF during the shock was expressed as a percentage of the fluorescence change during the S
1
upstroke action potential amplitude (the S
1
F
apa
), ie, ΔF/F
apa
%. In the macroscopic study, the magnitudes of ΔF/F
apa
% from recording spots 1 to 9, numbered from the left to the right ventricular electrodes, were 77±41%, 46±32%, 32±27%, 28±20%, 37±25%, 24±20%, 33±22%, 37±25%, and 59±29%, respectively (
P
<.05 among the nine spots). Depolarization or hyperpolarization could occur near either shock electrode with both shock polarities, but the magnitude of hyperpolarization was 1.8±0.9 times that of depolarization at the same recording spot when the shock polarity was reversed (
P
<.01). In the microscopic study, the change in ΔF/F
apa
% varied significantly over the microscopic regions examined. The maximum values of ΔF/F
apa
% for hyperpolarizing shocks during diastole reached only 7±10% of those for shocks during the plateau (
P
<.01). During diastole, the time until a new action potential occurred after the beginning of the shock was shorter when the membrane was depolarized (1.1±0.5 ms) than when it was hyperpolarized (12.8±9.1 ms,
P
<.01). Conclusions are as follows: (1) A shock can induce either hyperpolarization or depolarization. (2) Hyperpolarization or depolarization during a shock can occur near either the anodal or cathodal shock electrode. (3) Variation of ΔF/F
apa
% exists within a microscopic region. (4) The effects of a shock during an action potential plateau are different from those during diastole. (5) The different responses of the ΔF during a shock affect the excitation latency during diastole.
In Vivo Echocardiographic Detection of Enhanced Left Ventricular Function in Gene-Targeted Mice With Phospholamban Deficiency
Authors: Hoit Brian D. , Khoury Saeb F. , Kranias Evangelia G. , Ball Nancy , Walsh Richard A.
Published: June 2012

Abstract
We evaluated the ability of M-mode and Doppler echocardiography to assess left ventricular (LV) function reliably and repeatedly in mice and tested whether these techniques could detect physiological alterations in phospholamban (PLB)–deficient mice. Anesthetized wild-type mice (n=7) and mice deficient in PLB (n=8) were studied with two-dimensional guided M-mode and Doppler echocardiography using a 9-MHz imaging and 5- to 7.5-MHz Doppler transducer. Data were acquired in the baseline state and after intraperitoneal isoproterenol administration (2.0 μg/g IP). Interobserver and intraobserver variability and reproducibility were excellent. PLB-deficient mice were associated with significant (
P
<.05) increases in several physiological parameters (mean±SD) compared with wild-type control mice: normalized mean velocity of circumferential shortening (7.7±2.1 versus 5.5±1.0 circ/sec), peak aortic velocity (105±13 versus 75±9.2 cm/s), mean aortic acceleration (57±16 versus 31±4 m/s
2
), and peak early-diastolic transmitral velocity (80.0±7.2 versus 66.9±7.7 cm/s). LV dimensions, shortening fractions, heart rates, late diastolic transmitral (A) velocities, and early to late (E/A) diastolic velocity ratios were similar in both groups. Isoproterenol administration resulted in significant increases in Doppler indices of ventricular function in control but not PLB-deficient mice. These findings indicate that assessment of LV function can be performed noninvasively in mice under varying physiological conditions and that PLB regulates basal LV function in vivo.
l
-Arginine–Induced Conducted Signals Alter Upstream Arteriolar Responsivity to
l
-Arginine
Authors: Frame Mary D.S. , Sarelius Ingrid H.
Published: June 2012

