bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet-Derived Growth Factor Ligand and Receptor Expression in Response to Altered Blood Flow In Vivo
Authors: Mondy J. Sheppard , Lindner Volkhard , Miyashiro Jody K. , Berk Bradford C. , Dean Richard H. , Geary Randolph L.
Published: June 2012

Abstract
Blood flow and the tractive force shear stress are important determinants of artery caliber, and reduced shear predisposes arteries to intimal thickening and atherosclerosis. The molecular basis for shear-induced changes in artery wall structure is poorly defined. A number of factors associated with normal and pathological artery wall remodeling are induced by shear stress in endothelial cell cultures. These include platelet-derived growth factor (PDGF), a potent mitogen, chemoattractant, and vasoconstrictor. To determine whether similar changes occur in vivo, we examined the effects of reduced blood flow on endothelial cell PDGF expression and proliferation in the rat carotid artery. Branches of the right internal and external carotid arteries were ligated, reducing common carotid artery blood flow from 8.0±0.6 to 0.5±0.1 mL/min while increasing flow in the left carotid from 7.1±0.6 to 10.8±0.7 mL/min. Shear stress following the procedure was 1.4±0.2 and 33.4±1.1 dyne/cm
2
in carotids with reduced blood flow (RF) and increased blood flow (IF), respectively. Arteries were harvested 6, 24, 48, or 72 hours after ligation, perfusion-fixed, and opened longitudinally. Endothelial cell proliferation (bromodeoxyuridine [BrdU] labeling) was assessed en face at 24, 48, and 72 hours; expression of mRNA for PDGF-A and -B chains and PDGF α- and β-receptors (in situ hybridization) was determined at 6, 48, and 72 hours after unilateral flow reduction. RF induced endothelial cell proliferation, which peaked at 48 hours (RF BrdU labeling: 24 hours, 0.4±0.2%; 48 hours, 7.2±2.0%; and 72 hours, 4.1±0.6%; n=5). PDGF-B expression increased in RF compared with IF endothelium within 48 hours and persisted at 72 hours (percent labeling [RF/IF×100]: 6 hours, 76±20%; 48 hours, 395±179%; and 72 hours, 208±44%; n=3). PDGF-A expression was similarly increased in RF endothelium. In contrast, expression of PDGF α- and β-receptors was undetectable in RF and IF endothelium at all times. We conclude that endothelial cell PDGF ligand expression is induced by reduced shear stress in vivo and may play an important role in flow-mediated remodeling and atherogenesis.
Regulation of Matrix Metalloproteinase Expression in Human Vascular Smooth Muscle Cells by T Lymphocytes
Authors: Schönbeck Uwe , Mach François , Sukhova Galina K. , Murphy Curran , Bonnefoy Jean-Yves , Fabunmi Rosalind P. , Libby Peter
Published: June 2012

Abstract
Physical disruption of an atheromatous lesion often underlies acute coronary syndromes. Matrix-degrading enzymes, eg, matrix metalloproteinases (MMPs), may cause loss in mechanical integrity of plaque tissue that favors rupture. T lymphocytes accumulate at sites where atheromata rupture, but the mechanisms by which these immune cells may contribute to plaque destabilization are unknown. This study tested the hypothesis that the T-lymphocyte surface molecule CD40 ligand (CD40L), recently localized in atherosclerotic plaques, regulates the expression of MMPs in human vascular smooth muscle cells (SMCs), the most numerous cell type in arteries. We report here that stimulated human T lymphocytes induced the expression of the matrix-degrading enzymes, ie, interstitial collagenase (MMP-1), stromelysin (MMP-3), gelatinase B (MMP-9), and activated gelatinase A (MMP-2), in human vascular SMCs by cell contact via CD40 ligation, as demonstrated by Western blot analysis, zymography, and antibody neutralization. Recombinant human CD40L (rCD40L) induced de novo synthesis of MMP-1, MMP-3, and MMP-9 on vascular SMCs and stimulated the expression of these enzymes to a greater extent than did maximally effective concentrations of tumor necrosis factor-α or interleukin-1β, established agonists of MMP expression. Interferon gamma, another T-lymphocyte–derived cytokine, inhibited the induction of MMPs by rCD40L. Immunohistochemical analysis of human coronary atheromata colocalized MMP-1 and MMP-3 with CD40-positive SMCs. These results demonstrated that CD40 ligand, expressed on T lymphocytes, promoted the expression of matrix-degrading enzymes in vascular SMCs and thus established a new pathway of immune-modulated destabilization in human atheromata.
Enhanced Ca
2+
Current and Decreased Ca
2+
Efflux Restore Sarcoplasmic Reticulum Ca
2+
Content After Depletion
Authors: Trafford A. W. , Díaz M. E. , Negretti N. , Eisner D. A.
Published: June 2012

Abstract
[Ca
2+
]
i
was measured using the fluorescent indicator indo 1 in voltage-clamped ferret and rat ventricular myocytes. The Ca
2+
content of the sarcoplasmic reticulum (SR) was estimated from the integral of the Na
+
-Ca
2+
exchange current activated by caffeine. Refilling of the SR after caffeine removal was enhanced by stimulation. As the systolic Ca
2+
transient recovered, the integral of the L-type Ca
2+
current decreased and that of the Na
+
-Ca
2+
exchange tail current increased. For the early pulses, the gain of Ca
2+
via the Ca
2+
current is greater than the loss via the exchanger, and during steady state stimulation, the fluxes are equal. The difference in the integrals gives a measure of the net gain of cell Ca
2+
with each pulse. When these are summed, the calculated gain of cell Ca
2+
agrees well with the increase of SR Ca
2+
produced by stimulation, as measured from the caffeine-evoked currents. There was a nonlinear relationship between SR Ca
2+
content and the magnitude of the systolic Ca
2+
transient such that at high SR Ca
2+
content a given increase of content had a greater effect on the Ca
2+
transient than did an increase at low SR content. In conclusion, the effects of systolic Ca
2+
on the Ca
2+
current and Na
+
-Ca
2+
exchange current provide a means to regulate SR Ca
2+
content and thence the systolic Ca
2+
transient.
Ionic Remodeling Underlying Action Potential Changes in a Canine Model of Atrial Fibrillation
Authors: Yue Lixia , Feng Jianlin , Gaspo Rania , Li Gui-Rong , Wang Zhiguo , Nattel Stanley
Published: June 2012

