bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric Oxide Produced by Endothelial Cells Increases Production of Eicosanoids Through Activation of Prostaglandin H Synthase
Authors: Davidge Sandra T. , Baker Philip N. , McLaughlin Margaret K. , Roberts James M.
Published: June 2012

Abstract
The endothelium serves many functional roles, including the modulation of vascular smooth muscle tone through the release of vasoactive agents such as nitric oxide (NO) and the eicosanoids. We proposed that NO produced by endothelial cells would increase the production of eicosanoids through enhanced expression and/or activation of prostaglandin H synthase. NO and eicosanoid synthesis were stimulated in a bovine coronary microvessel endothelial cell line with the calcium ionophore A23187 (1 μmol/L). Our data demonstrated the following: (1) A23187 stimulated NO synthesis along with prostacyclin and thromboxane production. (2) Inhibition of NO synthesis with
N
G
-nitro-
l
-arginine methyl ester (0.1 mmol/L) significantly diminished both prostacyclin and thromboxane production. (3) Cells incubated with hemoglobin (2 μg/mL), which inactivates NO, decreased A23187-stimulated prostacyclin production, whereas cells incubated with superoxide dismutase (20 U/mL), which protects NO from superoxide anions, enhanced prostacyclin production. (4) Exogenous NO stimulated prostacyclin production. (5) The interaction of NO with prostacyclin persisted in the presence of excess exogenous arachidonic acid (100 μmol/L). (6) Cyclooxygenase activity in cell lysates increased in the first hour of NO stimulation. (7) NO stimulation of prostacyclin occurred within 1 hour and continued for 8 hours. (8) Neither constitutive nor inducible prostaglandin H synthase enzyme expression was altered by NO. (9) Cycloheximide (10 μmol/L) had no effect on A23187 stimulation of prostacyclin production. (10) Exogenous cGMP (10 μmol/L) or a phosphodiesterase inhibitor (1 mmol/L) did not affect prostacyclin production. These data indicate that stimulating synthesis of endogenous NO in cultured endothelial cells increased eicosanoid production through activation of prostaglandin H synthase.
Endothelium-Specific In Vivo Gene Transfer
Authors: Schulick Andrew H. , Dong Gang , Newman Kurt D. , Virmani Renu , Dichek David A.
Published: June 2012

Abstract
Targeted expression of genetic material within the vascular endothelium is potentially a powerful tool for the investigation of endothelial cell (EC) biology. We developed, optimized, and characterized an efficient somatic transgenic model of EC-specific gene transfer. Rat carotid arteries were infused with adenovirus expressing a β-galactosidase (β-gal) gene. The level and cell-type specificity of recombinant gene expression were measured by assaying β-gal activity in vessel extracts and by counting transduced cells in histological sections. Toxicity was evaluated by counting total ECs (3 days) and by measuring neointimal formation (14 days). Effects of transduction on the proliferation of vascular cells were measured with bromodeoxyuridine and [
3
H]thymidine. Maximum recombinant gene expression resulted from infusion of 1×10
10
to 1×10
11
plaque-forming units (pfu) per milliliter; ≈35% of luminal ECs were transduced. A high degree of EC specificity (90% to 98% of total transduced cells) was maintained over this range of virus concentrations. More highly concentrated virus resulted in loss of β-gal expression and a large decrease in luminal EC number (97% decrease,
P
<.001). Gene transfer at 4×10
10
pfu/mL was efficient, preserved EC integrity, and caused minimal neointimal formation. After gene transfer, there were early (3-day) increases in both EC and smooth muscle cell proliferation. At 14 days, only EC proliferation remained elevated (18% versus 1.4% in vehicle-infused arteries,
P
=.005). This animal model permits efficient highly EC-specific gene transfer. Vascular toxicity is minimal, although the EC proliferative index is elevated. This model will be useful in experiments that elucidate the biological role of EC gene products and define pathways of EC gene regulation and signal transduction in vivo.
Thrombin Regulates Expression of Monocyte Chemoattractant Protein-1 in Vascular Smooth Muscle Cells
Authors: Wenzel Ulrich O. , Fouqueray Bruno , Grandaliano Giuseppe , Kim Yong-Soo , Karamitsos Costantinos , Valente Anthony J. , Abboud Hanna E.
Published: June 2012

Abstract
Thrombin, a serine protease generated at sites of vascular injury, plays a role in the pathogenesis of atherosclerosis and restenosis after angioplasty. Adherence of monocytes to the endothelium and migration into the subendothelial space is an important early event in the pathogenesis of atherosclerosis. Monocyte chemoattractant protein 1 (MCP-1) may be an important mediator of monocyte recruitment to the tissue in this and other diseases. We have characterized the expression of MCP-1 in vascular smooth muscle cells (VSMCs) isolated from human renal artery and studied its regulation by thrombin. Serum-deprived cells release monocyte chemotactic activity that is neutralized (80%) by an MCP-1 antibody. The antibody recognized a 13- and 15-kD protein in smooth muscle cell–conditioned medium. Thrombin stimulates MCP-1 gene expression in a concentration- and time-dependent manner. An increase over basal levels was observed with concentrations of thrombin as low as 0.05 U/mL. The maximal effect occurred at 5 U/mL. The stimulatory effect was detected within 1 hour, reached a maximum at 3 hours, and was still present at 8 to 24 hours after the addition of thrombin. A concentration- and time-dependent effect of thrombin on MCP-1 gene expression was also found in rat VSMCs. The thrombin protease inhibitor hirudin blocked thrombin-induced MCP-1 expression. Thrombin stimulated the release of MCP-1 protein in conditioned medium of human VSMCs as measured by radioimmunoassay and chemotactic assay. Thrombin also increased monocyte chemotactic activity in short-term organ cultures of rat aortic rings and in first passage cells. The effect of thrombin on MCP-1 was mimicked by a thrombin receptor–activating peptide (NH
2
-Ser-Phe-Leu-Leu-Arg-Asn-Pro-COOH). These data describe an important biological activity of thrombin in VSMCs and provide a novel mechanism whereby locally released thrombin may contribute to the pathogenesis of atherosclerosis or restenosis after angioplasty.
Native Low-Density Lipoprotein Increases Endothelial Cell Nitric Oxide Synthase Generation of Superoxide Anion
Authors: Pritchard Kirkwood A. , Groszek Laura , Smalley David M. , Sessa William C. , Wu Mingdan , Villalon Patricio , Wolin Michael S. , Stemerman Michael B.
Published: June 2012