Abstract
Our purpose was to determine whether
l
-arginine was involved in vascular communication between downstream and upstream locations within a defined microvascular region. Arteriolar diameter was measured for the branches along a transverse arteriole in the superfused cremaster of anesthetized (pentobarbital sodium, 70 mg/kg IP) hamsters (N=53). The upstream branch arterioles dilated significantly to locally applied
l
-arginine (100 μmol/L pipette concentration) only if the downstream branches (≈1400 μm away) were preexposed. With exposure order downstream to upstream, diameter change was last branch, −3.8±1.5% (of baseline); third, +58.1±27%; first, +92±26% (n=5); with exposure order upstream to downstream: first branch, −0.4±3%; third, +5±11%; last, −5.6±7.5% (n=4). Thus, downstream preexposure to
l
-arginine altered the responsivity upstream to locally applied
l
-arginine. Downstream-applied
l
-arginine also induced a conducted vasodilation (+17.8±2.8%; n=14) 1327±166 μm upstream. This response was completely blocked by simultaneous sucrose (600 mOsm), halothane (0.0345%), or
N
ω
-nitro-
l
-arginine (L-NNA, 100 μmol/L) exposure to the feed vessel (second micropipette) midway between the downstream site of
l
-arginine exposure and the upstream observation site. An acetylcholine-induced conducted vasodilation (+18.1±2.6%, n=8) was also completely blocked by sucrose, halothane, or L-NNA. The change in responsivity upstream to locally applied
l
-arginine was not seen in the absence of a conducted vasodilation or when the conducted signal pathway was blocked after the conducted vasodilation was observed, and it could be triggered by a conducted response to acetylcholine as well as to
l
-arginine. Thus, the change in local responsivity upstream requires an ongoing conducted signal from downstream. Conducted signals likely play a dynamic role in the regulation of vascular responsivity within a defined microvascular region.
High- and Low-Affinity Sites for [
3
H]Dofetilide Binding to Guinea Pig Myocytes
Authors: Duff Henry J. , Feng Zhong-Ping , Sheldon Robert S.
Published: June 2012

Abstract
Dofetilide specifically blocks the rapid component of the delayed rectifier current (I
Kr
) at nanomolar concentrations in a saturable manner, suggesting the presence of a receptor. We characterized two [
3
H]dofetilide binding sites to ventricular myocytes from adult guinea pigs by using a conventional filter assay. Scatchard analysis revealed two binding sites with different affinities: a high-affinity site (
K
d
, 2.8±0.3×10
−8
mol/L; B
max
, 76±15 fmol/10
6
myocytes) and a low-affinity site (
K
d
, 1.64±0.4×10
−6
mol/L; B
max
, 1620±260 fmol/10
6
myocytes) (n=11). Kinetic studies showed that there were two dissociation rate constants for [
3
H]dofetilide (0.02±0.005 min
−1
[high-affinity site] and 0.22±0.064 min
−1
[low-affinity site], n=4), although the observed association rate constant is equally well fit to a single- or two-site model. The ability of known I
Kr
blockers to compete with [
3
H]dofetilide binding to both sites was assessed. E4031, clofilium, quinidine, and sotalol competed for binding at both sites. Disopyramide and NAPA only competed for a single binding site. The mean IC
50
values for inhibition of binding to both the high- and low-affinity binding sites correlated with their concentrations required to inhibit I
Kr
in electrophysiological studies. However, inhibition of [
3
H]dofetilide binding to the high-affinity site by class III antiarrhythmic drugs occurred at pharmacological concentrations, whereas suprapharmacological concentrations were required to inhibit binding to the low-affinity site. To demonstrate that the low-affinity site was not associated with I
Kr
, [
3
H]dofetilide binding was assessed in rat ventricular myocytes, which do not express I
Kr
electrophysiologically. In the rat, we observed specific binding of [
3
H]dofetilide only to the low-affinity site (
K
d
, 2.9±0.8×10
−7
mol/L; B
max
, 248±75 fmol/10
6
myocytes; n=9). In addition, low concentrations of solatol (50 μmol/L) inhibit [
3
H]dofetilide only at its high-affinity site without affecting its low-affinity site. Thus, it can be concluded that the high-affinity [
3
H]dofetilide binding site is associated with I
Kr
in guinea pig ventricular myocytes.
New Insights Into the Gating Mechanisms of Cardiac Ryanodine Receptors Revealed by Rapid Changes in Ligand Concentration
Authors: Sitsapesan Rebecca , Montgomery Richard A.P. , Williams Alan J.
Published: June 2012