Abstract
Rapid electrical activation, as occurs during atrial fibrillation (AF), is known to cause reductions in atrial refractoriness and in adaptation to heart rate of the atrial refractory period, which promote the maintenance of AF, but the underlying ionic mechanisms are unknown. In order to determine the cellular and ionic changes caused by chronic atrial tachycardia, we studied right atrial myocytes from dogs subjected to 1, 7, or 42 days of atrial pacing at 400/min and compared them with myocytes from sham-operated dogs (pacemaker inserted but not activated). Rapid pacing led to progressive increases in the duration of AF induced by bursts of 10-Hz stimuli (from 3±2 seconds in sham-operated dogs to 3060±707 seconds in dogs after 42 days of pacing,
P
<.001) and reduced atrial refractoriness and adaptation to rate of the atrial refractory period. Voltage-clamp studies showed that chronic rapid pacing did not alter inward rectifier K
+
current, rapid or slow components of the delayed rectifier current, the ultrarapid delayed rectifier current, T-type Ca
2+
current, or Ca
2+
-dependent Cl
−
current. In contrast, the densities of transient outward current (
I
to
) and L-type Ca
2+
current (
I
Ca
) were progressively reduced as the duration of rapid pacing increased, without concomitant changes in kinetics or voltage dependence. In keeping with in vivo changes in refractoriness, action potential duration (APD) and APD adaptation to rate were decreased by rapid pacing. The response of the action potential and ionic currents flowing during the action potential (as exposed by action-potential voltage clamp) to nifedipine in normal canine cells and in cells from rapidly paced dogs suggested that the APD changes in paced dogs were largely due to reductions in
I
Ca
. We conclude that sustained atrial tachycardia reduces
I
to
and
I
Ca
, that the reduced
I
Ca
decreases APD and APD adaptation to rate, and that these cellular changes likely account for the alterations in atrial refractoriness associated with enhanced ability to maintain AF in the model.
Alternatively Spliced IS6 Segments of the α
1C
Gene Determine the Tissue-Specific Dihydropyridine Sensitivity of Cardiac and Vascular Smooth Muscle L-Type Ca
2+
Channels
Authors: Welling Andrea , Ludwig Andreas , Zimmer Stephanie , Klugbauer Norbert , Flockerzi Veit , Hofmann Franz
Published: June 2012

Abstract
Dihydropyridines (DHPs) block the vascular smooth muscle L-type Ca
2+
channel at lower concentrations than the cardiac Ca
2+
channel, although their α
1
subunit, which binds the DHPs, is derived from the same gene. This α
1C
gene gives rise to several splice variants, among which the α
1C-b
variant is affected by lower concentrations of nisoldipine than the α
1C-a
variant. Functional expression of chimeras of α
1C-a
and α
1C-b
subunits demonstrated that the transmembrane segment IS6 is responsible for the different dihydropyridine sensitivity. Northern blot analysis showed that transcripts coding for the IS6 segment of the α
1C-a
subunit were expressed in heart but not in aorta, whereas the IS6 segment of the α
1C-b
subunit was expressed predominantly in vascular smooth muscle. In situ hybridization of rat heart sections confirmed this expression pattern of IS6 α
1C-a
and IS6 α
1C-b
in ventricular and smooth muscle myocytes, respectively. These results suggest that the different dihydropyridine sensitivities of cardiac and vascular L-type Ca
2+
channels are caused at least partially by the tissue-specific expression of alternatively spliced IS6 segments of the α
1C
gene.
Contribution of Glycogen and Exogenous Glucose to Glucose Metabolism During Ischemia in the Hypertrophied Rat Heart
Authors: Schönekess Brett O. , Allard Michael F. , Henning Sarah L. , Wambolt Richard B. , Lopaschuk Gary D.
Published: June 2012

Abstract
Although hypertrophied hearts have increased rates of glycolysis under aerobic conditions, it is controversial as to whether glucose metabolism during ischemia is altered in the hypertrophied heart. Because endogenous glycogen stores are a key source of glucose during ischemia, we developed a protocol to label the glycogen pool in hearts with either [
3
H]glucose or [
14
C]glucose, allowing for direct measurement of both glycogen and exogenous glucose metabolism during ischemia. Cardiac hypertrophy was produced in rats by banding the abdominal aorta for an 8-week period. Isolated hearts from aortic-banded and sham-operated rats were initially perfused under substrate-free conditions to decrease glycogen content to 40% of the initial pool size. Resynthesis and radiolabeling of the glycogen pool with [
3
H]glucose or [
14
C]glucose were accomplished in working hearts by perfusion for a 60-minute period with 11 mmol/L [
3
H]glucose or [
14
C]glucose, 0.5 mmol/L lactate, 1.2 mmol/L palmitate, and 100 μmol/mL insulin. Although glycolytic rates during the aerobic perfusion were significantly greater in hypertrophied hearts compared with control hearts, glycolytic rates from exogenous glucose were not different during low-flow ischemia. The contribution of glucose from glycogen was also not different in hypertrophied hearts compared with control hearts during ischemia (1314±665 versus 776±310 nmol · min
−1
· g dry wt
−1
, respectively). Glucose oxidation rates decreased during ischemia but were not different between the two groups. However, in both hypertrophied and control hearts, the ratio of glucose oxidation to glycolysis was greater for glucose originating from glycogen than from exogenous glucose. Our data demonstrate that glycogen is a significant source of glucose during low-flow ischemia, but the data do not differ between hypertrophied and control hearts.
A Role for Ca
2+
/Calmodulin-Dependent Protein Kinase II in the Mitogen-Activated Protein Kinase Signaling Cascade of Cultured Rat Aortic Vascular Smooth Muscle Cells
Authors: Abraham S. Thomas , Benscoter Holly A. , Schworer Charles M. , Singer Harold A.
Published: June 2012