Abstract
To examine mechanisms by which native low-density lipoprotein (n-LDL) perturbs endothelial cell (EC) release of superoxide anion (O
2
−
) and nitric oxide (NO), ECs were incubated with n-LDL at 240 mg cholesterol per deciliter for 4 days with media changes every 24 hours. n-LDL increases EC release of O
2
−
by more than fourfold and increases nitrite production by 57%. In the conditioned media from day-4 incubations, n-LDL increases total nitrogen oxides 20 times control EC (C-EC) levels. However, n-LDL did not alter EC NO synthase (eNOS) enzyme activity as measured by the [
3
H]citrulline assay.
N
ω
-Nitro-
l
-arginine methyl ester, a specific inhibitor of eNOS activity, increases C-EC release of O
2
−
by >300% but decreases LDL-treated EC (LDL-EC) release by >95%.
l
-Arginine inhibits the release of O
2
−
from LDL-ECs by >95% but did not effect C-EC release of O
2
−
. Indomethacin and SKF 525A partially attenuate LDL-induced increases in O
2
−
production by ≈50% and 30%, respectively. Thus, n-LDL increases O
2
−
and NO production, which increases the likelihood of the formation of peroxynitrite (ONOO
−
), a potent oxidant. n-LDL increases the levels of nitrotyrosine, a stable oxidation product of ONOO
−
, and tyrosine by ≈50%. In spite of this increase in oxidative metabolism, analysis of thiobarbituric acid substances reveals that no significant changes in the oxidation of n-LDL occur during the 24-hour incubations with ECs. These data indicate that n-LDL directly perturbs endothelial oxidative metabolism and uncouples
l
-arginine metabolism from NO release to increase eNOS generation of O
2
−
. Such changes may represent one of the earliest EC perturbations in atherogenesis.
Direct In Vivo Observation of Subendocardial Arteriolar Response During Reactive Hyperemia
Authors: Yada Toyotaka , Hiramatsu Osamu , Kimura Akihiro , Tachibana Hiroyuki , Chiba Yoko , Lu Songfang , Goto Masami , Ogasawara Yasuo , Tsujioka Katsuhiko , Kajiya Fumihiko
Published: June 2012

Abstract
To study the vasodilatory capacity of subendocardial (ENDO) arterioles, we evaluated the reactive hyperemic responses of ENDO as well as subepicardial (EPI) arterioles in 40 dogs by our needle-probe intravital microscope. We also examined the individual and combined effects of an ATP-sensitive K
+
channel blocker (glibenclamide, 200 μg/kg), an inhibitor of nitric oxide synthase (
N
G
-monomethyl-
l
-arginine [L-NMMA], 2 μmol/min, 20 minutes), and an adenosine-receptor antagonist (8-phenyltheophylline [8PT], 0.75 μmol/min, 15 minutes). The percent increase in end-diastolic diameter of ENDO arterioles was larger (
P
<.01) than that of EPI arterioles during reactive hyperemia, especially for the arterioles larger than 120 μm (
P
<.01). The diastolic-to-systolic vascular pulsation amplitude at the peak flow was greater in ENDO than EPI arterioles (25% versus 6%,
P
<.05). Compared with control conditions, the presence of both glibenclamide and L-NMMA suppressed the vasodilation responses of ENDO arterioles (
P
<.01 for both) and EPI arterioles (
P
<.05 for both). The effect of L-NMMA was greater in ENDO arterioles (
P
<.01), but that of glibenclamide was not different between ENDO and EPI arterioles. 8PT influenced the hyperemic response, although statistical significance was found only in the flow response. The effect of combined infusion of L-NMMA and glibenclamide with or without 8PT was greater than that of individual infusions in both ENDO and EPI arterioles. Conclusions are as follows: (1) The vasodilatory response of ENDO arterioles was even larger than that of EPI arterioles. Thus, the smaller flow reserve of ENDO arterioles may be caused by other factors, including the greater effects of myocardial compression and nitric oxide on the ENDO arterioles. (2) The vascular responses of ENDO and EPI arterioles were modulated by both endothelium-independent and -dependent vasodilative factors, and the effect of each factor including adenosine was associated with the effects of others.
Optical Mapping Reveals That Repolarization Spreads Anisotropically and Is Guided by Fiber Orientation in Guinea Pig Hearts
Authors: Kanai Anthony , Salama Guy
Published: June 2012