Abstract
We have developed a novel technique for incorporation of sheep cardiac sarcoplasmic reticulum (SR) Ca
2+
-release channels into planar phospholipid bilayers in order to investigate changes in [Ca
2+
] on a physiological time scale and have investigated whether the rate of change of cytosolic [Ca
2+
] has a direct effect on the gating of the cardiac SR Ca
2+
-release channel. Vesicles of heavy SR were incorporated into planar phospholipid bilayers painted on glass pipettes, and an established technique for rapid solution exchanges at excised membrane patches was modified to allow solution changes to be made at the bilayer within 10 ms. For a given change in [Ca
2+
], we demonstrate that the open probability (Po) is similar whether the cytosolic [Ca
2+
] is increased rapidly (10 ms) or slowly (1 s) and appears to be no different from the Po measured under steady state conditions that were recorded by using conventional bilayer techniques. We also demonstrate that no desensitization or inactivation occurs at −40 mV when the channel is activated by Ca
2+
alone or in the presence of other channel activators, ATP or EMD 41000. However, at +40 mV, rapid channel activation followed by inactivation was observed. The probability of such voltage-dependent inactivation appears to depend on the mechanism of channel activation.
Upregulation of Cardiac Angiotensin II AT
1
Receptors in Congenital Cardiomyopathic Hamsters
Authors: Lambert Chantal , Massillon Yolette , Meloche Sylvain
Published: June 2012

Abstract
Angiotensin II (Ang II) is a growth factor that stimulates protein synthesis and induces cellular hypertrophy in cardiac myocytes. To gain insight into the role of Ang II in cardiac hypertrophy, we examined the expression and subtype distribution of Ang II receptors in the ventricles of embryonic and of 25- to 350-day-old inbred control and cardiomyopathic (CHF 146) hamsters. Studies were also performed with heterozygous (cardiomyopathic×control) animals. Compared with the control hamsters, cardiomyopathic hamsters presented decreased body weights and increased ratios of ventricular weight to body weight in every adult group studied. Typical histological lesions appeared in the left ventricle of cardiomyopathic animals around 70 to 75 days, and their severity increased with time. Radioligand binding studies with cardiac ventricular membranes indicated that iodinated [Sar
1
,Ile
8
]Ang II (sarile) binds to a homogeneous population of sites in membranes derived from adult normal and cardiomyopathic animals. Competition curves using specific receptor subtype antagonists revealed that
125
I-sarile binding sites were exclusively of the AT
1
subtype in both groups of animals. Importantly, the density of AT
1
receptors was found to be significantly increased (90% augmentation at 70 to 75 days) in the ventricles of cardiomyopathic hamsters. This augmented expression was observed in all adult groups and was already present at 25 days, when no histological lesions were visible. The affinity of the receptor for losartan did not vary significantly between adult normal and cardiomyopathic animals (mean
K
d
, 19.6 and 16.7 nmol/L, respectively). No significant differences were observed in the total expression of Ang II receptors and in the proportion of AT
1
and AT
2
subtypes between the two groups of embryonic hearts. Heterozygous animals expressed an intermediate level of AT
1
receptor binding activity in their ventricles. Together, these results suggest that Ang II acting through the AT
1
receptor may play a critical role in the development and/or maintenance of cardiac hypertrophy.
Leukotriene C
4
/D
4
Induces P-Selectin and Sialyl Lewis
x
–Dependent Alterations in Leukocyte Kinetics In Vivo
Authors: Kanwar Samina , Johnston Brent , Kubes Paul
Published: June 2012

Abstract
The objective of this study was to assess the effect of leukotriene C
4
(LTC
4
) on the flux of rolling leukocytes, leukocyte rolling velocity, and leukocyte adhesion in postcapillary venules in vivo and to study the underlying molecular mechanisms involved. LTC
4
(20 nmol/L) induced a rapid and significant increase in leukocyte rolling flux that was inhibitable by an anti–P-selectin antibody and soluble sialyl Lewis
x
(sLe
x
). LTC
4
also induced a significant reduction in leukocyte rolling velocity, an event that was independent of P-selectin but entirely dependent on sLe
x
. This LTC
4
-induced reduction in leukocyte rolling velocity was independent of any hemodynamic alterations. Another P-selectin effector, histamine, did not affect leukocyte rolling velocity even at >5000 times the concentration of LTC
4
. Treatment with an anti–L-selectin antibody had no effect on the LTC
4
-induced increase in leukocyte rolling or reduction in rolling velocity. Inhibition of LTC
4
bioconversion to LTD
4
by pretreatment with
l
-serine (100 μmol/L) prevented the LTC
4
-induced increase in leukocyte rolling flux and the LTC
4
-induced reduction in leukocyte rolling velocity. A subtle, yet significant, increase in leukocyte adhesion was also observed with LTC
4
. Pretreatment with a platelet-activating factor receptor antagonist returned the LTC
4
-induced leukocyte rolling velocity to baseline levels. The addition of a very low concentration of platelet-activating factor (1 nmol/L) induced significant leukocyte adhesion in the presence of LTC
4
but not histamine. This study demonstrates that LTC
4
, via bioconversion to leukotriene D
4
, induces a P-selectin–dependent and sLe
x
-dependent increase in leukocyte rolling flux and a P-selectin–independent but sLe
x
-dependent reduction in leukocyte rolling velocity, a parameter that may play an essential role in subsequent leukocyte adhesion.
Effects of Lysophosphatidic Acid, a Novel Lipid Mediator, on Cytosolic Ca
2+
and Contractility in Cultured Rat Mesangial Cells
Authors: Inoue Chiyoko N. , Forster Hayley G. , Epstein Murray
Published: June 2012