Abstract
Exposure of cultured rat aortic vascular smooth muscle (VSM) cells to the Ca
2+
ionophore ionomycin produced an increase in extracellular signal–regulated kinase 1/2 (ERK1/2) activity that was maximal between 2 and 5 minutes but then declined to basal values within 20 minutes of stimulation. Elevation of [Ca
2+
]
i
in VSM cells leads to an even more rapid activation of Ca
2+
/calmodulin-dependent protein kinase II (CaM kinase II); thus, it was postulated that the Ca
2+
-dependent component of ERK1/2 activation was mediated by CaM kinase II. Transient ERK1/2 activation by ionomycin was almost completely abolished by pretreating cells with 30 μmol/L KN-93, a CaM kinase II inhibitor. Treatment of cells with KN-93 did not antagonize the ability of ionomycin to mobilize intracellular Ca
2+
but prevented CaM kinase II and ERK1/2 activation with almost identical potencies. Consistent with a role for Ca
2+
and calmodulin in intracellular Ca
2+
–induced activation of ERK, cells pretreated with calmodulin inhibitors (W-7 or calmidazolium) exhibited an attenuated ERK response to ionomycin. ERK1/2 activation in response to phorbol esters and platelet-derived growth factor were not significantly affected by KN-93, whereas the response to angiotensin II and thrombin were attenuated by 60% and 40%, respectively. Transient expression of wild-type δ
2
CaM kinase II in COS-7 cells resulted in increased ERK2 activity, whereas coexpression of wild-type and a kinase-negative mutant resulted in a diminution of this response. These data suggest that regulation of cellular responses by Ca
2+
-dependent pathways in VSM cells may be mediated in part by CaM kinase II–dependent activation of ERK1/2.
Angiotensin II–Induced Stimulation of Smooth Muscle α-Actin Expression by Serum Response Factor and the Homeodomain Transcription Factor MHox
Authors: Hautmann Martina B. , Thompson Maria M. , Swartz Ellen A. , Olson Eric N. , Owens Gary K.
Published: June 2012

Abstract
The objective of the present study was to examine the molecular mechanisms whereby angiotensin II (Ang II) stimulates smooth muscle (SM) α-actin expression in rat aortic smooth muscle cells (SMCs). Nuclear run-on analysis and transfection studies indicated that the effects of Ang II on SM α-actin were mediated at least in part at the transcriptional level. Transfection of various rat SM α-actin promoter/chloramphenicol acetyltransferase (CAT) constructs into SMCs demonstrated that the first 155 bp of the SM α-actin promoter was sufficient to confer maximal Ang II responsiveness, conferring an ≈4-fold increase in reporter activities in these SMCs compared with vehicle-treated SMCs. Mutation of either of two highly conserved CArG elements, designated A (−62) and B (−112), completely abolished Ang II–induced increases in reporter activity, whereas mutation of a homeodomain-like binding sequence at −145 (ATTA) reduced reporter activity by half. Results of EMSAs showed that nuclear extracts from Ang II–treated SMCs exhibited enhanced binding activity of serum response factor (SRF) to the CArG elements and of a homeo-domain factor, MHox, to the ATTA element. Northern analyses showed that Ang II also stimulated marked increases in MHox mRNA levels. Western analyses demonstrated that Ang II–induced increases in SRF binding were not due to increased SRF protein expression. Recombinant MHox markedly enhanced binding activity of SRF in EMSAs. Finally, MHox overexpression transactivated a SM α-actin promoter/CAT reporter construct by ≈3.5-fold in transient cotransfection studies. These results provide evidence for involvement of a homeodomain transcription factor, MHox, in Ang II–mediated stimulation of SM α-actin via a CArG/SRF-dependent mechanism.
Role of Angiotensin II in Activation of the JAK/STAT Pathway Induced by Acute Pressure Overload in the Rat Heart
Authors: Pan Jing , Fukuda Keiichi , Kodama Hiroaki , Makino Shinji , Takahashi Toshiyuki , Sano Motoaki , Hori Shingo , Ogawa Satoshi
Published: June 2012

Abstract
This study was designed to determine whether the JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) pathway is activated in cardiac hypertrophy induced in vivo by pressure overload in rats and to demonstrate whether angiotensin II is involved in the activation of the JAK/STAT pathway. Acute pressure overload was produced by constricting the abdominal aorta of Wistar rats. Immunoprecipitation–Western blot analysis revealed that pressure overload activated JAK1, JAK2, and Tyk2 as early as 5 minutes and that STAT1, STAT2, and STAT3 were tyrosine-phosphorylated rapidly after exposure to the pressure overload. Phosphorylation of STAT1 and STAT2 peaked in the early stage at 5 to 15 minutes, whereas that of STAT3 peaked in the late stage at 60 minutes. Gel mobility shift of the interferon gamma activation site/interferon alpha–stimulating response element was observed immediately after the aortic banding, whereas the band of
sis
-inducing element was shifted in the late stage at 60 minutes. Both cilazapril (angiotensin II–converting enzyme inhibitor) and E4177 (angiotensin II type 1 [AT
1
] receptor antagonist) significantly suppressed the phosphorylation of Tyk2 and partially inhibited the phosphorylation of JAK2, but neither affected JAK1. Coimmunoprecipitation of the AT
1
receptor with JAK2 or Tyk2 was clearly observed at 5 minutes and peaked at 15 minutes (20-fold the control value). These results indicate that the JAK/STAT pathway is activated by acute pressure overload in rats and that angiotensin II is involved in activating Tyk2, and partially activating JAK2, via the AT
1
receptor. Both angiotensin II–dependent and –independent pathways take part in activating the JAK/STAT pathway in the pressure-overloaded rat heart.
Transendothelial Neutrophil Migration
Authors: Cepinskas Gediminas , Noseworthy Ron , Kvietys Peter R.
Published: June 2012

