bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutral endopeptidase in the heart. Neutral endopeptidase inhibition prevents isoproterenol-induced myocardial hypoperfusion in rats by reducing bradykinin degradation.
Authors: Piedimonte G , Nadel J A , Long C S , Hoffman J I
Published: June 2012

Peptide mediators may play a role in the control of myocardial perfusion. We found immunohistochemical evidence of the peptide-degrading enzyme neutral endopeptidase (NEP) in cultured rat myocytes. Therefore, we examined the effect of an NEP inhibitor, phosphoramidon, on myocardial perfusion in rats after (1) stimulating sensory nerves with capsaicin and (2) inducing myocardial hypoperfusion with isoproterenol, with or without pretreatment with selective antagonists of the substance P (NK1) and bradykinin (B2) receptors. Three to five sequential determinations of myocardial blood flow were made in anesthetized rats by injecting 100,000 radionuclide-labeled microspheres suspended in 70% dextrose into the left ventricle. Phosphoramidon doubled coronary blood flow in response to a dose of capsaicin that was ineffective in the absence of the inhibitor. Isoproterenol (50 mg/kg IP) caused an immediate fall in blood pressure and coronary blood flow; after 20 minutes, flow had returned to normal but pressure was still subnormal. Administration of phosphoramidon reduced the recovery of blood pressure but greatly increased coronary blood flow. These changes were not altered by a substance P NK1 receptor blocker but were completely abolished by a selective bradykinin B2 receptor blocker. Our data indicate that (1) NEP is present in the rat myocardium, (2) sensory nerve-induced coronary vasodilation is markedly potentiated by NEP inhibition, (3) isoproterenol-induced myocardial hypoperfusion is prevented by NEP inhibition, and (4) this effect of NEP inhibition is due to reduced degradation of bradykinin.
Glucocorticoid induction of Kv1.5 K+ channel gene expression in ventricle of rat heart.
Authors: Takimoto K , Levitan E S
Published: June 2012

Multiple voltage-gated K+ channels contribute to the repolarization phases of the cardiac action potential and are targets of several antiarrhythmic drugs. The Kv1.5 K+ channel gene is expressed in the heart, and heterologous expression of this gene generates a slowly inactivating K+ current. Previously, we found that glucocorticoids specifically upregulate pituitary Kv1.5 gene expression. To test whether these steroids might also induce Kv1.5 gene expression in the heart, cardiac channel mRNA and protein were measured by RNase protection assay and by immunoblotting with antibody specific for the extracellular domain of Kv1.5 polypeptide. Kv1.5 mRNA and immunoreactive protein appeared to be more abundant in rat ventricle than atrium. Reduction of endogenous glucocorticoids by adrenalectomy decreased ventricular Kv1.5 mRNA approximately 8-fold, which was estimated by using cyclophilin mRNA as an internal control. Kv1.5 immunoreactive protein also decreased approximately 6-fold. Injection of dexamethasone into adrenalectomized rats acted within a day to increase ventricular Kv1.5 mRNA and immunoreactive protein approximately 50-fold and approximately 20-fold, respectively. In contrast, atrial Kv1.5 mRNA expression was unaffected by either adrenalectomy or injection of the glucocorticoid agonist. Furthermore, dexamethasone-induced upregulation was specific for Kv1.5, since whole-heart Kv1.4 and Kv2.1 mRNA levels, as well as ventricular Kv2.1 mRNA expression, were unchanged. Thus, dexamethasone specifically upregulates Kv1.5 K+ channel gene expression in rat ventricle but not atrium. Glucocorticoids may affect excitability of ventricular myocytes and the efficacy of clinically useful drugs by changing the expression of the Kv1.5 K+ channel.
Catheter-mediated pulmonary vascular gene transfer and expression.
Authors: Muller D W , Gordon D , San H , Yang Z , Pompili V J , Nabel G J , Nabel E G
Published: June 2012

