bluuebunny/crossref_metadata_2025 huggingface dataset:
Complement Gene Expression by Rabbit Heart
Authors: Yasojima Koji , Kilgore Kenneth S. , Washington Ruth A. , Lucchesi Benedict R. , McGeer Patrick L.
Published: June 2012

Abstract
—Activation of the complement system has been implicated in the pathogenesis of myocardial ischemia/reperfusion injury. It has always been assumed that liver is the primary source of complement components. In the present study, we used the reverse-transcriptase polymerase chain reaction technique to establish that the mRNAs for complement proteins C3 and C9 are expressed in rabbit heart. Rabbit liver, brain, spleen, and kidney were also shown to express C3 and C9 mRNAs. We used Western blotting to establish that these mRNAs in heart are translated into the corresponding proteins. We further established that dramatic upregulation of the mRNAs occurred in Langendorff-perfused isolated hearts subjected to ischemia and reperfusion. C3 mRNA was always expressed at higher levels than was C9 mRNA, but C9 mRNA showed greater upregulation under stress. Compared with levels in control hearts subjected to 5 minutes of normoxic perfusion, hearts subjected to 0.5 hours of ischemia followed by 1 hour of reperfusion had a 4.72-fold increase in C3 mRNA and a 19.5-fold increase in C9 mRNA. By contrast, C3 mRNA in hearts subjected to 3.5 hours of normoxic perfusion showed no change, and those subjected to 3.5 hours of ischemia showed only a 1.72-fold increase, whereas C9 mRNA levels increased by 5.17-fold after 3.5 hours of normoxic perfusion and 12.5-fold after 3.5 hours of ischemia. The results of this study demonstrate for the first time that heart tissue is capable of expressing genes and proteins of the complement system, although it is not yet known which cell types are responsible. They further demonstrate that ischemia and reperfusion of the heart promotes a rapid upregulation of the mRNAs encoding the complement proteins C3 and C9 and that these abnormal levels considerably exceed those of normal liver. These observations are consistent with the hypothesis that local production of complement proteins may contribute significantly to the degree of ischemic injury to the myocardium and that complement expression is augmented by reperfusion.
Atypical Protease-Activated Receptor Mediates Endothelium-Dependent Relaxation of Human Coronary Arteries
Authors: Hamilton Justin R. , Nguyen Paul B. , Cocks Thomas M.
Published: June 2012

Abstract
—Protease-activated receptors (PARs) are a family of G protein–coupled receptors activated by a tethered ligand sequence within the amino terminal that are revealed by site-specific proteolysis. The thrombin-sensitive PAR-1 and trypsin-activated PAR-2 mediate endothelium-dependent vascular relaxation in a number of species. Because both thrombin and trypsin-like enzymes have been implicated in coronary artery disease, the purpose of this study was to investigate whether similar receptors are present in human coronary arteries. Thrombin (0.001 to 0.1 U/mL) and trypsin (0.001 to 1 U/mL) caused concentration- and endothelium-dependent relaxations of human coronary artery ring segments suspended in organ chambers for isometric tension recording and contracted with the thromboxane A
2
mimetic U46619. These relaxations were dependent on the catalytic activity of each enzyme and were inhibited by the NO synthase inhibitor
N
G
-nitro-
l
-arginine (100 μmol/L) and the NO scavenger oxyhemoglobin (20 μmol/L). The synthetic PAR-1 tethered ligand sequence SFLLRN-NH
2
(0.01 to 10 μmol/L) also caused endothelium-dependent relaxation of U46619-contracted human coronary arteries; however, the equivalent PAR-2 ligand SLIGKV-NH
2
caused almost no relaxation. In addition, desensitization to either thrombin or trypsin resulted in cross-desensitization to the other enzyme but had only a minimal affect on the response to SFLLRN-NH
2
. Therefore, we conclude that human coronary artery endothelial cells possess a PAR-1–like receptor that is potently activated by thrombin, trypsin, and SFLLRN-NH
2
to cause NO-mediated vascular relaxation. Once cleaved, this receptor is recycled in a truncated form, able to respond to exogenous application of only its tethered ligand sequence, suggesting the presence of another endogenous activator possibly acting independently of receptor cleavage.
Nitric Oxide Inhibits α
2
-Adrenoceptor–Mediated Endothelium-Dependent Vasodilation
Authors: Thorin Eric , Huang Paul L. , Fishman Mark C. , Bevan John A.
Published: June 2012

Abstract
—This study was designed to investigate the interaction between the NO/
l
-arginine pathway and the α
2
-adrenoceptor–mediated endothelium-dependent vasorelaxation. Reactivity of isolated resistance mesenteric arterial segments from mice lacking the gene for constitutive endothelial NO synthase (eNOS
−
mice, n=14) and from their wild-type controls (WT mice, n=46) was studied in isometric conditions in the presence of indomethacin (blocker of cyclooxygenase). Oxymetazoline (OXY, 0.01 to 30 μmol/L; a selective α
2
-adrenoceptor agonist) induced an endothelium-dependent relaxation of eNOS
−
but not WT arteries preconstricted either with phenylephrine or serotonin. In the presence of
N
ω
-nitro-
l
-arginine (l-NNA, 100 μmol/L), an inhibitor of NOS, OXY induced an endothelium-dependent relaxation of WT mesenteric arteries. l-NNA had no effect on the relaxation caused by OXY in eNOS
−
arterial rings. Therefore, the relaxation caused by OXY was independent of NO formation. To demonstrate the inhibitory role of NO on the α
2
-adrenoceptor–mediated relaxation, subthreshold (0.1 nmol/L) to threshold (1 nmol/L) concentrations of sodium nitroprusside (donor of NO) were added to l-NNA–treated arteries before OXY challenges: in these conditions, the α
2
-adrenoceptor–mediated relaxation of eNOS
−
and WT arteries was inhibited. OXY-induced relaxation was restored on readdition of methylene blue (1 μmol/L, inhibitor of guanylate cyclase), suggesting that cGMP may be the mechanism of inhibition of the α
2
-adrenergic pathway in the presence of NO. Finally, OXY-mediated relaxation was blocked by tetraethylammonium (1 mmol/L) but not glibenclamide (1 μmol/L), suggesting the involvement of an endothelium-derived hyperpolarizing factor that activates Ca
2+
-activated K
+
channels. In conclusion, α
2
-adrenoceptor activation caused relaxation of isolated murine mesenteric arteries that was functionally blocked by NO through a mechanism that may involve activation of the soluble guanylate cyclase and cGMP formation. The endothelium-dependent α
2
-adrenoceptor–mediated relaxation is likely to be due to an endothelium-derived hyperpolarizing factor, whose release and/or production is reduced by concurrent NO formation.
Nonuniformity of Endothelial Constitutive Nitric Oxide Synthase Distribution in Cardiac Endothelium
Authors: Andries Luc J. , Brutsaert Dirk L. , Sys Stanislas U.
Published: June 2012