Abstract
During an acute inflammatory response polymorphonuclear leukocytes (PMNs) adhere to and emigrate across the venular microvasculature. There is general agreement on the mechanisms involved in PMN adhesive interactions. However, the mechanisms by which PMNs migrate across the endothelial lining remain controversial, particularly with respect to the role of elastase. In the present study, we used human umbilical vein endothelial cells (HUVECs) and PMNs to test the hypothesis that the relative role of PMN-derived elastase may be dependent on the degree of HUVEC retraction within monolayers. A high (10
−7
mol/L), but not a low (10
−10
mol/L), concentration of platelet-activating factor (PAF) caused HUVEC retraction of sufficient magnitude to increase transendothelial protein movement. Elastase inhibitors prevented PMN transendothelial migration in response to the low, but not the high, concentration of PAF. These findings suggest that PMN migration across confluent endothelial cells is elastase dependent, whereas PMN migration across retracted endothelial cells is elastase independent. However, under the latter condition (high concentration of PAF), the two endogenous proteases, α
2
-macroglobulin and α
1
-antitrypsin, could interfere with PAF-induced PMN transendothelial migration. Thus, as the concentration of PAF is increased, migrating PMNs use other proteases, in addition to elastase. We also noted that transendothelial protein movement is closely coupled to PMN migration.
Dilated Cardiomyopathy in Transgenic Mice With Cardiac-Specific Overexpression of Tumor Necrosis Factor-α
Authors: Kubota Toru , McTiernan Charles F. , Frye Carole S. , Slawson Susan E. , Lemster Bonnie H. , Koretsky Alan P. , Demetris A. Jake , Feldman Arthur M.
Published: June 2012

Abstract
The failing human heart expresses tumor necrosis factor-α (TNF-α). However, its pathophysiological significance is not clear. We previously reported that robust overexpression of TNF-α in the murine heart causes lethal myocarditis. In this study, we modified the transgene to reduce the production of TNF-α by preserving the destabilizing sequence in TNF-α cDNA. Expression was driven by the murine α-myosin heavy chain promoter. Use of this modified construct allowed us to establish a murine transgenic line (TG). TG offspring were examined at 6, 12, and 24 weeks. All showed a significantly higher heart weight–to–body weight ratio. Northern blot analysis confirmed the expression of transgene in the heart, and enzyme-linked immunosorbent assay demonstrated the presence of TNF-α protein. The TG heart demonstrated a mild, diffuse, lymphohistiocytic interstitial inflammatory infiltrate. Cardiomyocyte necrosis and apoptosis were present but not abundant. Magnetic resonance imaging showed that the TG heart was significantly dilated with reduced ejection fraction. Although the left ventricular dP/dt
max
was not different at baseline, its responsiveness to isoproterenol was significantly blunted in TG. Atrial natriuretic factor was expressed in the TG ventricle. A group of TG died spontaneously, and subsequent autopsies revealed exceptional dilatation of the heart, increased lung weight, and pleural effusion, suggesting that they died of congestive heart failure. The cumulative mortality rate at 6 months was 23%. In conclusion, the mouse overexpressing TNF-α recapitulated the phenotype of congestive heart failure. This provides a novel model to elucidate the role of this cytokine in the development of congestive heart failure.
Increased Expression of Interleukin-1β and Monocyte Chemotactic and Activating Factor/Monocyte Chemoattractant Protein-1 in the Hypertrophied and Failing Heart With Pressure Overload
Authors: Shioi Tetsuo , Matsumori Akira , Kihara Yasuki , Inoko Moriaki , Ono Koh , Iwanaga Yoshitaka , Yamada Takehiko , Iwasaki Atsushi , Matsushima Kouji , Sasayama Shigetake
Published: June 2012

Abstract
Studies on the effects of proinflammatory cytokines on the heart suggest that they play some roles in the pathogenesis of congestive heart failure (CHF). To determine the involvement of proinflammatory cytokine in cardiac hypertrophy and CHF induced by mechanical overload, we investigated the expression of interleukin (IL)-1β and monocyte chemotactic and activating factor (MCAF)/monocyte chemoattractant protein-1 (MCP-1) in the left ventricle (LV) of Dahl salt-sensitive (DS) rats that showed hypertrophy of the LV induced by hypertension and subsequently developed CHF. The IL-1β mRNA content in the LV of DS rats increased 3.9-fold when LV hypertrophy developed, and the increase reached 6.2-fold at the CHF stage compared with that of age-matched Dahl salt-resistant (DR) rats. The amount of IL-1β in the LV was positively correlated with the LV weight/body weight ratio. Most of the IL-1β immunoreactivity was localized in the endothelial cells and interstitial macrophages. The mRNA levels of MCAF in the LV increased 3.6-fold at 11 weeks and reached 4.8-fold at the CHF stage relative to the age-matched DR rats. MCAF protein was localized to the endothelial cells and interstitial macrophages. In DS rats, the number of interstitial macrophages increased diffusely throughout the LV. We suggest that increased chemokine expression, macrophage infiltration, and proinflammatory cytokine expression play some role in the pathogenesis of cardiac hypertrophy and failure induced by chronic mechanical overload.
Electrophysiological Characterization of an Alternatively Processed ERG K
+
Channel in Mouse and Human Hearts
Authors: Lees-Miller James P. , Kondo Colleen , Wang Li , Duff Henry J.
Published: June 2012