The study and treatment of pulmonary diseases may be greatly facilitated by in vivo expression of specific recombinant genes in the pulmonary vasculature and lung parenchyma. To evaluate the feasibility of gene transfer to the pulmonary vasculature, cationic liposomes and adenoviral vectors encoding a human placental alkaline phosphatase (hpAP) gene were delivered into a pulmonary artery of 24 pigs by percutaneous right heart catheterization. Pulmonary tissue was harvested within 20 minutes or 5, 14, or 28 days later and was analyzed for gene transfer and expression. Five days after exposure to liposomes or adenoviral vectors, transfer of DNA and expression of mRNA were demonstrated in transfected lung tissue. Recombinant alkaline phosphatase protein was observed in both the vasculature and in alveolar septa but not in the bronchi. Expression of hpAP protein was observed at 5 days, was diminished at 14 days, and was absent 28 days after gene transfer with both liposome and adenoviral vectors. No major adverse effects of gene expression were detected by histological examination of the transfected lung segments compared with control segments. Gene transfer to the lung by either vector was not associated with significant biochemical abnormalities or histological changes 5, 14, or 28 days later in other organs, including carotid artery, heart, liver, spleen, kidney, skeletal muscle, ovary, and testes. These studies demonstrate that after intravascular gene delivery to the lung, recombinant genes are expressed in the vasculature and alveoli. This approach may provide a useful model for the experimental study of pulmonary vascular diseases, including pulmonary fibrosis and pulmonary thrombosis disorders.
des-Arg9-bradykinin produces tone-dependent kinin B1 receptor-mediated responses in the pulmonary vascular bed.
Authors: DeWitt B J , Cheng D Y , Kadowitz P J
Published: June 2012

Responses to des-Arg9-bradykinin, a selective kinin B1 receptor agonist, were characterized in the pulmonary vascular bed of the intact-chest cat. Injections of des-Arg9-bradykinin into the perfused lobar artery under low-resting tone conditions caused dose-related increases in lobar arterial pressure; whereas in the same experiment under elevated tone conditions, injections of the B1 agonist caused dose-related decreases in lobar arterial pressure. Vasoconstrictor responses to des-Arg9-bradykinin under low-tone conditions and vasodilator responses under elevated-tone conditions were antagonized by des-Arg9,[Leu8]-bradykinin, a kinin B1 receptor antagonist, whereas responses under low- and high-tone conditions were not altered by Hoe 140, a kinin B2 receptor antagonist. Vasoconstrictor responses to des-Arg9-bradykinin under low-tone conditions were attenuated by phentolamine, prazosin, and reserpine but not by sodium meclofenamate, suggesting that release of catecholamines and activation of alpha-adrenergic receptors are involved. Pulmonary vasodilator responses under elevated-tone conditions were inhibited by N omega-nitro-L-arginine methyl ester, suggesting that des-Arg9-bradykinin stimulates the release of nitric oxide, whereas meclofenamate and U-37883A, a nonsulfonylurea ATP-sensitive K+ channel antagonist, did not alter vasodilator responses to the B1 receptor agonist. These results suggest that vasoconstrictor responses to des-Arg9-bradykinin under low-tone conditions are mediated by the activation of kinin B1 receptors, the release of catecholamines within the lung, and the activation of alpha-adrenergic receptors, whereas vasodilator responses under elevated tone conditions are mediated by activation of B1 receptors and the release of nitric oxide from the endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)
Inhibition of sympathetic vasoconstriction is a major principle of vasodilation by nitric oxide in vivo.
Authors: Zanzinger J , Czachurski J , Seller H
Published: June 2012