Abstract
—Endocardial endothelium and endothelium of coronary vessels produce NO. Histochemical methods have suggested that coronary arterial endothelial cells contain more endothelial constitutive NO synthase (ecNOS) than does coronary venous endothelium. We have further investigated the distribution of ecNOS in cardiac endothelium using immunofluorescence and en face confocal microscopy of rat heart. In endocardial endothelium, confocal microscopy revealed distinct ecNOS labeling of peripheral cell borders, cytoplasmic labeling, and labeling of the Golgi complexes. Labeling of the cell borders and of the Golgi complexes was confirmed by double staining for ecNOS and for platelet and endothelial cell adhesion molecule or Golgi 58k protein, respectively. Cytoplasmic labeling was strongest in coronary arterial endothelium. The size of the ecNOS-labeled Golgi complexes decreased from coronary arterial endothelial cells (8.63±0.39 μm
2
, mean±SE of 5 rats) to endocardial endothelium (7.07±0.61 μm
2
) and to coronary venous endothelium (3.65±0.20 μm
2
). In addition, pixel intensity of ecNOS labeling was higher in arterial endothelial cells than in venous endothelial cells. Endothelium of myocardial capillaries also contained small ecNOS-labeled Golgi complexes. No correlation was observed between endothelial cell surface area and Golgi complex size. Caveolin-1 labeling was strongest in capillaries and did not coincide completely with ecNOS labeling in endocardial and venous endothelium. These results suggest that endocardial and coronary arterial endothelium in the rat have a higher synthetic activity and might express more ecNOS than is expressed by cardiac venous and capillary endothelium. The observed heterogeneity in ecNOS distribution might be related to the specific mechanochemical environment and function of each endothelial compartment.
Neural Crest Cell Contribution to the Developing Circulatory System
Authors: Bergwerff Maarten , Verberne Marlies E. , DeRuiter Marco C. , Poelmann Robert E. , Gittenberger-de-Groot Adriana C.
Published: June 2012

Abstract
—In this study, the distribution patterns of neural crest (NC) cells (NCCs) in the developing vascular system of the chick were thoroughly studied and examined for a correlation with smooth muscle cell differentiation and vascular morphogenesis. For this purpose, we performed long-term lineage tracing using quail-chick chimera techniques and premigratory NCC infection with a replication-incompetent retrovirus containing the LacZ reporter gene in combination with immunohistochemistry. Results indicate that NCC deposition around endothelial tubes is influenced by anteroposterior positional information from the pharyngeal arterial system. NCCs were shown to be among the first cells to differentiate into primary smooth muscle cells of the arch arteries. At later stages, NCCs eventually differentiated into adventitial fibroblasts and smooth muscle cells and nonmuscular cells of the media and intima. NCCs were distributed in the aortic arch and pulmonary arch arteries and in the brachiocephalic and carotid arteries. The coronary and pulmonary arteries and the descending aorta, however, remained devoid of NCCs. A new finding was that the media of part of the anterior cardinal veins was also determined to be NC-derived. NC-derived elastic arteries differed from non-NC elastic vessels in their cellular constitution and elastic fiber organization, and the NC appeared not to be involved in designating a muscular or elastic artery. Boundaries between NC-infested areas and mesodermal vessel structures were mostly very sharp and tended to coincide with marked changes in vascular morphology, with the exception of an intriguing area in the aortic and pulmonary trunks.
Action of Tropoelastin and Synthetic Elastin Sequences on Vascular Tone and on Free Ca
2+
Level in Human Vascular Endothelial Cells
Authors: Faury Gilles , Garnier Stéphanie , Weiss Anthony S. , Wallach Jean , Fülöp Tamàs , Jacob Marie-Paule , Mecham Robert P. , Robert Ladislas , Verdetti Jean
Published: June 2012