Abstract
Guinea pig hearts were stained with a voltage-sensitive dye and imaged on a photodiode array to record fluorescent action potentials (APs) from 124 sites. Activation and repolarization patterns were recorded from the epicardium during stimulation at different loci and correlated with the underlying fiber architecture. Endocardial APs were recorded by inserting a light guide into the ventricular cavity or by dissecting out the ventricular free wall to expose the endocardium. In hearts paced on the right atrium to simulate sinus rhythm, activation emerged synchronously over a large area of the ventricular epicardium and spread laterally in 5 to 7 ms. The apparent longitudinal and transverse velocities were 2.66±0.11 and 1.65±0.09 m/s (n=12). In contrast, repolarization began near the apex on the endocardium and spread transmurally in 6±1.3 ms (n=12) and then anisotropically along the epicardium in 25 to 30 ms with apparent maximum (0.53±0.11 m/s) and minimum (0.31±0.10 m/s) repolarization velocities that aligned with the longitudinal and transverse axes of epicardial fibers. When paced on the epicardium, activation of intact hearts (n=12) and perfused sheets (n=8) was anisotropic, with longitudinal (0.85±0.05 m/s) and transverse (0.44±0.04 m/s) conduction velocities that aligned with the epicardial fiber orientation. When activation was initiated at different sites on the epicardium, repolarization always began near the apex and exhibited patterns similar to those obtained under right atrial pacing, but with slower longitudinal (0.41±0.09 m/s) and transverse (0.23±0.07 m/s) repolarization velocities (n=18). In sheets stretched parallel to the longitudinal axis of surface fibers, AP durations (APDs) increased as a function of fiber length, from the length at zero developed tension to 120% of the length at maximum developed tension (L
max
). Spatial distributions of APDs did not change during stretches along the rising phase of the length-tension curve. In sheets stretched to 50% of L
max
, APDs were shorter and more homogeneous on the endocardium (mean APD, 188 ms; ΔAPD, 195−186=9 ms) than on the epicardium (mean APD, 204 ms; ΔAPD, 212−186=26 ms; n=8). In guinea pig hearts, activation is rapid; therefore, repolarization depends primarily on intrinsic spatial heterogeneities of APDs. Consequently, repolarization begins at endocardial cells with the shortest APDs and spreads transmurally and then anisotropically on the surface according to the epicardial cell orientation.
Fluid Shear Stress Stimulates Mitogen-Activated Protein Kinase in Endothelial Cells
Authors: Tseng Hennessey , Peterson Timothy E. , Berk Bradford C.
Published: June 2012

Abstract
Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. Because mitogen-activated protein (MAP) kinases have been shown to be activated by physical forces, we measured the phosphorylation and enzyme activity of MAP kinase to identify the signal events involved in the endothelial cell response to fluid shear stress. Flow at physiological shear stress (3.5 to 117 dynes/cm
2
) activated 42-kD and 44-kD MAP kinases present in cultured bovine aortic endothelial cells, with maximal effect at 12 dynes/cm
2
. Activation of a G protein was necessary, as demonstrated by complete inhibition by the nonhydrolyzable GDP analog GDP-βS. Activation of protein kinase C (PKC) was required, as shown by inhibiting PKC with staurosporine or downregulating PKC with phorbol 12,13-dibutyrate. Both Ca
2+
-dependent and -independent PKC activity, measured by translocation and substrate phosphorylation, increased in response to flow. However, MAP kinase activation was not dependent on Ca
2+
mobilization, since Ca
2+
chelation had no inhibitory effect. On the basis of these findings, it is proposed that flow activates two signal-transduction pathways in endothelial cells. One pathway is Ca
2+
dependent and involves activation of phospholipase C and increases in intracellular Ca
2+
. A new pathway, described in the present study, is Ca
2+
independent and involves a G protein and increases in PKC and MAP kinase activity.
Expression of cGMP-Dependent Protein Kinase I and Phosphorylation of Its Substrate, Vasodilator-Stimulated Phosphoprotein, in Human Endothelial Cells of Different Origin
Authors: Draijer Richard , Vaandrager Arie B. , Nolte Christine , de Jonge Hugo R. , Walter Ulrich , van Hinsbergh Victor W.M.
Published: June 2012

Abstract
Previous studies demonstrated that the thrombin-induced permeability of endothelial cell monolayers is reduced by the elevation of cGMP. In the present study, the presence of cGMP-dependent protein kinase (cGMP-PK) immunoreactivity and activity in various types of human endothelial cells (ECs) and the role of cGMP-PK in the reduction of thrombin-induced endothelial permeability was investigated. cGMP-PK type I was demonstrated in freshly isolated ECs from human aorta and iliac artery as well as in cultured ECs from human aorta, iliac vein, and foreskin microvessels. Addition of the selective cGMP-PK activator 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), an established cGMP-PK substrate, which is localized at cell-cell contact sites of confluent ECs. cGMP-PK type I expression decreased during serial passage of ECs, which correlated with a diminished ability of 8-pCPT-cGMP to induce VASP phosphorylation. Preincubation of aorta and microvascular EC monolayers with 8-pCPT-cGMP caused a 50% reduction of the thrombin-stimulated permeability, as determined by measuring the peroxidase passage through EC monolayers on porous filters. Furthermore, the thrombin-induced rise in cytoplasmic [Ca
2+
]
i
was strongly attenuated by the cGMP-PK activator in fura 2-loaded aorta ECs. In contrast, cGMP-PK could not be demonstrated in freshly isolated and cultured human umbilical vein ECs. Incubation of umbilical vein ECs with 8-pCPT-cGMP did not cause VASP phosphorylation and had no effect on the thrombin-induced increases in cytoplasmic Ca
2+
and endothelial permeability. These data indicate that cGMP-PK type I is expressed in various types of human macrovascular and microvascular ECs but is absent or expressed in very low amounts in umbilical vein ECs. cGMP-PK type I expression in ECs may be important in the regulation of endothelial permeability and the release of factors involved in vasoregulation and hemostasis.
Regulation of Very-Low-Density Lipoprotein Receptor in Hypertrophic Rat Heart
Authors: Masuzaki Hiroaki , Jingami Hisato , Matsuoka Naoki , Nakagawa Osamu , Ogawa Yoshihiro , Mizuno Megumi , Yoshimasa Yasunao , Yamamoto Tokuo , Nakao Kazuwa
Published: June 2012