The objective of this study was to determine whether vasodilator effects of nitric oxide (NO) can be explained by the inhibition of vasoconstriction caused by peripheral sympathetic nerve activity (SNA) in vivo. For this purpose, we studied the effects of systemic inhibition of NO synthesis during experimental variation of SNA in anesthetized cats. Intravenous infusion of NG-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg) in baroreceptor-intact animals (n = 6) caused increases in mean arterial blood pressure (MAP) from 105.8 +/- 3.4 to 192.0 +/- 4.3 mm Hg that were associated with slight decreases in preganglionic SNA recorded from the white ramus of the third thoracic segment. Higher SNA appeared in completely baroreceptor-denervated cats (n = 10) than in the intact cats, but no changes in nerve activity occurred after the subsequent administration of L-NAME. In contrast, MAP increased from 123.3 +/- 4.0 to 245.8 +/- 5.1 mm Hg. In baroreceptor-denervated cats, reversible suppression of peripheral SNA produced by cooling of the ventral surface of the rostral ventrolateral medulla oblongata (RVLM) caused significant hypotension (61.1 +/- 2.6 mm Hg) and almost completely reversed the hypertension caused by L-NAME (76.0 +/- 3.7 mm Hg). Intravenous administration of the alpha 1-adrenergic receptor antagonist prazosin after L-NAME reduced MAP to a similar extent. In contrast, hypertension induced by angiotensin II could not be reversed by RVLM cooling. The pressor effects of intravenously administered noradrenaline during RVLM cooling were markedly potentiated by L-NAME and attenuated by the NO-donor compound S-nitroso-N-acetylpenicillamine (SNAP).(ABSTRACT TRUNCATED AT 250 WORDS)
Apoptosis (Programmed Cell Death) in Arteries of the Neonatal Lamb
Authors: Cho Aesim , Courtman David W. , Langille B. Lowell
Published: June 2012

Abstract
We have examined whether cell death contributes to postnatal remodeling of arteries in lambs. First, abdominal aortic smooth muscle cell proliferation rates fell from 2.87±0.08% per day at 3 days of age to 1.75±0.15% per day at 21 days. These proliferation rates would yield a 50% increase in DNA content in the absence of cell death. No increase in DNA content was observed (
P
<.05 for predicted versus measured accumulation); therefore, significant cell death was inferred. The same analysis did not indicate high cell-death rates in the carotid, renal, or iliac arteries; however, cell death was detected in situ by end-labeling partially degraded DNA with terminal deoxynucleotidyl transferase or by nuclear labeling with propidium iodide, a fluorescent dye that permeates only nonviable cells. Nuclei were labeled in all arteries, although labeling was most frequent in the abdominal aorta, a vessel that regresses substantially after birth. Cell death was apoptotic because DNA extracted from arteries and end-labeled with [
32
P]dCTP produced a series of low molecular weight bands (DNA ladder) on an agarose gel, a hallmark of apoptosis. The ladder was strong for neonatal abdominal aorta but weak for other arteries. Only weak laddering was observed for fetal abdominal aortas in late gestation, confirming that high apoptosis rates in this vessel were initiated after birth. Intense DNA ladders and frequent in situ labeling indicated high rates of apoptosis in the postnatal intra-abdominal umbilical artery, another vessel that regresses after birth. We conclude that apoptosis contributes to postpartum arterial remodeling . This contribution is greatest in arteries that regress after birth.
Continuous Nitric Oxide Inhalation Reduces Pulmonary Arterial Structural Changes, Right Ventricular Hypertrophy, and Growth Retardation in the Hypoxic Newborn Rat
Authors: Roberts Jesse D. , Roberts Carole T. , Jones Rosemary C. , Zapol Warren M. , Bloch Kenneth D.
Published: June 2012

Abstract
Breathing low oxygen levels for several weeks produces progressive pulmonary artery hypertension and smooth muscle hypertrophy and hyperplasia in many species. Because nitric oxide (NO) is an important regulator of pulmonary vascular tone, we examined whether the continuous inhalation of low levels of NO gas would attenuate pulmonary arterial structural changes in hypoxic rat pups. Nine-day-old rat pups and their mothers continuously breathed at F
io
2
0.21 or 0.10 with or without adding 20 ppm (by volume) NO for 2 weeks. Lung tissue was obtained for vascular morphometric analysis, and the hearts were dissected to measure right ventricular weight and levels of mRNA encoding rat atrial natriuretic factor (rANF). In addition, femur and skull length were radiographically determined. Breathing at F
io
2
0.10 for 14 days increased pulmonary arterial wall thickness and the proportion of muscular arteries in the lung periphery. Right ventricular weight and right ventricular rANF gene expression increased, whereas body weight and skeletal growth were reduced (all
P
<.05). Continuous inhalation of 20 ppm NO at F
io
2
0.10 for 2 weeks decreased hypoxic pulmonary vascular structural changes and somatic growth retardation and prevented the increase of right ventricular weight and right ventricular rANF mRNA levels. These observations suggest that chronically breathing NO attenuates pulmonary vascular smooth muscle hypertrophy and/or hyperplasia and extension into distal arterial walls, right ventricular hypertrophy, and growth retardation of newborns breathing at a low oxygen level.
Inositol Phosphate Release and Metabolism During Myocardial Ischemia and Reperfusion in Rat Heart
Authors: Anderson Karen E. , Dart Anthony M. , Woodcock Elizabeth A.
Published: June 2012