Abstract
—The elastic properties of extensible tissues such as arteries and skin are mainly due to the presence of elastic fibers whose major component is the extracellular matrix protein elastin. Pathophysiological degradation of this protein leads to the generation of elastin peptides that have been identified in the circulation in the ng/mL to μg/mL range. Similar concentrations of an elastin peptide preparation (κ-elastin) were previously demonstrated to induce, among other biological actions, a dose- and endothelium-dependent vasorelaxation mediated by the elastin/laminin receptor and by endothelial NO production. To determine the elastin sequence(s) responsible for vasomotor activity and to learn more about possible signaling pathways, we have compared the action of different concentrations (10
−13
to 10
−7
mol/L) of recombinant human tropoelastin, eight synthetic elastin peptides, and a control peptide (VPVGGA) on both rat aortic ring tension and [Ca
2+
]
i
of cultured human umbilical vein endothelial cells. No vasoactivity could be detected for VPVGGA and for the elastin-related sequences VGVGVA, PGVGVA, and GVGVA. Tropoelastin, VGV, PGV, and VGVAPG were found to induce an endothelium- and dose-dependent vasorelaxation and to increase endothelial [Ca
2+
]
i
, whereas PVGV and VGVA produced these effects only at low concentration (10
−11
mol/L). A likely candidate for mediating the elastin peptide–related effects is the elastin/laminin receptor, since the presence of lactose strongly inhibited the vasoactivity associated with these compounds. Our results show that although the flanking amino acids modulate its activity, VGV seems to be the core sequence recognized by the elastin receptor.
Depressed Heart Rate Variability and Arterial Baroreflex in Conscious Transgenic Mice With Overexpression of Cardiac G
sα
Authors: Uechi Masami , Asai Kuniya , Osaka Motohisa , Smith Amelia , Sato Naoki , Wagner Thomas E. , Ishikawa Yoshihiro , Hayakawa Hirokazu , Vatner Dorothy E. , Shannon Richard P. , Homcy Charles J. , Vatner Stephen F.
Published: June 2012

Abstract
—Recently, we developed a transgenic mouse with cardiac-specific G
sα
overexpression (TG mouse), which exhibits enhanced postsynaptic β-adrenergic receptor signaling, ultimately developing a cardiomyopathy. The goal of the present study was to determine whether cardiac G
sα
overexpression alters autonomic cardiovascular control, which could shed light on the mechanism responsible for the later development of cardiomyopathy. Mean arterial pressure was increased (
P
<.05) in conscious, chronically instrumented TG mice (123±1 mm Hg) compared with age-matched wild-type (WT) control mice (103±1 mm Hg). Respiratory frequency was increased (
P
<.05) in TG mice (269±26/min) compared with WT mice (210±20/min). By use of telemetric techniques, baseline heart rate (HR) was elevated (
P
<.05) in conscious, untethered TG mice (696±13 bpm) compared with WT mice (568±28 bpm). Intrinsic HR, after propranolol and atropine or after ganglionic blockade with hexamethonium, was not different between TG and WT mice. Both the normal minute-to-minute and circadian variations of HR observed in WT mice were markedly blunted in TG mice. HR variability was assessed by the time-domain and frequency-domain methods. At baseline, time-domain analysis indices were reduced (
P
<.05) in TG mice compared with WT mice. Although the low frequency (LF) component was higher (
P
<.05) than the high frequency (HF) component in WT mice, the LF component was less (
P
<.05) than the HF component in TG mice. In addition, arterial baroreflex regulation of HR was markedly blunted in TG mice in response to both nitroglycerin-induced hypotension and phenylephrine-induced hypertension. The reduced LF/HF ratio in TG mice was surprising in view of enhanced β-adrenergic signaling and may be due to reduced neural tone secondary to the elevated arterial pressure or alterations in arterial baroreflex control. Dobutamine infusion in WT mice also resulted in depressed HR variability. The combination of elevated baseline HR, arterial pressure, and respiratory frequency suggests that enhanced β-adrenergic signaling in TG mice results in reduced HR variability, in terms of both minute-to-minute variability and the lack of circadian variations in HR. The lack of normal HR variability in general and the failure of HR to decline, even during sleep, may actually be critical mechanisms contributing to the ultimate development of cardiomyopathy in these animals.
Angiotensin II–Nitric Oxide Interaction on Sympathetic Outflow in Conscious Rabbits
Authors: Liu Jun-Li , Murakami Hiroshi , Zucker Irving H.
Published: June 2012

Abstract
—Increasing evidence suggests that endogenous NO inhibits sympathetic outflow in anesthetized animals. However, in a recent study from this laboratory, we were unable to find any evidence of increased renal sympathetic nerve activity (RSNA) in response to blockade of NO synthesis in conscious rabbits. Because angiotensin II (Ang II) increases sympathetic outflow, one factor for this discrepancy may be the difference in the resting level of Ang II, which may be lower in well-trained conscious animals. In the present study, the effects of blockade of NO synthesis with
N
ω
-nitro-
l
-arginine methyl ester (L-NAME, 30 mg/kg IV) on resting RSNA with and without a background intravenous infusion of Ang II (10 ng · kg
−1
· min
−1
) was investigated in conscious rabbits. Intravenous administration of L-NAME (30 mg/kg) caused an increase in mean arterial blood pressure (MAP, from 80.4±2.9 to 92.8±2.5;
P
=.0001) and a decrease in RSNA (from 100±0% to 53.4±8.6%,
P
=.0016). When the elevated blood pressure was returned to control by infusion of hydralazine (0.01 to 0.06 mg · kg
−1
· min
−1
), RSNA returned to the level before L-NAME administration. During a sustained infusion of Ang II (10 ng · kg
−1
· min
−1
), L-NAME increased MAP from 89.2±2.9 to 109.0±4.3 mm Hg (
P
=.0101) and decreased RSNA from 100.0±0% to 53.7±7.5% (
P
=.0013). Under this circumstance, however, when the MAP was returned to the level that existed before the administration of L-NAME, RSNA increased significantly above the level that existed before the administration of L-NAME (164.5±17.7% versus 100±0%,
P
=.0151). The enhancement of the sympathetic response by Ang II was completely blocked by the AT
1
receptor antagonist, losartan. In contrast, during a background infusion of phenylephrine, which increased MAP to the same level as produced by Ang II, L-NAME had no effect on RSNA when MAP was returned to the control level.
N
ω
-Nitro-
d
-arginine methyl ester had no effect on MAP and RSNA. Intravenous infusion of Ang II alone for 75 minutes had no effect on RSNA when MAP was returned to control levels. These data suggest that an elevated level of Ang II is critical for the inhibitory effect of NO on sympathetic outflow in conscious rabbits and imply that these two substances have a major impact on the regulation of sympathetic outflow.
Ca
2+
-Independent Activation of the Endothelial Nitric Oxide Synthase in Response to Tyrosine Phosphatase Inhibitors and Fluid Shear Stress
Authors: Fleming Ingrid , Bauersachs Johann , Fisslthaler Beate , Busse Rudi
Published: June 2012