Abstract
Mutants of HERG, the human form of ERG (the
ether-a-go-go
–related K
+
channel gene), are responsible for some forms of the long-QT syndrome, an abnormality of cardiac repolarization. HERG was cloned from brain and has properties similar but not identical to the rapidly activating component of the native cardiac K
+
channel current (
I
Kr
). We identified in the mouse an alternatively processed form of ERG (MERG B) that is expressed abundantly in heart but only in trace amounts in brain. MERG B has a unique 36–amino acid NH
2
-terminal domain that is strongly basic and considerably shorter than the 376–amino acid NH
2
-terminal domain of HERG. When expressed in
Xenopus
oocytes, the kinetics of activation and deactivation of the MERG B current were best fit by a biexponential function, with the fast components dominant over the slow components. The fast component of activation had a mean τ value of 163±16 ms at −20 mV and 8±4 ms at +20 mV (n=4). The fast component of deactivation had a mean τ value of 145±29 ms at −20 mV and 12±4 ms at −90 mV (n=4). The MERG B current was blocked by the selective
I
Kr
blocker, dofetilide, with an IC
50
of 54 nmol/L. In addition, we isolated HERG B, the human homologue of MERG B, which has electrophysiological characteristics qualitatively similar to those of MERG B. We have identified ERG B, an alternatively processed isoform of the ERG gene, expressed selectively in heart and with electrophysiological characteristics similar to those of native cardiac
I
Kr
.
Dual Role for Nitric Oxide in the Regulation of Plasma Volume and Albumin Escape During Endotoxin Shock in Conscious Rats
Authors: Filep János G. , Delalandre Aline , Beauchamp Micheline
Published: June 2012

Abstract
To assess the role of nitric oxide (NO) produced by the constitutive (cNOS) and inducible NO synthase (iNOS) in the regulation of vascular functions, we compared the effects of aminoguanidine, a relatively selective inhibitor of iNOS, and
N
G
-nitro-
l
-arginine methyl ester (L-NAME), a nonselective NOS inhibitor on blood pressure, plasma volume, and albumin escape during the early and delayed phases of endotoxin shock in conscious, chronically catheterized rats. Red blood cell volume and plasma volume were determined by using chromium-51–tagged erythrocytes and iodine-125–labeled albumin, respectively. Injection of lipopolysaccharide (LPS) 10 mg/kg IV resulted in a fall in blood pressure, hemoconcentration, and increased total-body albumin escape, which is reflected by a 25% reduction in plasma volume. When LPS was injected into animals pretreated with L-NAME (7.4 μmol/kg IV 15 minutes before LPS), losses in plasma volume and albumin escape were significantly greater than in rats that received LPS alone, despite that L-NAME attenuated the hypotensive action of LPS. Aminoguanidine pretreatment (162 μmol/kg) had no effect on the early responses to LPS, whereas it was as potent as L-NAME in reversing hypotension when injected 70 minutes after LPS. Aminoguanidine treatment also prevented further losses in plasma volume and markedly attenuated total-body and organ albumin escape rates elicited by LPS. L-NAME produced only a slight attenuation of LPS-induced losses in plasma volume and albumin escape in most organs studied, whereas it potentiated albumin extravasation in the lung. These results demonstrate that inhibition of cNOS potentiates, whereas inhibition of iNOS markedly attenuates, losses in plasma volume and albumin escape elicited by LPS, and suggest that selective inhibitors of iNOS may be more effective than nonselective inhibitors of all forms of NOS in the therapy of septic shock.
Potentiation of the Actions of Bradykinin by Angiotensin I–Converting Enzyme Inhibitors
Authors: Minshall Richard D. , Tan Fulong , Nakamura Fumiaki , Rabito Sara F. , Becker Robert P. , Marcic Branislav , Erdös Ervin G.
Published: June 2012

Abstract
Part of the beneficial effects of angiotensin I–converting enzyme (ACE) inhibitors are due to augmenting the actions of bradykinin (BK). We studied this effect of enalaprilat on the binding of [
3
H]BK to Chinese hamster ovary (CHO) cells stably transfected to express the human BK B
2
receptor alone (CHO-3B) or in combination with ACE (CHO-15AB). In CHO-15AB cells, enalaprilat (1 μmol/L) increased the total number of low-affinity [
3
H]BK binding sites on the cells at 37°C, but not at 4°C, from 18.4±4.3 to 40.3±11.9 fmol/10
6
cells (
P
<.05;
K
d
, 2.3±0.8 and 5.9±1.3 nmol/L; n=4). Enalaprilat preserved a portion of the receptors in high-affinity conformation (
K
d
, 0.17±0.08 nmol/L; 8.1±0.9 fmol/10
6
cells). Enalaprilat decreased the IC
50
of [Hyp
3
-Tyr(Me)
8
]BK, the BK analogue more resistant to ACE, from 3.2±0.8 to 0.41±0.16 nmol/L (
P
<.05, n=3). The biphasic displacement curve of the binding of [
3
H]BK also suggested the presence of high-affinity BK binding sites. Enalaprilat (5 nmol to 1 μmol/L) potentiated the release of [
3
H]arachidonic acid and the liberation of inositol 1,4,5-trisphosphate (IP
3
) induced by BK and [Hyp
3
-Tyr(Me)
8
]BK. Moreover, enalaprilat (1 μmol/L) completely and immediately restored the response of the B
2
receptor, desensitized by the agonist (1 μmol/L [Hyp
3
-Tyr(Me)
8
]BK); this effect was blocked by the antagonist, HOE 140. Finally, enalaprilat, but not the prodrug enalapril, decreased internalization of the receptor from 70±9% to 45±9% (
P
<.05, n=7). In CHO-3B cells, enalaprilat was ineffective. ACE inhibitors in the presence of both the B
2
receptor and ACE enhance BK binding, protect high-affinity receptors, block receptor desensitization, and decrease internalization, thereby potentiating BK beyond blocking its hydrolysis.
Role of Tissue Renin in the Regulation of Aldosterone Biosynthesis in the Adrenal Cortex of Nephrectomized Rats
Authors: Volpe Massimo , Gigante Bruna , Enea Iolanda , Porcellini Antonio , Russo Rosaria , Lee Min Ae , Magri Paola , Condorelli Gerolama , Savoia Carmine , Lindpaintner Klaus , Rubattu Speranza
Published: June 2012