Abstract
A detailed study of the effects of global myocardial ischemia and reperfusion on inositol phosphate release and metabolism has been undertaken by using isolated perfused rat hearts. Ischemia for longer than 5 minutes caused a cessation of inositol phosphate production, with inositol phosphates initially present accumulating as isomers of inositol monophosphate. This inhibition was independent of norepinephrine. In contrast, 2-minute reperfusion following 20-minute ischemia produced a rapid and transient release of inositol phosphates that was dependent on the release of norepinephrine and mediated by α
1
-adrenergic receptors. By a number of criteria, this reperfusion response was different from the norepinephrine response in normoxic tissue. First, total release of inositol phosphates was greater (466±37 compared with 345±29 cpm/mg protein,
P
<.05). Second, inositol 1,4,5-trisphosphate was released with postischemic reperfusion (103±18 to 207±11 pmol/mg protein), whereas release was not detected in normoxic myocardium. In agreement with this, neomycin (0.5 and 5 mmol/L) inhibited inositol phosphate release only under reperfusion conditions. Third, the reperfusion response, unlike the response in nonischemic tissue, required extracellular Ca
2+
. Longer periods of reperfusion resulted in a return to a pattern of inositol phosphate release that was not different from that seen in normoxic tissue. The rapid and transient release of inositol 1,4,5-trisphosphate at 2-minute postischemic reperfusion provides an explanation for the enhanced role of α
1
-adrenergic receptors under these conditions and suggests an important role for this compound in initiating reperfusion-induced pathological events.
The Stochastic Nature of Cardiac Propagation at a Microscopic Level
Authors: Spach Madison S. , Heidlage J. Francis
Published: June 2012

Abstract
The object of this study is to present evidence that the myocardial architecture creates inhomogeneities of electrical load at the cellular level that cause cardiac propagation to be stochastic in nature; ie, the excitatory events during propagation are constantly changing and disorderly in the sense of varying intracellular events and delays between cells. At a macroscopic level, however, these stochastic events become averaged and appear consistent with a continuous medium. We examined this concept in a two-dimensional (2D) model of myocardial architecture by exploring whether experimentally observed V̇
max
variability reflected different patterns of intracellular excitation events and junctional delays. The patterns of V̇
max
variability at randomly chosen intracellular sites were similar experimentally and in the 2D model. The 2D cellular model produced marked variability in gap junction delays; however, on the average, different gap junctions were used for cell-to-cell charge flow during conduction in different directions. During longitudinal propagation (LP), the velocity increased from the proximal to the distal end of each myocyte, and V̇
max
was lowest proximally, increased to a maximum at the distal fourth of the cell, and decreased distally. Transverse propagation (TP) produced rapid intracellular conduction with variable intracellular excitation sequences. TP V̇
max
was greater than LP V̇
max
in most subcellular regions, but near the ends of some myocytes, a reversed “TP>LP V̇
max
” relation occurred. Total charge carried by the sodium current varied inversely with V̇
max
, demonstrating feedback effects of cellular loading on the subcellular sodium current and the kinetics of the sodium channels. The results suggest that the stochastic nature of normal propagation at a microscopic level provides a considerable protective effect against arrhythmias by reestablishing the general trend of wave-front movement after small variations in excitation events occur.
Effects of Antisense c-
myb
Oligonucleotides on Vascular Smooth Muscle Cell Proliferation and Response to Vessel Wall Injury
Authors: Villa Augusto E. , Guzman Luis A. , Poptic Earl J. , Labhasetwar Vinod , D’Souza Stanley , Farrell Catherine L. , Plow Edward F. , Levy Robert J. , DiCorleto Paul E. , Topol Eric J.
Published: June 2012