Abstract
Lysophosphatidic acid (LPA), the smallest and structurally simplest phospholipid, has the potential to modulate cellular signaling in diverse tissues and cell types, including fibroblasts. In the present study, we assessed the effects of LPA on cultured rat glomerular mesangial cells. Quantitative changes of [Ca
2+
]
i
in response to LPA were measured in monolayers of mesangial cells loaded with the fluorescent Ca
2+
indicator fura 2. LPA (10 nmol/L to 100 μmol/L) increased [Ca
2+
]
i
in a dose-dependent manner and evoked inositol trisphosphate formation. LPA (1 μmol/L to 30 μmol/L) also elicited a marked, albeit transient, contractile response in mesangial cells cultured on collagen gel, as assessed by a decrease in cell surface area (CSA). The contraction persisted for 5 minutes (CSA decreased by 31%), whereupon the mesangial cells gradually relaxed with a consequent increase in CSA. Pretreatment of mesangial cells with isradipine (1 μmol/L), a dihydropyridine-sensitive Ca
2+
channel blocker, completely blocked LPA-induced contraction. Isradipine also decreased resting [Ca
2+
]
i
levels as well as the peak and the subsequently sustained [Ca
2+
]
i
levels induced by LPA, suggesting that the contractile effects of LPA are dependent on Ca
2+
entry through voltage-gated Ca
2+
channels. Finally, LPA stimulated an increase in both prostaglandin E
2
synthesis and cAMP accumulation, indicating that these mediators may modulate the contractile effects of LPA. Our study is the first demonstration that exogenous LPA induces mesangial cell contraction and suggests that the contraction is mediated by mobilization of intracellular Ca
2+
by activation of the phosphoinositide cascade as well as by promotion of Ca
2+
entry across the plasma membrane.
Regional Expression of Natriuretic Peptide Receptors During the Formation of Arterial Neointima in the Rabbit
Authors: Brown J. , Chen Q.
Published: June 2012

Abstract
In vitro evidence suggests that natriuretic peptide receptors (NPR)-B and NPR-C inhibit vascular smooth muscle (VSM) proliferation. NPR-B is guanylate cyclase-coupled and selectively activated by C-type natriuretic peptide (CNP)-(1-22). NPR-C is not guanylate cyclase-coupled and, unlike NPR-B, avidly binds atrial natriuretic peptide (ANP)-(1-28) as well as CNP-(1-22). Here, we investigate these receptors during the VSM proliferation and neointimal formation found 5, 7, and 20 days after compressing the central ear artery of the rabbit. Receptors were mapped autoradiographically using [
125
I-Tyr
0
]CNP-(1-22), which binds NPR-B and NPR-C, and
125
I-ANP-(1-28), which binds NPR-C and NPR-A, another guanylate cyclase-coupled receptor. Normal tunica media had NPR-B–like binding sites, and the level of these did not change significantly after compression. Consistent with this, CNP-(1-22) stimulated cGMP production equally with membranes from normal or damaged arteries and was more effective than ANP-(1-28). Neointima, which became evident 5 to 7 days after arterial damage, expressed NPR-C–like sites and no detectable NPR-B–like binding. NPR-C–like sites also appeared on the media for the first time between 5 and 7 days after compression. Immunohistochemistry for proliferating cell nuclear antigen revealed widespread mitosis in VSM at 5 days after compression, but mitosis was virtually restricted to the neointima at and beyond 7 days after compression. Thus, whereas levels of NPR-B did not change significantly after arterial injury and NPR-A was not detected, NPR-C–like receptors were unregulated as mitosis declined in the media and as a prominent neointima formed.