Abstract
—Fluid shear stress enhances NO formation via a Ca
2+
-independent tyrosine kinase inhibitor–sensitive pathway. In the present study, we investigated the effects of the protein tyrosine phosphatase inhibitor phenylarsine oxide and of fluid shear stress on endothelial NO production as well as on the membrane association and phosphorylation of the NO synthase (NOS) III. Phenylarsine oxide (10 μmol/L) induced an immediate and maintained NO-mediated relaxation of isolated rabbit carotid arteries, which was insensitive to the removal of extracellular Ca
2+
and the calmodulin antagonist calmidazolium. This phenylarsine oxide–induced vasodilatation was unaffected by genistein but abrogated by the tyrosine kinase inhibitor erbstatin A. Incubation of native or cultured endothelial cells with phenylarsine oxide resulted in a time-dependent tyrosine phosphorylation of mainly Triton X-100–insoluble (cytoskeletal) proteins, along with a parallel change in the detergent solubility of NOS III, such that the enzyme was recovered in the cytoskeletal fraction. A similar, though slightly delayed, phenomenon was also observed after the application of fluid shear stress but not in response to any receptor-dependent agonist. Although Ca
2+
-independent NO formation was sensitive to erbstatin A, phenylarsine oxide treatment was associated with the tyrosine dephosphorylation of NOS III rather than its hyperphosphorylation. Proteins that also underwent redistribution in response to the tyrosine phosphatase inhibitor included paxillin, phospholipase C-γ
1
, mitogen-activated protein kinase, and the tyrosine kinases Src and Fyn. We envisage that fluid shear stress and tyrosine phosphatase inhibitors may alter the conformation and/or protein coupling of NOS III, facilitating its interaction with specific phospholipids, proteins, and/or protein kinases that enhance/maintain its Ca
2+
-independent activation.
Response of Intra-acinar Pulmonary Microvessels to Hypoxia, Hypercapnic Acidosis, and Isocapnic Acidosis
Authors: Yamaguchi Kazuhiro , Suzuki Koichi , Naoki Katsuhiko , Nishio Kazumi , Sato Nagato , Takeshita Kei , Kudo Hiroyasu , Aoki Takuya , Suzuki Yukio , Miyata Atsushi , Tsumura Harukuni
Published: June 2012

Abstract
—To elucidate the differential reactivity of pulmonary microvessels in the acini to hypoxia, excessive CO
2
, and increased H
+
, we investigated changes in the diameter of precapillary arterioles, postcapillary venules, and capillaries in isolated rat lungs on exposure to normocapnic hypoxia (2% O
2
), normoxic hypercapnia (15% CO
2
), and isocapnic acidosis (0.01 mol/L HCl). Microvascular diameters were precisely examined using a real-time confocal laser scanning luminescence microscope coupled to a high-sensitivity camera with an image intensifier. Measurements were made under conditions with and without indomethacin or
N
ω
-nitro-
l
-arginine methyl ester to assess the importance of vasoactive substances produced by cyclooxygenase (COX) or NO synthase (NOS) as it relates to the reactivity of pulmonary microvessels to physiological stimuli. We found that acute hypoxia contracted precapillary arterioles that had diameters of 20 to 30 μm but did not constrict postcapillary venules of similar size. COX- and NOS-related vasoactive substances did not modulate hypoxia-elicited arteriolar constriction. Hypercapnia induced a distinct venular dilatation closely associated with vasodilators produced by COX but not by NOS. Arterioles were appreciably constricted in isocapnic acidosis when NOS, but not COX, was suppressed, whereas venules showed no constrictive response even when both enzymes were inhibited. Capillaries were neither constricted nor dilated under any experimental conditions. These findings suggest that reactivity to hypoxia, CO
2
, and H
+
is not qualitatively similar among intra-acinar microvessels, in which COX- and NOS-associated vasoactive substances function differently.
Inactivation of Voltage-Gated Cardiac K
+
Channels
Authors: Rasmusson Randall L. , Morales Michael J. , Wang Shimin , Liu Shuguang , Campbell Donald L. , Brahmajothi Mulugu V. , Strauss Harold C.
Published: June 2012