Abstract
To elucidate the regulation of very-low-density lipoprotein (VLDL) receptor, we have studied its gene expression in the heart of spontaneously hypertensive rats–stroke prone (SHR-SP, an animal model for hypertension-induced cardiac hypertrophy) compared with Wistar-Kyoto rats. RNase protection assay showed that ventricular VLDL receptor mRNA falls to 41% of normal levels at 4 weeks, when hypertension is not yet fully developed, and drops further to 14% at 13 weeks, when cardiac hypertrophy is established. Lipoprotein lipase mRNA decreases in parallel with VLDL receptor mRNA. In cultured neonatal rat ventricular cardiomyocytes, VLDL receptor mRNA decreases in parallel with the process of cardiocyte hypertrophy during the 24 hours after treatment with 10
−8
mol/L endothelin-1, falling to 40% of the initial value. These results demonstrate that there is downregulation of VLDL receptor gene expression in cardiac hypertrophy both in vivo and in vitro and suggest that the regulation of the VLDL receptor is possibly linked with the switch in energy substrate from lipid to glucose known to occur in cardiac hypertrophy.
Locally Produced Tumor Necrosis Factor-α Mediates Interleukin-2Induced Lung Injury
Authors: Rabinovici R. , Feuerstein G. , Abdullah F. , Whiteford M. , Borboroglu P. , Sheikh E. , Phillip D.-R. , Ovadia P. , Bobroski L. , Bagasra O. , Neville L.F.
Published: June 2012

Abstract
Interleukin (IL)-2–induced microvascular lung injury is an experimental paradigm commonly used to investigate the pathogenesis of the adult respiratory distress syndrome. Since tumor necrosis factor-α (TNF-α) is known to induce such an injury in vivo and since TNF-α is involved in other models of lung injury, we postulated that it might also mediate pulmonary toxicity after IL-2 administration. The present study tested this hypothesis by evaluating the effect of TNF-α inhibition on IL-2–induced lung injury in the rat. Recombinant human IL-2 (10
6
U IV per rat, n=6) elevated lung water, myeloperoxidase activity, and protein accumulation in bronchoalveolar lavage fluid and induced tissue hypoxia. Also, IL-2 enhanced lung tissue TNF-α mRNA and peptide (1543±496 pg/g lung wet weight) localized to alveolar macrophages by in situ hybridization. In marked contrast, IL-2 failed to affect serum TNF-α, which remained at undetectable levels. Pretreatment with anti–TNF-α monoclonal antibody (25 mg/kg IV, n=7) or the TNF-α synthesis inhibitor rolipram (200 μg/kg IV, n=7) attenuated lung injury and reverted tissue hypoxia. Furthermore, TNF-α inhibition prevented the upregulation of lung tissue IL-1β, IL-6, cytokine-induced neutrophil chemoattractant, and E-selectin (ELAM-1) but not intercellular adhesion molecule-1 mRNAs in response to IL-2. These data imply that locally produced TNF-α mediates IL-2–induced lung inflammation and tissue injury and point to the potential utilization of TNF-α inhibitors in treating the pulmonary toxicity of IL-2 immunotherapy.
Preferential Differentiation of P19 Mouse Embryonal Carcinoma Cells Into Smooth Muscle Cells
Authors: Suzuki Toru , Kim Hyo-Soo , Kurabayashi Masahiko , Hamada Hiroshi , Fujii Hideta , Aikawa Masanori , Watanabe Masafumi , Watanabe Noboru , Sakomura Yasunari , Yazaki Yoshio , Nagai Ryozo
Published: June 2012

Abstract
Investigation of the molecular mechanisms that control smooth muscle cell (SMC) development and differentiation is a prerequisite in understanding the regulatory mechanisms of physiological and pathological SMC-associated vascular processes. The pluripotent murine embryonal carcinoma P19 cell, whose developmental potential resembles that of early embryonic cells, can develop into cell types derived from the neuroectoderm, mesoderm, and endoderm. In the present study, we have shown a unique strategy to enhance SMC differentiation in P19 cells. Under chemical induction of high concentrations of retinoic acid (1 μmol/L), P19 cells showed optimum differentiation into SMCs. Because the P19 cells thus induced also showed differentiation into neuronal cells, a strategy to block neuronal lineage differentiation was developed using a stable transformant antisense RNA construct against Brn-2, a neuronal lineage–specific POU-domain transcription factor; thus, by specifically inhibiting neuronal differentiation, enhanced SMC differentiation by P19 cells was attained. SMC expression was confirmed by immunohistochemical staining, RNA analysis (RNase protection assay), and protein analysis (Western blot) using SMC-specific markers (eg, SM1 and calponin) and α-smooth muscle actin. Our results show that the pathway of SMC differentiation may provide an in vitro system useful in the investigation of SMC regulatory mechanisms (eg, transcriptional regulation) and in the further understanding of SMC development and differentiation.
Protein Kinase C Activates ATP-Sensitive K
+
Current in Human and Rabbit Ventricular Myocytes
Authors: Hu Keli , Duan Dayue , Li Gui-Rong , Nattel Stanley
Published: June 2012