Abstract
The process of restenosis after arterial balloon dilatation has been demonstrated to involve smooth muscle cell hyperplasia. Initial reports with antisense oligonucleotides directed against the proto-oncogene c-
myb
suggest marked in vitro specificity and in vivo efficacy. In the present study, we sought to confirm and extend the hypothesis that antisense to c-
myb
results in a specific antiproliferative effect with a comprehensive assessment by using different oligonucleotide preparations, different species, and tissue and cellular uptake experiments. Phosphorothioate-protected oligonucleotides representing the appropriate sequence for antisense to c-
myb
and multiple controls were used to inhibit proliferation of platelet-derived growth factor– and fetal bovine serum–stimulated rat, dog, and human aortic smooth muscle cells in vitro and neointimal proliferation in the rat carotid injury model. In vitro experiments using identical culture conditions in rat, dog, and human aortic smooth muscle cells failed to show specificity as well as consistency in growth inhibitory effects that could be attributed to an antisense mechanism. Proliferation of smooth muscle cell growth in culture was consistently inhibited with oligomers containing a contiguous 4-guanosine residue motif. In vivo, the rat carotid injury neointimal hyperplasia was similar for antisense c-
myb
(0.095±0.009 mm
2
) and sense c-
myb
(0.090±0.009 mm
2
). Fluorescent-labeled oligonucleotides were present in tissue after local delivery via pluronic gel, and their activity rapidly declined over a 72-hour period. Our findings point to the potential nonspecificity and lack of consistency of the antisense oligonucleotide to c-
myb
in vitro and in vivo. An alternative nonantisense mechanism for the inhibition of smooth muscle cell proliferation, involving contiguous 4-guanosine residues, is proposed.
Coenzyme A Glutathione Disulfide
Authors: Schlüter Hartmut , Meissner Michael , van der Giet Marcus , Tepel Martin , Bachmann Jürgen , Groß Isolde , Nordhoff Eckhard , Karas Michael , Spieker Claus , Witzel Herbert , Zidek Walter
Published: June 2012

Abstract
The adrenal gland is involved in the regulation of vascular tone by secretion of vasoactive agents such as catecholamines, neuropeptide Y, or endogenous ouabain. A further potent vasoconstrictor is isolated from bovine adrenal glands and is identified by chromatography, mass spectrometry, UV spectroscopy, and enzymatic cleavage as coenzyme A glutathione disulfide (CoASSG). CoASSG is found in chromaffin granules of adrenal glands and is released from adrenal medulla slices by carbachol. At a concentration of 10
−12
mol/L CoASSG increases renal vascular resistance. Intra-aortic injection of 5×10
−10
mol CoASSG increases blood pressure in the intact animal. Besides its vasopressor properties, this substance potentiates the effects of angiotensin II on vascular tone. It is concluded that CoASSG could play a role in blood pressure regulation not only by direct effects but also by modulation of the action of angiotensin II.
Increased In Vivo Expression and Production of Endothelin-1 by Porcine Cardiomyocytes Subjected to Ischemia
Authors: Tønnessen Theis , Giaid Adel , Saleh Dina , Naess Pål A. , Yanagisawa Masashi , Christensen Geir
Published: June 2012

Abstract
Circulating levels of the endothelium-derived vasoconstrictor peptide endothelin-1 (ET-1) are increased in association with myocardial ischemia and infarction. The present study investigates whether ET-1 is synthesized and produced locally in the ischemic heart. Sixteen pigs were divided into three groups. In the first group, the left anterior descending coronary artery was occluded for 90 minutes, followed by 150 minutes of reperfusion (group A, n=8). Two additional groups were subjected to 90 minutes (group B, n=4) or 240 minutes (group C, n=4) of ischemia without reperfusion. Biopsies from the nonischemic and ischemic myocardium were rapidly obtained from the beating heart and subsequently examined by Northern blot, in situ hybridization, and immunohistochemistry. Arterial plasma ET-1 was measured before ischemia and at the end of the experiments. Northern blot revealed a twofold increase in ET-1 mRNA in the ischemic myocardium compared with the nonischemic myocardium. In situ hybridization showed a considerable increase in ET-1 mRNA over the ischemic cardiomyocytes. Substantial ET-1–like immunoreactivity (ET-1-ir) was detected in cardiomyocytes in the ischemic region. In contrast, little or no ET-1-ir or mRNA was detected in nonischemic cardiomyocytes. Both in the ischemic and nonischemic regions, little ET-1-ir was detected in vascular endothelial cells or vascular smooth muscle cells. There was no difference in the intensity and distribution of ET-1 mRNA expression or ET-1-ir among experimental groups A, B, and C. Arterial plasma ET-1 was increased only in group A, the reperfused group. In conclusion, these findings provide a definitive evidence for a de novo synthesis of ET-1 by cardiomyocytes subjected to ischemia in vivo.
Calcium Sensitivity of Isometric Tension Is Increased in Canine Experimental Heart Failure
Authors: Wolff Matthew R. , Whitesell Larry F. , Moss Richard L.
Published: June 2012