Abstract
The aim of the study was to investigate whether the adrenal renin-angiotensin system plays an independent role in the regulation of mineralocorticoid biosynthesis in the adrenal gland and to explore the mechanisms of this action. Twelve-week-old male Sprague-Dawley rats were studied: 22 rats were maintained on a regular diet; 27 and 22 rats received a low salt diet with and without treatment, respectively, with the angiotensin II (Ang II) AT
1
-subtype receptor antagonist losartan (10 mg/kg per day). A fraction of each group of rats underwent bilateral nephrectomy (n=12, 15, and 10, respectively) and was killed 48 hours later. In an additional group of 24 (12 intact and 12 nephrectomized) rats, the effects of the Ang II AT
2
-subtype receptor antagonist
PD123319
were investigated. In intact rats, plasma renin activity (PRA) and adrenal renin activity and expression were progressively raised by salt restriction and losartan, whereas aldosterone synthase mRNA and plasma aldosterone (PA) levels were increased by salt restriction and reduced by losartan. Forty-eight hours after nephrectomy, PRA fell to undetectable levels; in contrast, adrenal renin expression, assessed by semiquantitative reverse-transcriptase polymerase chain reaction (using GAPDH as a standard for gene expression), showed an 18-fold increase and was further increased after salt restriction and losartan (all
P
<.05). Also, adrenal renin activity was raised after nephrectomy and further increased after salt restriction (
P
<.05) and losartan. Cytochrome P450 aldosterone synthase expression in the adrenal cortex was stimulated by nephrectomy alone and by nephrectomy combined with low salt intake (
P
<.05), with consequent increases in PA concentrations. In losartan-treated salt-restricted nephrectomized rats, cytochrome P450 aldosterone synthase expression (
P
<.05 versus nephrectomy alone and nephrectomy plus salt restriction) and PA concentrations were diminished (
P
<.05) in spite of the observed increases of adrenal renin expression. The AT
2
-receptor antagonism did not significantly affect PRA, adrenal renin, and aldosterone biosynthesis and production in either intact or nephrectomized salt-restricted rats. These results demonstrate that the adrenal renin-angiotensin system plays an independent role in the regulation of mineralocorticoid biosynthesis in vivo. This action is mediated primarily via the Ang II AT
1
-subtype receptors.
Experimental Diabetes Is Associated With Functional Activation of Protein Kinase Cε and Phosphorylation of Troponin I in the Heart, Which Are Prevented by Angiotensin II Receptor Blockade
Authors: Malhotra Ashwani , Reich David , Reich Daniel , Nakouzi Antonio , Sanghi Vinay , Geenen David L. , Buttrick Peter M.
Published: June 2012

Abstract
A cardiomyopathy that is characterized by an impairment in diastolic relaxation and a loss of calcium sensitivity of the isolated myofibril has been described in chronic diabetic animals and humans. To explore a possible role for protein kinase C (PKC)–mediated phosphorylation of myofibrillar proteins in this process, we characterized the subcellular distribution of the major PKC isoforms seen in the adult heart in cardiocytes isolated from diabetic rats and determined patterns of phosphorylation of the major regulatory proteins, including troponin I (TnI). Rats were made diabetic with a single injection of streptozotocin, and myocardiocytes were isolated and studied 3 to 4 weeks later. In nondiabetic animals, 76% of the PKCε isoform was located in the cytosol and 24% was particulate, whereas in diabetic animals, 55% was cytosolic and 45% was particulate (
P
<.05). PKCδ, the other major PKC isoform seen in adult cardiocytes, did not show a change in subcellular localization. In parallel, TnI phosphorylation was increased 5-fold in cardiocytes isolated from the hearts of diabetic animals relative to control animals (
P
<.01). The change in PKCε distribution and in TnI phosphorylation in diabetic animals was completely prevented by rendering the animals euglycemic with insulin or by concomitant treatment with a specific angiotensin II type-1 receptor (AT
1
) antagonist. Since PKC phosphorylation of TnI has been associated with a loss of calcium sensitivity of intact myofibrils, these data suggest that angiotensin II receptor–mediated activation of PKC may play a role in the contractile dysfunction seen in chronic diabetes.
Enhanced Na
+
-Ca
2+
Exchange in the Infarcted Heart
Authors: Litwin Sheldon E. , Bridge John H. B.
Published: June 2012

Abstract
Cellular Ca
2+
regulation is abnormal in diseased hearts. We designed this study to assess the role of the Na
+
-Ca
2+
exchanger in excitation-contraction coupling in surviving myocardium of the infarcted heart. We measured cellular contractions and whole-cell currents in single left ventricular myocytes isolated from the hearts of rabbits with healed myocardial infarction (MI). Eight weeks after MI, rabbits had left ventricular dysfunction without overt heart failure. Myocytes isolated from regions adjacent to the infarcted zone were significantly longer than cells from control hearts. At low stimulation rates (0.5 Hz), the amplitude of field-stimulated contractions was increased (11.6±0.5% versus 10.2±0.6% resting cell length), whereas the time to peak shortening and action potential duration were prolonged in the MI cells. When stimulation frequency was increased to 2.0 Hz, cellular shortening did not change or decreased in myocytes from infarcted hearts, whereas control cells had a positive shortening-interval relationship. Cells from infarcted hearts had a significantly decreased (31%) L-type Ca
2+
current (
I
Ca
) density but no change in the current-voltage relationship or the kinetics of
I
Ca
inactivation. Maximal Na
+
-Ca
2+
exchange current density was significantly increased (32%) in the cells from infarcted hearts. Sarcoplasmic reticulum (SR) Ca
2+
content during a stable train of contractions, as estimated from caffeine-induced inward currents, was slightly increased (
P
=NS) in the MI myocytes. To determine whether Na
+
-Ca
2+
exchange influenced SR Ca
2+
content, cells were clamped at potentials between −70 and +90 mV for 400 ms. The amplitude of the contraction during a subsequent clamp step to +10 mV was then measured as an index of SR loading that occurred during the preceding clamp step. Steps to positive potentials produced greater augmentation of the subsequent contraction in MI than in control myocytes. In myocytes from the infarcted heart, increased activity of the Na
+
-Ca
2+
exchanger may promote Ca
2+
entry or decrease Ca
2+
extrusion. This relative augmentation of inward Ca
2+
flux by the exchanger may enhance SR Ca
2+
loading and thus support contractility that would otherwise be impaired as a result of decreased Ca
2+
current. However, Ca
2+
influx by the exchanger may contribute to the prolongation of contractions in myocytes from infarcted hearts.
Mevalonate-Dependent Inhibition of Transendothelial Migration and Chemotaxis of Human Peripheral Blood Neutrophils by Pravastatin
Authors: Dunzendorfer Stefan , Rothbucher Dorothea , Schratzberger Peter , Reinisch Norbert , Kähler Christian M. , Wiedermann Christian J.
Published: June 2012