Abstract
—Inactivation is the process by which an open channel enters a stable nonconducting conformation after a depolarizing change in membrane potential. Inactivation is a widespread property of many different types of voltage-gated ion channels. Recent advances in the molecular biology of K
+
channels have elucidated two mechanistically distinct types of inactivation, N-type and C-type. N-type inactivation involves occlusion of the intracellular mouth of the pore through binding of a short segment of residues at the extreme N-terminal. In contrast to this “tethered ball” mechanism of N-type inactivation, C-type inactivation involves movement of conserved core domain residues that result in closure of the external mouth of the pore. Although C-type inactivation can show rapid kinetics that approach those observed for N-type inactivation, it is often thought of as a slowly developing and slowly recovering process. Current models of C-type inactivation also suggest that this process involves a relatively localized change in conformation of residues near the external mouth of the permeation pathway. The rate of C-type inactivation and recovery can be strongly influenced by other factors, such as N-type inactivation, drug binding, and changes in [K
+
]
o
. These interactions make C-type inactivation an important biophysical process in determining such physiologically important properties as refractoriness and drug binding. C-type inactivation is currently viewed as arising from small-scale rearrangements at the external mouth of the pore. This review will examine the multiplicity of interactions of C-type inactivation with N-terminal–mediated inactivation and drug binding that suggest that our current view of C-type inactivation is incomplete. This review will suggest that C-type inactivation must involve larger-scale movements of transmembrane-spanning domains and that such movements contribute to the diversity of kinetic properties observed for C-type inactivation.
Thy-1, a Novel Marker for Angiogenesis Upregulated by Inflammatory Cytokines
Authors: Lee Wen -Sen , Jain Mukesh K. , Arkonac Burak M. , Zhang Dorothy , Shaw Shyh-Yu , Kashiki Saori , Maemura Koji , Lee Shwu-Luan , Hollenberg Norman K. , Lee Mu-En , Haber Edgar
Published: June 2012

Abstract
—We identified the cell surface glycoprotein Thy-1 on the endothelium of newly formed blood vessels in four models of angiogenesis in adult rats. Anti–Thy-1 staining showed that Thy-1 was upregulated in adventitial blood vessels after balloon injury to the carotid artery. Preabsorption with a rat Thy-1–Ig fusion construct eliminated all immunoreactivity and thus confirmed the specificity of the Thy-1 staining. Thy-1 was also expressed in the endothelium of small blood vessels formed after tumor implantation in the cornea, in periureteral vessels formed after ligation of the renal artery, and in small blood vessels of the uterus formed during pregnancy. In contrast with its expression during adult angiogenesis, Thy-1 was not expressed in the endothelium of blood vessels during embryonic angiogenesis. In vitro, the inflammatory cytokines interleukin-1β and tumor necrosis factor-α upregulated Thy-1 mRNA by 8- and 14-fold, respectively. Vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor-β, and platelet-derived growth factor-BB had no effect on Thy-1 mRNA. Thus, Thy-1 appears to be a marker of adult but not embryonic angiogenesis. The upregulation of Thy-1 by cytokines but not growth factors indicates the importance of inflammation in the pathogenesis of adult angiogenesis.
Dynamic Ca
2+
-Induced Inward Rectification of K
+
Current During the Ventricular Action Potential
Authors: Zaza Antonio , Rocchetti Marcella , Brioschi Angelica , Cantadori Annalisa , Ferroni Arnaldo
Published: June 2012

Abstract
—Inward rectification, an important determinant of cell excitability, can result from channel blockade by intracellular cations, including Ca
2+
. However, mostly on the basis of indirect arguments, Ca
2+
-mediated rectification of inward rectifier K
+
current (
I
K1
) is claimed to play no role in the mammalian heart. The present study investigates Ca
2+
-mediated
I
K1
rectification during the mammalian ventricular action potential. Guinea pig ventricular myocytes were patch-clamped in the whole-cell configuration. The action potential waveform was recorded and then applied to reproduce normal excitation under voltage-clamp conditions. Subtraction currents obtained during blockade of K
+
currents by either 1 mmol/L Ba
2+
(
I
Ba
) or K
+
-free solution (
I
0K
) were used to estimate
I
K1
. Similar time courses were observed for
I
Ba
and
I
0K
; both currents were strongly reduced during depolarization (inward rectification). Blockade of L-type Ca
2+
current by dihydropyridines (DHPs) increased systolic
I
Ba
and
I
0K
by 50.7% and 254.5%, respectively. β-Adrenergic stimulation, when tested on
I
0K
, had an opposite effect; ie, it reduced this current by 66.5%. Ryanodine, an inhibitor of sarcoplasmic Ca
2+
release, increased systolic
I
Ba
by 47.7%, with effects similar to those of DHPs. Intracellular Ca
2+
buffering (BAPTA-AM) increased systolic
I
Ba
by 87.7% and blunted the effect of DHPs. Thus,
I
K1
may be significantly reduced by physiological Ca
2+
transients determined by both Ca
2+
influx and release. Although Ca
2+
-induced effects may represent only a small fraction of total
I
K1
rectification, they are large enough to affect excitability and repolarization. They may also contribute to facilitation of early afterdepolarizations by conditions increasing Ca
2+
influx.
Antibody to Thrombin Receptor Inhibits Neointimal Smooth Muscle Cell Accumulation Without Causing Inhibition of Platelet Aggregation or Altering Hemostatic Parameters After Angioplasty in Rat
Authors: Takada Masahiro , Tanaka Hiroshi , Yamada Toshie , Ito Osamu , Kogushi Motoji , Yanagimachi Mamoru , Kawamura Takanori , Musha Takashi , Yoshida Fusayo , Ito Masashi , Kobayashi Hiroko , Yoshitake Shinji , Saito Isao
Published: June 2012

Abstract
—An antibody was raised in rabbits against SFFLRNPSEDTFEQF peptide, which is an NH
2
-terminal peptide of the thrombin-cleaved rat thrombin receptor. In vitro, the antibody inhibited rat smooth muscle cell proliferation but had no effect on rat platelet aggregation or clotting time. These data indicate that the antibody is a specific blocker of the thrombin receptor–signaling pathway in rat smooth muscle cells but does not work as a blocker in rat platelets, suggesting the existence of a second thrombin receptor in the platelets. Using an in vivo balloon catheter–induced injury model in rats, we examined the effect of the anti-rat thrombin receptor IgG on intimal smooth muscle cell accumulation 2 weeks after angioplasty. Analysis of the ratio of intimal to medial cross-sectional areas showed that injection of immune IgG resulted in 43.7% and 53.1% reduction (
P
<0.01) of neointimal smooth muscle cell accumulation compared with saline and nonimmune IgG treatment, respectively. Moreover, the injection of immune IgG caused a significant decrease of thrombin receptor mRNA expression and also 40.5% and 43.0% decreases (
P
<0.01) of the proliferating cell nuclear antigen (PCNA) index in the intima compared with the PCNA index after saline and nonimmune IgG treatment, respectively. The suppression of the PCNA index was also observed in the immune IgG–treated group at an early stage after angioplasty. These results suggest that thrombin receptor activation is involved in the proliferation and accumulation of neointimal smooth muscle cells induced by balloon injury.
Genistein Increases the Sensitivity of Cardiac Ion Channels to β-Adrenergic Receptor Stimulation
Authors: Hool Livia C. , Middleton Lisa M. , Harvey Robert D.
Published: June 2012