Abstract
To examine the role of alterations in myofibrillar function in chronic heart failure, we determined isometric tension–pCa relations in permeabilized myocardium from a canine model of dilated cardiomyopathy (DCM) produced by chronic rapid pacing. In the initial series of experiments, seven dogs were paced at 250 beats per minute for 28.9±7.0 days, resulting in ventricular dilatation and reduced ejection fractions by echocardiography and elevated intracardiac filling pressures. Isometric tension–pCa relations were measured by using mechanically disrupted and permeabilized myocyte-sized preparations obtained from left ventricular biopsies before (n=11) and after (n=10) chronic rapid pacing–induced heart failure. Resting sarcomere length (SL) was set at 2.35 μm, and preparations had low end compliance (SL was 2.23±0.03 μm during maximal activation). Passive tension (2.1±1.0 versus 2.4±0.6 mN/mm
2
) and maximal Ca
2+
-activated tension (25.9±9.3 versus 27.8±6.8 mN/mm
2
) were similar for control and DCM preparations, respectively. However, the calcium sensitivity of isometric tension was increased in failing myocardium (pCa
50
5.95±0.11 [DCM] versus 5.83±0.10 [control],
P
=.001). Treatment of myofibrillar preparations with the catalytic subunit of protein kinase A decreased calcium sensitivity of tension to a greater degree in failing preparations (shift of pCa
50
from 6.04±0.06 to 5.75±0.09, n=7) than in nonfailing preparations (5.91±0.08 to 5.74±0.07, n=8), and isometric tension–pCa relations in the two groups were not significantly different after protein kinase A treatment. These data suggest that the increased calcium sensitivity in DCM may be due at least in part to a reduction of the adrenergically mediated phosphorylation of myofibrillar regulatory proteins. This increased calcium sensitivity of isometric tension may partially compensate for decreases in systolic calcium transients in DCM but may also contribute to the diastolic dysfunction that accompanies this condition.
Arachidonic Acid–Dependent Phosphorylation of Troponin I and Myosin Light Chain 2 in Cardiac Myocytes
Authors: Damron Derek S. , Darvish Ahmad , Murphy LeeAnn , Sweet Wendy , Moravec Christine S. , Bond Meredith
Published: June 2012

Abstract
Recent evidence has suggested that arachidonic acid (AA) may be an important signaling molecule in cardiac excitation-contraction coupling. We previously showed that AA and endothelin-1 (ET) inhibit distinct K
+
channels via protein kinase C–dependent pathways in rat ventricular myocytes. In addition, we demonstrated that Ca
2+
transients in populations of fura 2–loaded myocytes were potentiated by AA and ET via activation of protein kinase C. In this study, we have used suspensions of [
32
P]orthophosphate (
32
P
i
)–labeled rat ventricular myocytes to study the effects of AA and ET at the level of the myofilaments. After a 10-minute incubation of the labeled cells with phorbol 12-myristate 13-acetate (PMA), AA, or ET in the presence or absence of the protein kinase C inhibitor calphostin C, the myofibrillar proteins were separated by PAGE. Measurement of unloaded cell shortening using video edge detection in single electrically stimulated myocytes was also used to assess the effects of AA and ET on myocyte contractility. Incubation with either PMA, AA, or ET resulted in similar increases in
32
P
i
incorporation into troponin I (TnI) and myosin light chain 2 (MLC2), which was inhibited by preincubation with the protein kinase C antagonist calphostin C. In addition, the ability of these agonists to stimulate phosphorylation of TnI or MLC2 did not require extracellular Ca
2+
or intact intracellular Ca
2+
stores. The effects of AA and ET together on phosphorylation of TnI or MLC2 were not additive. These results show that AA and ET phosphorylate myofibrillar proteins in intact cardiac myocytes via a calphostin C–inhibitable Ca
2+
-independent pathway and that AA, like ET, stimulates a positive inotropic effect in cardiac myocytes. Thus, phosphorylation of cardiac myofilaments by AA and/or ET, in addition to phosphorylation-dependent inhibition of distinct K
+
channels, may represent an important signaling pathway by which AA and ET influence the inotropic state of the heart.
Forskolin Stimulates Swelling-Induced Chloride Current, Not Cardiac Cystic Fibrosis Transmembrane-Conductance Regulator Current, in Human Cardiac Myocytes
Authors: Oz Mehmet C. , Sorota Steve
Published: June 2012