Abstract
Pravastatin, a hydrophilic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, has been reported to beneficially affect atherogenesis, plaque stability, and transient myocardial ischemia in significant coronary artery disease by influencing lipid metabolism and by intracellular signaling via mevalonate pathway products other than cholesterol. Leukocytes are implicated to play a pathophysiological role in these events. We were interested in finding out whether pravastatin could affect transendothelial migration (TEM), chemotaxis, and respiratory burst activity of the neutrophil ex vivo. In addition, effects on monocyte and T-lymphocyte chemotaxis were tested. For TEM assays, monolayers of human umbilical vein endothelial cells (HUVECs) were grown to confluence on polycarbonate filters bearing 5-μm pores in Transwell (Costar) culture plate inserts. Chemotaxis experiments were performed using modified Boyden chambers with cellulose nitrate micropore filters. Respiratory burst activity was measured fluorometrically. Treatment of neutrophils and monocytes with pravastatin at 2 to 200 μmol/L and 10 to 1000 μmol/L, respectively, significantly decreased chemotaxis triggered by fMet-Leu-Phe. This effect was abolished in the presence of mevalonic acid (500 μmol/L); no effect of pravastatin was seen on T-lymphocyte chemotaxis triggered by interleukin-8. Preincubation of neutrophils with pravastatin (200 μmol/L) also resulted in a significant reduction in the number of neutrophils that transmigrated a tumor necrosis factor–stimulated or lipopolysaccharide-stimulated HUVEC monolayer. At none of the concentrations tested (2 pmol/L to 200 μmol/L) did pravastatin affect neutrophil respiratory burst activity. We conclude that pravastatin may alter monocyte chemotaxis and neutrophil-endothelial interactions in migratory responses at concentrations obtained in vivo with cholesterol-lowering doses.
Increased Myogenic Tone and Diminished Responsiveness to ATP-Sensitive K
+
Channel Openers in Cerebral Arteries From Diabetic Rats
Authors: Zimmermann Paul A. , Knot Harm J. , Stevenson Andrá S. , Nelson Mark T.
Published: June 2012

Abstract
Diabetes mellitus has profound adverse effects on vascular and, in particular, endothelial function. Although pressure-induced constriction (“myogenic tone”) is a major contributor to the regulation of blood flow, little is known about the effects of diabetes on this response. Diabetes has been shown to diminish the dilation of cerebral arteries to synthetic ATP-sensitive K
+
(K
ATP
) channel openers. In this study, we explored the effects of diabetes induced in rats by streptozotocin on cerebral artery (250 to 300 μm) myogenic tone and on vasodilations to the synthetic K
ATP
channel openers pinacidil and levcromakalim. Elevation of intravascular pressure caused a graded membrane potential depolarization and constriction, which was greater in arteries from diabetic rats compared with normal rats (at 60 mm Hg, 5 mV more depolarized and 22 μm more constricted). Pressurized arteries (at 60 mm Hg) from diabetic rats were 5- to 15-fold less sensitive to pinacidil and levcromakalim than were control arteries (EC
50
values for pinacidil and levcromakalim were 1.4 and 0.6 μmol/L, respectively, in diabetic animals and 0.3 and 0.04, respectively, in control animals;
P
<.05). Removal of the endothelium or addition of a NO synthase inhibitor,
N
G
-nitro-
l
-arginine (LNNA), in control arteries decreased the sensitivity to K
ATP
channel openers and depolarized and constricted control arteries to levels similar to those observed in arteries from diabetic animals. Sodium nitroprusside caused a membrane potential hyperpolarization and enhanced the response to pinacidil in arteries from diabetic animals. Removal of the endothelium or LNNA had little effect on the apparent K
ATP
channel opener sensitivity, the membrane potential, and pressure-induced constrictions of arteries from diabetic animals. The results are consistent with the hypothesis that this type of diabetes leads to a decrease in tonic NO release from the endothelium, which in turn causes membrane potential depolarization and vasoconstriction, resulting in a diminished response to K
ATP
channel openers.
Mathematical Model of an Adult Human Atrial Cell
Authors: Nygren A. , Fiset C. , Firek L. , Clark J. W. , Lindblad D. S. , Clark R. B. , Giles W. R.
Published: June 2012

Abstract
—We have developed a mathematical model of the human atrial myocyte based on averaged voltage-clamp data recorded from isolated single myocytes. Our model consists of a Hodgkin-Huxley–type equivalent circuit for the sarcolemma, coupled with a fluid compartment model, which accounts for changes in ionic concentrations in the cytoplasm as well as in the sarcoplasmic reticulum. This formulation can reconstruct action potential data that are representative of recordings from a majority of human atrial cells in our laboratory and therefore provides a biophysically based account of the underlying ionic currents. This work is based in part on a previous model of the rabbit atrial myocyte published by our group and was motivated by differences in some of the repolarizing currents between human and rabbit atrium. We have therefore given particular attention to the sustained outward K
+
current (
I
sus
), which putatively has a prominent role in determining the duration of the human atrial action potential. Our results demonstrate that the action potential shape during the peak and plateau phases is determined primarily by transient outward K
+
current,
I
sus
, and L-type Ca
2+
current (
I
Ca,L
) and that the role of
I
sus
in the human atrial action potential can be modulated by the baseline sizes of
I
Ca,L
,
I
sus
, and the rapid delayed rectifier K
+
current. As a result, our simulations suggest that the functional role of
I
sus
can depend on the physiological/disease state of the cell.
Recombinant Murine Interleukin-12 Facilitates Induction of Cardiac Myosin–Specific Type 1 Helper T Cells in Rats
Authors: Okura Yuji , Takeda Kazuyoshi , Honda Shigeru , Hanawa Haruo , Watanabe Hisami , Kodama Makoto , Izumi Tohru , Aizawa Yoshifusa , Seki Shuhji , Abo Toru
Published: June 2012