Abstract
—The whole-cell patch-clamp technique was used to monitor the effects of genistein, a tyrosine kinase inhibitor, on membrane currents recorded from isolated guinea pig ventricular myocytes. Under control conditions, genistein (50 μmol/L) did not activate the latent cAMP-regulated Cl
−
current (
I
Cl
). However, in the presence of a subthreshold concentration (1 nmol/L) of the β-adrenergic agonist isoproterenol (Iso), genistein caused a near-maximal activation of this current. In the absence of genistein, Iso activated
I
Cl
with an EC
50
of 5 nmol/L. In the presence of genistein, Iso activated
I
Cl
with an EC
50
of 0.3 nmol/L. This facilitatory effect was not observed in the presence of daidzein (50 μmol/L), an analogue of genistein that only weakly inhibits tyrosine kinase activity. Furthermore, peroxovanadate, a potent inhibitor of phosphotyrosine phosphatase activity, inhibited
I
Cl
activated by Iso alone, and it blocked the stimulatory effect of genistein in the presence of Iso. To determine whether the stimulatory effect of genistein was specific for
I
Cl
, we also studied its action on the cAMP-regulated delayed rectifier K
+
current (
I
K
) and L-type Ca
2+
current (
I
Ca-L
) present in these cells. Basal
I
K
and
I
Ca-L
were partially (≈30% to 40%) inhibited by genistein. However, this inhibitory effect was mimicked by daidzein, suggesting that inhibition of tyrosine kinase activity is not involved. In addition to the nonspecific inhibitory effect, genistein also caused a significant increase in the β-adrenergic sensitivity of the unblocked cationic currents. In the absence of genistein, 1 nmol/L Iso had no effect on either
I
K
or
I
Ca-L
. However, in the presence of genistein, 1 nmol/L Iso significantly increased the magnitude of both currents. These results suggest that tyrosine kinase activity may play an important role in regulating β-adrenergic responsiveness of the heart.
Differential Regulation of Protease Activated Receptor-1 and Tissue Plasminogen Activator Expression by Shear Stress in Vascular Smooth Muscle Cells
Authors: Papadaki Maria , Ruef Johannes , Nguyen Kytai Truong , Li Fengzhi , Patterson Cam , Eskin Suzanne G. , McIntire Larry V. , Runge Marschall S.
Published: June 2012

Abstract
—Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm
2
), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm
2
), mRNA expression was decreased (to 29% of stationary control;
P
<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control;
P
<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control;
P
<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface–associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca
2+
] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.
Reduced Nitric Oxide Production and Altered Myocardial Metabolism During the Decompensation of Pacing-Induced Heart Failure in the Conscious Dog
Authors: Recchia Fabio A. , McConnell Patrick I. , Bernstein Robert D. , Vogel Traci R. , Xu Xiaobin , Hintze Thomas H.
Published: June 2012

Abstract
—The aim of the present study was to determine whether cardiac nitric oxide (NO) production changes during the progression of pacing-induced heart failure and whether this occurs in association with alterations in myocardial metabolism. Dogs (n=8) were instrumented and the heart paced until left ventricular end-diastolic pressure reached 25 mm Hg and clinical signs of severe failure were evident. Every week, hemodynamic measurements were recorded and blood samples were withdrawn from the aorta and the coronary sinus for measurement of NO metabolites, O
2
content, free fatty acids (FFAs), and lactate and glucose concentrations. Cardiac production of NO metabolites or consumption of O
2
or utilization of substrates was calculated as coronary sinus-arterial difference times coronary flow. In end-stage failure, occurring at 29±1.6 days, left ventricular end-diastolic pressure was 25±1 mm Hg, left ventricular systolic pressure was 92±3 mm Hg, mean arterial pressure was 75±2.5 mm Hg, and dP/dt
max
was 1219±73 mm Hg/s (all
P
<0.05). These changes in hemodynamics were associated with a fall of cardiac NO metabolite production from 0.37±0.16 to −0.28±0.13 nmol/beat (
P
<0.05). O
2
consumption and lactate uptake did not change significantly from control, while FFA uptake decreased from 0.16±0.03 to 0.05±0.01 μEq/beat and glucose uptake increased from −2.3±7.0 to 41±10 μg/beat (
P
<0.05). The cardiac respiratory quotient also increased significantly by 28%. In 14 normal dogs the same measurements were performed at control and 1 hour after we injected 30 mg/kg of nitro-
l
-arginine, a competitive inhibitor of NO synthase. O
2
consumption increased from 0.05±0.002 mL/beat at control to 0.071±0.003 mL/beat after nitro-
l
-arginine, while FFA uptake decreased from 0.1±0.01 to 0.06±0.01 μEq/beat, lactate uptake increased from 0.15±0.04 to 0.31±0.03 μmol/beat, glucose uptake increased from 8.2±5.0 to 35.4±9.5 μg/beat, and RQ increased by 23% (all
P
<0.05). Our results indicate that basal cardiac production of NO falls below normal levels during cardiac decompensation and that there are shifts in substrate utilization. This switch in myocardial substrate utilization also occurs after acute pharmacological blockade of NO production in normal dogs.
Transient and Prolonged Increase in Endothelial Permeability Induced by Histamine and Thrombin
Authors: Amerongen Geerten P. van Nieuw , Draijer Richard , Vermeer Mario A. , van Hinsbergh Victor W. M.
Published: June 2012