Abstract
Mediators involved in ischemic preconditioning, such as adenosine and norepinephrine, can activate protein kinase C (PKC), and a variety of observations suggest that both PKC and ATP-sensitive K
+
current (I
KATP
) play essential roles in ischemic preconditioning. PKC is therefore a candidate to link receptor binding to I
KATP
activation, but it has not been shown whether and how PKC can activate I
KATP
in the heart. The present study was designed to determine whether PKC can activate I
KATP
in rabbit and human ventricular myocytes. Under conditions designed to minimize Na
+
and Ca
2+
currents, dialysis of rabbit ventricular myocytes with pipette solutions containing reduced [ATP] elicited I
KATP
, with a 50% effective concentration (EC
50
) of 260 μmol/L. In cells that failed to show I
KATP
under control conditions, superfusion with 1 μmol/L phorbol 12,13-didecanoate (PDD) elicited I
KATP
in a fashion that depended on pipette [ATP], with an [ATP] EC
50
of 601 μmol/L. PDD-induced I
KATP
activation was concentration dependent, with an EC
50
of 7.1 nmol/L. The highly selective PKC inhibitor bisindolylmaleimide totally prevented I
KATP
activation by PDD, and in blinded experiments, 1 μmol/L PDD elicited I
KATP
in eight of nine cells, whereas its non–PKC-stimulating analogue 4α-PDD failed to elicit I
KATP
in any of the five cells tested (
P
=.003). Similar experiments were conducted in human ventricular myocytes and showed that 0.1 μmol/L PDD elicited I
KATP
at pipette [ATP] of 100 and 400 μmol/L (five of five cells at each concentration) but not at 1 mmol/L [ATP] (none of five cells). We conclude that PKC activates I
KATP
in rabbit and human ventricular myocytes by reducing channel sensitivity to intracellular ATP. This finding has potentially important implications for understanding the mechanisms of ischemic preconditioning.
Regulated Expression of the Ets-1 Transcription Factor in Vascular Smooth Muscle Cells In Vivo and In Vitro
Authors: Hultgårdh-Nilsson Anna , Cercek Bojan , Wang Jian-Wei , Naito Shinji , Lövdahl Cecilia , Sharifi Behrooz , Forrester James S. , Fagin James A.
Published: June 2012

Abstract
Ets-1 regulates the transcription of several genes encoding extracellular matrix proteins (ie, osteopontin and tenascin) as well as enzymes involved in degradation and remodeling of the extracellular matrix (ie, stromelysin and urokinase plasminogen activator). In the present study, we investigated the regulation of c-ets-1 in cultured rat vascular smooth muscle cells as well as in the arterial wall after balloon injury in vivo. Serum-starved smooth muscle cells exposed to serum for various time points express a major c-ets-1 mRNA transcript of 5.3 kb and minor bands of 4.0 and 2.5 kb with a peak at 2 hours after stimulation. These effects were concentration dependent. Western blotting revealed an increase in 55- and 40-kD immunoreactive ets-1 proteins in cells treated with serum for 2 hours, and binding to an oligonucleotide containing the ets-1 consensus
cis
-acting motif was demonstrated by electrophoretic mobility shift assay. Ets-1 mRNA abundance was induced with a peak at 2 hours after stimulation with platelet-derived growth factor-BB and with angiotensin II. There was a distinct increase of ets-1 immunoreactivity in the inner layer of the media 2 hours after balloon catheter injury of rat arteries, which declined after 6 hours and returned to the basal level 1 day after vessel wall damage. Arterial c-ets-1 mRNA content was induced with an identical time course. These findings suggest that c-ets-1 may be of importance in the mitogenic signaling pathway of smooth muscle cells grown in culture. In addition, ets-1 may play a role in the activation of smooth muscle cells in vivo after mechanical injury of the vessel wall. Because the ets-1 transcription factor activates the gene expression of a number of mRNA species involved in matrix deposition and degradation, these data are compatible with a role for ets-1 in vascular remodeling and/or cell migration.
Effect of Membrane Potential on the Initiation of Acetylcholine-Induced Ca
2+
Transients in Isolated Guinea Pig Coronary Myocytes
Authors: Ganitkevich Vladimir Y. , Isenberg Gerrit
Published: June 2012

Abstract
The muscarinic stimulation of single voltage-clamped coronary arterial smooth muscle cells of the guinea pig was used to evaluate the effect of membrane potential on the inositol 1,4,5-tris-phosphate (IP
3
)–mediated changes of ionized [Ca
2+
] in the cytoplasm (Ca
2+
transient) measured with indo 1. When applied at the membrane potential of −50 mV, 10 μmol/L acetylcholine (ACh) induced a [Ca
2+
]
i
increase after the mean latency of 2.6±0.9 s. The latency was reduced to 1.1±0.3 s when the same dose was applied at a holding potential of +50 mV. In paired experiments in the same cells, the latency of response at +50 mV was reduced by a factor of 2.2±0.3 compared with the response at −50 mV. Supramaximal [ACh] (100 μmol/L) induced Ca
2+
transients with a 0.4±0.1-s latency, which was independent of membrane potential. When applied repetitively at −50 mV, ACh induced Ca
2+
transients with a progressively reduced amplitude and slower rate of rise. Depolarization to +50 mV accelerated the rate of rise of the Ca
2+
transient by a factor of 3.4±0.4 without affecting the amplitude. The modulation of the initiation of Ca
2+
transient by a 100-mV depolarization can be explained by an approximately threefold increase in the rate of IP
3
accumulation.
Identification of Protein Kinase C Isoforms in Rat Mesenteric Small Arteries and Their Possible Role in Agonist-Induced Contraction
Authors: Ohanian Vasken , Ohanian Jacqueline , Shaw Linda , Scarth Sylvia , Parker Peter J. , Heagerty Anthony M.
Published: June 2012

Abstract
We have identified immunologically the protein kinase C (PKC) isoforms present in rat mesenteric small arteries, defined their distribution between particulate and soluble fractions, and studied their involvement in phorbol ester–induced contraction. Our analysis revealed the presence of the Ca
2+
-dependent PKCs (α and γ), Ca
2+
-independent PKCs (δ and ε), and the atypical isoform (ζ). PKCβ could not be detected, whereas PKCγ is likely to be of neural origin. All isoforms exhibited different distributions: PKCα, PKCε, and PKCζ were found in both particulate and soluble fractions. In contrast, PKCδ was mainly in the particulate fraction, and PKCγ was in the soluble fraction. Phorbol esters, which activate PKC and cause smooth muscle contraction, downregulated only the α and δ isoforms. This was associated with a parallel loss of contractile response to phorbol ester. The force developed to submaximal concentrations of noradrenaline was decreased after phorbol dibutyrate pretreatment, although the sensitivity and maximal response were unchanged. Phorbol ester pretreatment did not affect the contractile response to vasopressin. The sensitivity to non–receptor-mediated contraction, caused by K
+
in the presence of prazosin, was slightly reduced by 4α- and 4β-phorbol ester pretreatment. Maximal tension in response to this agonist was not affected. We conclude that PKCα and/or PKCδ is necessary for phorbol ester–mediated contraction but is not essential for noradrenaline-, vasopressin-, or K
+
-induced contraction, demonstrating differences in the mechanisms involved in the contractile response between these agents.
Passive Load and Angiotensin II Evoke Differential Responses of Gene Expression and Protein Synthesis in Cardiac Myocytes
Authors: Kent Robert L. , McDermott Paul J.
Published: June 2012