Abstract
—Autoimmunity after viral myocarditis is considered to be one of the causes of dilated cardiomyopathy. Cytokines are assumed to play an important role in the pathogenesis. We recently reported that interleukin (IL)-2 and interferon (IFN)-γ mRNA are expressed in the myocardium of rats with experimental autoimmune myocarditis (EAM). However, the role of cytokines in autoimmune myocardial injury in detail is still not clear. Reverse transcription–polymerase chain reaction identified IL-12 (p40) mRNA in antigen-presenting cells in the initial phase of EAM. Cardiac myosin–specific T lymphocytes (MSTLs) were cultured with cardiac myosin peptide (CMP) in the presence of IL-2 and/or IL-12 and were transferred to other naive rats. The results showed that EAM could be effectively induced by transfer of MSTLs cultured with IL-12, whereas transfer of MSTLs cultured with IL-2 was less effective. However, IL-2 acts synergistically with IL-12, and MSTLs cultured with both cytokines most efficiently induce EAM. In vitro experiments showed that MSTLs cultured with both IL-12 and IL-2 produced a much greater amount of IFN-γ than did MSTLs cultured with either IL-12 or IL-2 alone. The amount of IFN-γ production was correlated with pathogenicity of MSTLs. Transfer experiments after sorting further demonstrated that the transfer was affected by CD4
+
helper T (Th) cells but not by CD8
+
cytotoxic T lymphocytes. IL-12 and IL-2 synergistically enhance the pathogenicity of MSTLs. Furthermore, a type 1 Th (Th1) cytokine, IFN-γ, which is a potent regulatory cytokine of autoimmunity, is produced by MSTLs. IL-12 and IL-2 potentiate the expansion of cardiac myosin–specific Th1 cells and play an important role in the development of autoimmune myocardial injury.
α
1
-Adrenergic Stimulation of Sarcolemmal Na
+
-H
+
Exchanger Activity in Rat Ventricular Myocytes
Authors: Yokoyama Hiroyuki , Yasutake Masahiro , Avkiran Metin
Published: June 2012

Abstract
—α
1
-Adrenoceptor (α
1
-AR) stimulation increases sarcolemmal Na
+
-H
+
exchanger (NHE) activity. The present study was designed to determine the role(s) of α
1
-AR subtype(s) in mediating this response. As an index of NHE activity, acid efflux rates (
J
H
s) were determined in single rat ventricular myocytes loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after 2 consecutive intracellular acid pulses in bicarbonate-free medium.
J
H
at pH
i
6.90 did not change significantly during the second pulse relative to the first in control cells but increased in a dose-dependent manner when the second pulse occurred in the presence of phenylephrine (nonselective α
1
-AR agonist) or A61603 (α
1A
-AR–selective agonist), with EC
50
values of 1.24 μmol/L and 3.6 nmol/L, respectively (both agonists given together with 1 μmol/L atenolol). Stimulation of NHE activity by 10 μmol/L phenylephrine was inhibited in a dose-dependent manner by the competitive antagonists prazosin, WB4101, and 5-methylurapidil, with IC
50
values of 12, 32, and 149 nmol/L, respectively. Analyses of the relative EC
50
and IC
50
values obtained (and
K
i
values estimated from the antagonist IC
50
s) in relation to the relative potencies of these agents at native rat α
1
-AR subtypes and their relative affinities for recombinant rat α
1
-ARs suggest that α
1
-adrenergic stimulation of sarcolemmal NHE activity is likely to be mediated selectively by the α
1A
-AR.
Flow Rate–Modulated Dissolution of Fibrin With Clot-Embedded and Circulating Proteases
Authors: Komorowicz Erzsébet , Kolev Krasimir , Léránt István , Machovich Raymund
Published: June 2012

Abstract
—The efficiency of plasmin, miniplasmin, and neutrophil leukocyte elastase in fibrin digestion is well characterized in static systems. Since in vivo the components of the fibrinolytic system are permanently exposed to flow, we have developed two in vitro models and studied the effect of shear forces on fibrin dissolution with these proteases. Cylindrical nonocclusive fibrin clots are perfused at various flow rates through their preformed axial channel, and dissolution of fibrin is followed by measuring the absorbance of degradation products released into the circulating fluid phase. In one experimental setting, fibrin surface is degraded with enzymes applied in the recirculating fluid phase; in another setting, clots containing gel-embedded proteases are perfused with enzyme-free buffer. As shear rate at fibrin surface is changed from 25 to 500 s
−1
, the rate of product release by recirculated enzymes increases 2.8-, 2.9-, and 4-fold for plasmin, miniplasmin, and porcine pancreatic elastase, respectively. Buffer-perfused fibrin containing gel-embedded plasmin or miniplasmin is disintegrated by shear forces at a relatively early stage of dissolution, and this disassembly is related to the formation of fragment Y (150 kDa) and fragment D (100 kDa) fibrin degradation products. Fibrin clots degraded by incorporated polymorphonuclear leukocyte elastase, which yields different degradation products, do not disassemble abruptly, even at the highest shear rate (500 s
−1
). Our results suggest that fibrin surface degradation is accelerated with increasing shear rate and that plasmin or miniplasmin embedded in the clot promotes the release of particular clot remnants into the circulating phase, whereas polymorphonuclear leukocyte elastase does not.