Abstract
—In the present study, we differentiated between short- and long-term effects of vasoactive compounds on human endothelial permeability in an in vitro model. Histamine induced a rapid and transient (<3 minutes) decrease in barrier function, as evidenced by a decreased transendothelial electrical resistance and an increased passage of
22
Na ions. This increase in permeability was inhibited completely by chelation of intracellular calcium ions by BAPTA-AM and inhibition of calmodulin activity and myosin light chain (MLC) phosphorylation. The presence of serum factors prolonged the barrier dysfunction induced by histamine. Thrombin by itself induced a prolonged barrier dysfunction (>30 minutes) as evidenced by an increased passage of peroxidase and 40 kDa dextran. It was dependent only partially on calcium ions and calmodulin. The protein tyrosine kinase inhibitors genistein and herbimycin A, but not the inactive analogue daidzein, inhibited to a large extent the increase in permeability induced by thrombin. Genistein and BAPTA-AM inhibited the thrombin-induced permeability in an additive way, causing together an almost complete prevention of the thrombin-induced increase in permeability. Inhibition of protein tyrosine kinase was accompanied by a decrease in MLC phosphorylation and a reduction in the extent of F-actin fiber and focal attachment formation. Inhibition of
Rho
A by C3 transferase toxin reduced both the thrombin-induced barrier dysfunction and MLC phosphorylation. Genistein and C3 transferase toxin did not elevate the cellular cAMP levels. No evidence was found for a significant role of protein kinase C in the thrombin-induced increase in permeability or in the accompanying MLC phosphorylation. These data indicate that in endothelial cell monolayers that respond to histamine in a physiological way, thrombin induces a prolonged increase in permeability by “calcium sensitization,” which involves protein tyrosine phosphorylation and
Rho
A activation.
Targeted Overexpression of the Sarcoplasmic Reticulum Ca
2+
-ATPase Increases Cardiac Contractility in Transgenic Mouse Hearts
Authors: Baker Debra L. , Hashimoto Katsuji , Grupp Ingrid L. , Ji Yong , Reed Thomas , Loukianov Evgenij , Grupp Gunter , Bhagwhat Ajit , Hoit Brian , Walsh Richard , Marban Eduardo , Periasamy Muthu
Published: June 2012

Abstract
—Cardiac hypertrophy and heart failure are known to be associated with a reduction in Ca
2+
-ATPase pump levels of the sarcoplasmic reticulum (SR). To determine whether, and to what extent, alterations in Ca
2+
pump numbers can affect contraction and relaxation parameters of the heart, we have overexpressed the cardiac SR Ca
2+
-ATPase specifically in the mouse heart using the α-myosin heavy chain promoter. Analysis of 2 independent transgenic lines demonstrated that sarco(endo)plasmic reticulum Ca
2+
-ATPase isoform (SERCA2a) mRNA levels were increased 3.88±0.4-fold and 7.90±0.2-fold over those of the control mice. SERCA2a protein levels were increased by 1.31±0.05-fold and 1.54±0.05-fold in these lines despite high levels of mRNA, suggesting that complex regulatory mechanisms may determine the SERCA2a pump levels. The maximum velocity of Ca
2+
uptake (V
max
) was increased by 37%, demonstrating that increased pump levels result in increased SR Ca
2+
uptake function. However, the apparent affinity of the SR Ca
2+
-ATPase for Ca
2+
remains unchanged in transgenic hearts. To evaluate the effects of overexpression of the SR Ca
2+
pump on cardiac contractility, we used the isolated perfused work-performing heart model. The transgenic hearts showed significantly higher myocardial contractile function, as indicated by increased maximal rates of pressure development for contraction (+dP/dt) and relaxation (–dP/dt), together with shortening of the normalized time to peak pressure and time to half relaxation. Measurements of intracellular free calcium concentration and contractile force in trabeculae revealed a doubling of Ca
2+
transient amplitude, with a concomitant boost in contractility. The present study demonstrates that increases in SERCA2a pump levels can directly enhance contractile function of the heart by increasing SR Ca
2+
transport.
Overexpression of the Cardiac Na
+
/Ca
2+
Exchanger Increases Susceptibility to Ischemia/Reperfusion Injury in Male, but Not Female, Transgenic Mice
Authors: Cross Heather R. , Lu Liyan , Steenbergen Charles , Philipson Kenneth D. , Murphy Elizabeth
Published: June 2012