Abstract
This study introduced an improved model of loaded adult cardiocytes to address a proposed requirement for angiotensin II (Ang II) in the transduction pathway between load on the cardiac myocyte and its early anabolic responses of gene expression and acceleration of protein synthesis. The isolated cardiocytes were subjected to passive load by step increments of stretch and responded with proportional acceleration of protein synthesis in both adult and neonatal cardiocytes; this response was unaltered by 1 μmol/L [Sar
1
,Ile
8
]Ang II, an antagonist peptide to Ang II. Ang II from 1 nmol/L to 10 μmol/L did not increase protein synthesis after 4 hours in adult cardiocytes nor at 100 nmol/L in neonatal cardiocytes. However, 100 nmol/L Ang II did increase [
3
H]phenylalanine incorporation into neonatal cardiocyte protein over a 24-hour period by 10%, whereas passive load increased [
3
H]phenylalanine incorporation into protein by 30%, which was not blocked by [Sar
1
,Ile
8
]Ang II. Thus, the anabolic effect of load does not require Ang II to increase either 4-hour protein synthesis in both adult and neonatal cardiocytes or 24-hour [
3
H]phenylalanine incorporation into protein in neonatal cardiocytes. The genetic response of the cardiocyte to load was examined by assessing c-
fos
and Na
+
-Ca
2+
exchanger mRNA levels, because these are rapidly expressed at the onset of cardiac pressure overload. The c-
fos
mRNA was increased fourfold within 1 hour after 100 nmol/L Ang II treatment of either adult or neonatal cardiocytes. This c-
fos
induction was blocked by [Sar
1
,Ile
8
]Ang II. One hour after loading of adult cardiocytes, induction of c-
fos
expression was increased threefold; this was also blocked by [Sar
1
,Ile
8
]Ang II. Thus, load-induced c-
fos
expression was Ang II dependent in adult cardiocytes. In contrast, exchanger mRNA levels were increased threefold 1 hour after loading of adult cardiocytes, but this increased expression was not blocked by [Sar
1
,Ile
8
]Ang II. For additional comparison, c-
fos
expression was induced by Ang II and phorbol myristate acetate, which did not induce exchanger expression; conversely, exchanger expression was induced by veratridine, which did not increase c-
fos
expression. Thus, separate c-
fos
and exchanger expression pathways can be differentiated in adult cardiocytes. This study demonstrated that Ang II is not required for load to initiate the anabolic processes of accelerated protein synthesis or enhanced Na
+
-Ca
2+
exchanger gene expression in cardiocytes; however, load induced c-
fos
expression is Ang II dependent.
Differential Response to Na
+
Channel Blockade, β-Adrenergic Stimulation, and Rapid Pacing in a Cellular Model Mimicking the SCN5A and HERG Defects Present in the Long-QT Syndrome
Authors: Priori Silvia G. , Napolitano Carlo , Cantù Francesco , Brown Arthur M. , Schwartz Peter J.
Published: June 2012

Abstract
The long-QT syndrome (LQTS) is a hereditary disorder characterized by an abnormally prolonged QT interval and by life-threatening arrhythmias. Recently, two of the genes responsible for LQTS have been identified: SCN5A, a voltage-dependent Na
+
channel on chromosome 3 (LQT3), and HERG, responsible for the rapid component of the delayed rectifier current (I
Kr
), on chromosome 7 (LQT2). We developed an in vitro model to attempt reproduction of the expected alterations in LQT3 and LQT2 patients. Guinea pig ventricular myocytes were exposed to anthopleura toxin A (anthopleurin), an inhibitor of the inactivation of the Na
+
current, and to dofetilide, a selective blocker of I
Kr
. Both interventions significantly prolonged action potential duration (APD), by 54±13 and 62±16 ms, respectively. Cells pretreated with anthopleurin significantly shortened APD in response to mexiletine, isoproterenol, and rapid pacing (from 264±38 to 226±32 ms after mexiletine,
P
<.001). On the contrary, cells exposed to dofetilide did not shorten the APD after mexiletine and even prolonged it after initial exposure to isoproterenol (from 280±25 to 313±20 ms,
P
<.001); during rapid pacing, APD was shortened but less (38±9 versus 60±11 ms,
P
<.05) than in anthopleurin-treated cells. This study shows that a cellular model for LQTS, based on the recent advances in molecular genetics, can provide adequate “phenotypes” of prolonged repolarization amenable to the testing of interventions of potential clinical relevance. We found differential responses to Na
+
channel blockade, to β-adrenergic stimulation, and to rapid pacing according to specific pretreatment with either anthopleurin (to mimic LQT3) or dofetilide (to mimic LQT2). These different responses in myocytes bear striking similarities with the differential response to analogous interventions in LQTS patients with mutations on the SCN5A and HERG genes.
Effect of Endothelin-1 on Actomyosin ATPase Activity
Authors: McClellan George , Weisberg Andrea , Winegrad Saul
Published: June 2012

Abstract
Endothelin is a powerful inotropic peptide that increases isometric force in isolated papillary muscle and the extent of shortening in isolated single cardiac myocytes. Its mechanism of action has been variously attributed to increased Ca
2+
activation, increased Ca
2+
sensitivity of the contractile proteins, and increased intracellular pH, but the physiological function of the changes in cardiac performance remains obscure. In this study, the effects of endothelin-1 on both force development and the kinetics of contraction have been examined. Isometric force, actomyosin ATPase activity, and unloaded shortening velocity were measured. The effects were dose dependent. From 1 to 50 pmol/L endothelin-1 did not alter force development in isolated trabeculae with intact endothelial cells, but actomyosin ATPase activity was increased. Between 100 pmol/L and 10 nmol/L endothelin-1 raised isometric force, decreased actomyosin ATPase activity, and decreased unloaded shortening velocity. The reduction in ATPase activity was progressively enhanced as sarcomere length was increased from 1.9 to 2.4 μm. These results indicate that the effects of endothelin-1 on the force of contraction and the rate of ATP hydrolysis are not tightly coupled and are changed in the opposite directions by endothelin-1 over most of its effective-dose range. This raises the possibility that endothelin-1 may increase the economy of contraction. A novel function of endothelin may be the modulation of the efficiency of contraction, particularly when increased preload raises the contractile work of the heart.
α
1
-Adrenergic Activation Inhibits β-Adrenergic–Stimulated Unitary Ca
2+
Currents in Cardiac Ventricular Myocytes
Authors: Chen Long , El-Sherif Nabil , Boutjdir Mohamed
Published: June 2012

We have previously shown that whole-cell L-type Ca
2+
current that was stimulated through β-adrenergic receptors was negatively modulated by α
1
-adrenergic activation. In the present study, we investigated the kinetic basis of this modulation at the single-channel level in adult rat ventricular myocytes using Ba
2+
as the charge carrier. Unitary current sweeps were evoked by 300-ms depolarizing pulses to 0 mV, from a holding potential of −50 mV at 0.5 Hz. During control conditions, the ensemble-averaged current amplitude was 0.18±0.01 pA (n=7). To achieve β-adrenergic stimulation (β effect), cells were superfused with norepinephrine (10 μmol/L) in the presence of prazosin (10 μmol/L), an α
1
-adrenergic blocker. β-Adrenergic stimulation enhanced ensemble-averaged current (from 0.18±0.01 to 0.75±0.04 pA,
P
<.05, n=7), increased the open-time constants, and decreased the closed-time constants. To activate α
1
-receptors while maintaining the β-adrenergic stimulation, cells were superfused with norepinephrine alone (α
1
+β effects). α
1
-Adrenergic activation reduced ensemble-averaged current (from 0.75±0.04 to 0.41±0.03 pA,
P
<.05, n=7), decreased open-time constants, and increased closed-time constants. α
1
-Adrenergic activation also inhibited ensemble-averaged currents stimulated by a low concentration (10 μmol/L) of 8-bromo-cAMP but not by (−)Bay K 8644 (1 μmol/L). Calphostin C (1 μmol/L), a specific inhibitor of protein kinase C, attenuated α
1
-adrenergic inhibition on β-adrenergic–stimulated unitary currents. We conclude that α
1
-adrenergic activation exerts an inhibitory effect on β-adrenergic–stimulated unitary Ba
2+
current at the single-channel level. The shortening of the open-time and the lengthening of the closed-time constants and the increase in blank sweeps may explain the inhibition of the Ca
2+
-channel activity and the reduction in whole-cell Ca
2+
current previously reported. This inhibition is in part mediated through the protein kinase C pathway.
Angiotensin II Type 2 Receptor–Mediated Regulation of Rat Neuronal K
+
Channels
Authors: Martens Jeffrey R. , Wang Desuo , Sumners Colin , Posner Philip , Gelband Craig H.
Published: June 2012

We have previously shown that angiotensin II (Ang II), via AT
2
receptors, increases whole-cell K
+
current in cultured rat hypothalamus and brain stem neurons. We have now investigated the AT
2
receptor–mediated effects of Ang II on the activity of single delayed rectifier K
+
channels in cell-attached membrane patches. In control recordings (bath, 5.4 mmol/L K
+
; pipette, 140 mmol/L K
+
), two voltage-dependent channels were recorded with conductances of 34±4 and 56±6 pS, respectively (n=6). When patches were excised, the channels reversed near a membrane potential expected for a K
+
channel. In cell-attached patches (−40 mV), Ang II (100 nmol/L) increased open probability of the 56-pS K
+
channel from 0.03±0.01 to 0.21±0.05 (n=3). The selective AT
2
receptor antagonist PD 123319 (1 μmol/L) but not the AT
1
receptor antagonist losartan (1 μmol/L) blocked the actions of Ang II (n=3). The selective AT
2
receptor agonist CGP 42112 (100 nmol/L) produced similar effects to Ang II. Kinetic analysis of the Ang II effect showed that open-time histograms were best fit by two exponential functions. Ang II increased both open-time constants relative to control (control, τ
1
=0.9±0.1 milliseconds, τ
2
=2.3±0.3 milliseconds; Ang II, τ
1
=3.1±0.4 milliseconds, τ
2
=12.1±2.4 milliseconds), and PD 123319 blocked this effect (n=3). The closed-time histogram was not affected by Ang II, PD 123319, or losartan. These results suggest that activation of AT
2
receptors modulates rat hypothalamus and brain stem neuronal whole-cell K
+
current by increasing the open probability of a 56-pS K
+
channel.
Role of Endothelium-Derived Nitric Oxide in the Modulation of Canine Myocardial Mitochondrial Respiration In Vitro
Authors: Xie Yi-Wu , Shen Weiqun , Zhao Gong , Xu Xiaobin , Wolin Michael S. , Hintze Thomas H.
Published: June 2012