Abstract
—Influx of Ca
2+
into myocytes via Na
+
/Ca
2+
exchange may be stimulated by the high levels of intracellular Na
+
and the changes in membrane potential known to occur during ischemia/reperfusion. This increased influx could, in turn, lead to Ca
2+
overload and injury. Overexpression of the cardiac Na
+
/Ca
2+
exchanger therefore may increase susceptibility to ischemia/reperfusion injury. To test this hypothesis, the hearts of male and female transgenic mice, overexpressing the Na
+
/Ca
2+
exchange protein, and hearts of their wild-type littermates, were perfused with Krebs-Henseleit buffer and subjected to 20 minutes of ischemia and 40 minutes of reperfusion. Preischemic left ventricular developed pressures and +dP/dt
max
, as well as −dP/dt
min
, were higher in the male transgenic hearts compared with wild-type, implying a role for Na
+
/Ca
2+
exchange in the contraction, as well as the relaxation, phases of the cardiac beat. Postischemic function was lower in male transgenic than in male wild-type hearts (7±2% versus 32±6% of preischemic function), but there was no difference between female transgenic and female wild-type hearts, both at ≈30% of preischemic function. To assess whether this male/female difference was due to female-specific hormones such as estrogen, the hearts of bilaterally ovariectomized and sham-operated transgenic females were subjected to the same protocol. The functional recoveries of ovariectomized female transgenic hearts were lower (17±3% of preischemic function) than those of wild-type and sham-operated transgenic females. The lower postischemic functional recovery in the male transgenic and female ovariectomized transgenic hearts correlated with lower recoveries of the energy metabolites, ATP and phosphocreatine, as measured by
31
P nuclear magnetic resonance spectroscopy. Alternans were observed during reperfusion in male transgenic and female ovariectomized transgenic hearts only, consistent with intracellular Ca
2+
overload. Western analyses showed that alterations in the expression of the Na
+
/Ca
2+
exchange or L-type Ca
2+
channel proteins were not responsible for the protection observed in the female transgenic hearts. In conclusion, in males, overexpression of the Na
+
/Ca
2+
exchanger reduced postischemic recovery of both contractile function and energy metabolites, indicating that the Na
+
/Ca
2+
exchanger may play a role in ischemia/reperfusion injury. From the studies of females, however, it appears that this exacerbation of ischemia/reperfusion injury by overexpression of the Na
+
/Ca
2+
exchanger can be overcome partially by female-specific hormones such as estrogen.
Expression of Tissue Factor Pathway Inhibitor in Vascular Smooth Muscle Cells and Its Regulation by Growth Factors
Authors: Caplice Noel M. , Mueske Cheryl S. , Kleppe Laurel S. , Peterson Timothy E. , Broze George J. , Simari Robert D.
Published: June 2012

Abstract
—Tissue factor pathway inhibitor (TFPI) in vivo is thought to be synthesized mainly by endothelial cells. To date, no significant regulator of TFPI synthesis has been described. Vascular smooth muscle cells (VSMC) express tissue factor in vitro and in vivo, which may contribute to vascular thrombosis. We hypothesized that VSMC might also express TFPI. To determine this, we examined growth-arrested coronary VSMC in culture and found that VSMC secreted an amount of TFPI similar to that seen in endothelial cells. Immunohistochemistry of normal human coronary arteries showed TFPI staining throughout the media and intima of the vessel with localization to VSMC and endothelial cells. To determine regulation of TFPI expression in VSMC, we examined the effects of serum stimulation on TFPI secretion and found that FBS induced a 5-fold increase in TFPI antigen and activity levels in conditioned medium at 48 hours (
P
<0.001) when compared with serum-free conditions. A similar stimulatory effect was seen with 10% pooled human serum. Moreover, epidermal growth factor and platelet-derived growth factor-B increased TFPI secretion by 4- to 5-fold and 2- to 3-fold, respectively (
P
<0.05), and these growth factors accounted for ≈50% of the TFPI secretion effects of human serum. The serum effect was associated with a 3-fold increase in TFPI mRNA 24 hours after release from growth arrest and a 50% decrease in TFPI secretion after treatment with actinomycin D. Taken together, this study suggests that there is significant TFPI expression in VSMC in culture and in VSMC within the intima and media of the normal coronary artery wall. We present the first evidence for TFPI regulation by serum in VSMC and more specifically by its constituent growth factors, epidermal growth factor and platelet-derived growth factor-B.
Persistent Activation of a Swelling-Activated Cation Current in Ventricular Myocytes From Dogs With Tachycardia-Induced Congestive Heart Failure
Authors: Clemo Henry F. , Stambler Bruce S. , Baumgarten Clive M.
Published: June 2012

Abstract
—The hypothesis that cellular hypertrophy in congestive heart failure (CHF) modulates mechanosensitive (ie, swelling- or stretch-activated) channels was tested. Digital video microscopy and amphotericin–perforated-patch voltage clamp were used to measure cell volume and ion currents in ventricular myocytes isolated from normal dogs and dogs with rapid ventricular pacing–induced CHF. In normal myocytes, osmotic swelling in 0.9× to 0.6× isosmotic solution (296 mOsm/L) was required to elicit an inwardly rectifying swelling-activated cation current (
I
Cir,swell
) that reversed near –60 mV and was inhibited by 10 μmol/L Gd
3+
, a mechanosensitive channel blocker. Block of
I
Cir,swell
by Gd
3+
simultaneously reduced the volume of normal cells in hyposmotic solutions by up to ≈10%, but Gd
3+
had no effect on volume in isosmotic solution. In contrast,
I
Cir,swell
was persistently activated under isosmotic conditions in CHF myocytes, and Gd
3+
decreased cell volume by ≈8%. Osmotic shrinkage in 1.1× to 1.5× isosmotic solution inhibited both
I
Cir,swell
and Gd
3+
-induced cell shrinkage in CHF cells, whereas osmotic swelling only slightly increased
I
Cir,swell
. The
K
0.5
and Hill coefficient for Gd
3+
block of
I
Cir,swell
and Gd
3+
-induced cell shrinkage were estimated as ≈2.0 μmol/L and ≈1.9, respectively, for both normal and CHF cells. In both groups, the effects of Gd
3+
on current and volume were blocked by replacing bath Na
+
and K
+
and were linearly related with varying Gd
3+
concentration and the degree of cell swelling. CHF thus altered the set point for and caused persistent activation of
I
Cir,swell
. This current may contribute to dysrhythmias, hypertrophy, and altered contractile function in CHF and may be a novel target for therapy.