The mechanism responsible for the regulation of cardiac function by endogenous nitric oxide (NO) remains unclear. In this investigation, O
2
consumption by freshly isolated myocardial muscle segments from the left ventricular free wall of canine hearts was quantified by a Clark-type O
2
electrode at 37°C.
S
-nitroso-
N
-acetylpenicillamine (SNAP, 9±3% to 50±8%), bradykinin (BK, 14±3% to 30±5%), or carbachol (CCh, 15±4% to 29±4%) significantly attenuated tissue O
2
consumption at doses of 10
−7
to 10
−4
mol/L (mean±SE,
P
<.05). The effects of BK and CCh, but not SNAP, were blocked by 10
−4
mol/L
N
G
-nitro-
l
-arginine, consistent with both BK and CCh stimulating NO biosynthesis and with SNAP decomposing to release NO, respectively. Similar doses of 8-Br-cGMP caused a respiratory inhibition, but to a lesser extent (9±2% to 14±6%). A mitochondrial uncoupler, 2,4-dinitrophenol (at 1 mmol/L), blocked the effects of 8-Br-cGMP, but not those of SNAP, BK, or CCh, suggesting that the major site of action of NO is on mitochondrial electron transport. Myocardial muscle from dogs with pacing-induced heart failure had a basal O
2
consumption rate of 251±21 nmol·min
−1
·g
−1
, which was 54% higher than the rate seen in muscle from normal healthy canine hearts. The inhibitory effects of BK and CCh on O
2
consumption were not observed in failing cardiac tissue, but SNAP showed an unaltered inhibitory effect. Therefore, our results indicate that NO released from microvascular endothelium by BK, stimulation of muscarinic receptors, and perhaps flow velocity may play an important physiological role in the control of cardiac mitochondrial respiration, and the loss of this regulatory function may contribute to the development of heart failure.
Monocyte- and Cytokine-Induced Downregulation of Angiotensin-Converting Enzyme in Cultured Human and Porcine Endothelial Cells
Authors: Papapetropoulos Andreas , Antonov Alexander , Virmani Renu , Kolodgie Frank D. , Munn David H. , Marczin Nandor , Ryan James W. , Gerrity Ross G. , Catravas John D.
Published: June 2012

We investigated the effects of monocytes on endothelial cell (EC) ectoenzyme activity. Coculture of human aortic ECs with human monocytes (2×10
5
monocytes per 2-cm
2
well) led to a decrease in EC angiotensin-converting enzyme (ACE) activity (64.5±3.5% of control) but not aminopeptidase N, aminopeptidase P, and 5′-nucleotidase activities. Similar results were obtained using human umbilical vein EC–human monocyte and porcine aortic EC–porcine monocyte cocultures. The decrease in ACE activity was monocyte concentration and coculture time dependent, reaching a maximum of 65% decrease in activity at 120 hours. Monocyte-mediated reduction in ACE activity did not require cell to cell contact, since exposure of ECs to conditioned medium from cocultures (CCCM) or from monocyte cultures (MCM) produced a decrease in ACE activity similar to that observed in EC-monocyte cocultures. Exogenously added tumor necrosis factor (TNF)-α and interleukin (IL)-1α, two known secretory products of monocytes, simulated the effects of monocytes on ACE activity. Western blot analysis revealed a decrease in the amount of ACE protein in TNF-α–treated and CCCM-treated ECs compared with control ECs. Both TNF-α and IL-1α were present in CCCM and MCM but not EC-conditioned medium. Incubation of the cocultures with a mixture of neutralizing antibodies against TNF-α and IL-1 totally abolished the monocyte-induced decrease in ACE activity. In conclusion, monocytes decrease ACE activity in cultured ECs through the release of cytokines such as TNF-α and IL-1.
In Vivo Collagen Turnover Following Experimental Balloon Angioplasty Injury and the Role of Matrix Metalloproteinases
Authors: Strauss Bradley H. , Robinson Ranga , Batchelor Wayne B. , Chisholm Robert J. , Ravi Grama , Natarajan Madhu K. , Logan Richard A. , Mehta Shamir R. , Levy Daniel E. , Ezrin Alan M. , Keeley Fred W.
Published: June 2012

Extracellular matrix formation is the major component of the restenosis lesion that develops after balloon angioplasty. Although ex vivo studies have shown that the synthesis of collagen is stimulated early after balloon angioplasty, there is a delay in accumulation in the vessel wall. The objectives of this study were to assess collagen turnover and its possible regulation by matrix metalloproteinases (MMPs) in a double-injury iliac artery rabbit model of restenosis. Rabbits were killed at four time points (immediately and at 1, 4, and 12 weeks) after balloon angioplasty. In vivo collagen synthesis and collagen degradation were measured after a 24-hour incubation with [
14
C]proline. Arterial extracts were also run on gelatin zymograms to determine MMP (gelatinase) activity. Collagen turnover studies were repeated in a group of 1-week postangioplasty rabbits that were treated with daily subcutaneous injections of either a nonspecific MMP inhibitor, GM6001 (100 mg/kg per day), or placebo. Collagen synthesis and degradation showed similar temporal profiles, with significant increases in the balloon-injured iliac arteries compared with control nondilated contralateral iliac arteries immediately after angioplasty and at 1 and 4 weeks. Peak collagen synthesis and degradation occurred at 1 week and were increased (approximately four and three times control values, respectively). Gelatin zymography was consistent with the biochemical data by showing an increase of a 72-kD gelatinase (MMP-2) in the balloon-injured side immediately after the second injury, peaking at 1 week, and still detectable at 4 and 12 weeks (although at lower levels). In balloon-injured arteries, the MMP inhibitor reduced both collagen synthesis and degradation. Overall, at 1 week after balloon angioplasty, GM6001 resulted in a 33% reduction in collagen content in balloon-injured arteries compared with placebo (750±143 to 500±78 μg hydroxyproline per segment,
P
<.004), which was associated with a nonsignificant 25% reduction in intimal area. Our data suggest that degradation of newly synthesized collagen is an important mechanism regulating collagen accumulation and that MMPs have an integral role in collagen turnover after balloon angioplasty.