Abstract
Whole-cell patch clamp was used to look for cystic fibrosis transmembrane-conductance regulator (CFTR)–like chloride currents in calcium-tolerant human cardiac myocytes. Potassium-containing solutions were used initially. Steady state currents were measured with hyperpolarizing ramps (−16.25 mV/s). Peak net inward currents during voltage steps from −50 to +5 mV were used as an index of L-type calcium current. Isoproterenol (1 μmol/L) or forskolin (10 μmol/L) were used in attempts to evoke CFTR-like chloride current. No forskolin- or isoproterenol-induced steady state current was found in any of 17 atrial cells from seven patients in the absence of cell swelling. Every cell exhibited a large increase in net inward current in response to forskolin, suggesting that cAMP-dependent stimulation of L-type calcium current was present. Swelling with osmotic stress induced an outwardly rectifying steady state current with a reversal potential close to the chloride equilibrium potential. Once this current was activated, exposure to forskolin caused a further increase that subsided on washout (four of four cells, two patients). The atrial swelling-induced current was studied in more detail by using cesium-containing solutions. The current was determined to be a chloride current because the reversal potential was sensitive to changes in intracellular chloride and outward currents were blocked by 150 μmol/L DIDS. Ventricular cells were isolated from five failing human hearts. No CFTR-like current was found in any of 17 cells. In eight of eight ventricular cells, a swelling-induced current was found. The amplitude of the swelling-induced current was enhanced by forskolin. In human ventricular cells, outward swelling-induced currents were inhibited by DIDS. The present results are in apparent conflict with mRNA measurements made by others. Our results suggest that significant amounts of functional channels do not accumulate despite the reported presence of cardiac CFTR mRNA in human heart. However, human myocardium does express a swelling-induced chloride current that can be stimulated by forskolin.
Effect of Graded Hypoxia on the Induction and Function of Inducible Nitric Oxide Synthase in Rat Mesangial Cells
Authors: Archer Stephen L. , Freude Kimberly A. , Shultz Pamela J.
Published: June 2012

Abstract
Inducible nitric oxide synthase (iNOS) catalyzes the formation of nitric oxide (NO) from
l
-arginine and O
2
. Although some O
2
is required for this reaction, it is uncertain whether biologically relevant levels of hypoxia alter this pathway. We examined the effects of graded hypoxia on several steps in the iNOS pathway in lipopolysaccharide (LPS)-stimulated rat glomerular mesangial cells: induction of iNOS mRNA, NO synthesis, NO oxidation to nitrite (NO
2
−
) and nitrate (NO
3
−
), and accumulation of cGMP. Cultured cells were incubated for 24 hours in airtight flasks containing O
2
(21%, 10%, 2.5%, and 0%), CO
2
(5%), and N
2
(balance), resulting in media P
o
2
levels of 140±3, 85±1, 46±3 (moderate hypoxia), and 32±5 (severe hypoxia) mm Hg, respectively. During normoxia (P
o
2
, 85 to 140 mm Hg) LPS increased iNOS mRNA with associated increases in NO synthesis, NO
2
−
and NO
3
−
accumulation, and intracellular cGMP levels. In the absence of LPS, there was minimal NO synthesis and no detectable iNOS mRNA. Even during severe hypoxia, LPS elevated NO
2
−
and NO
3
−
relative to levels in unstimulated cells (
P
<.05), although to a lesser extent than during normoxia (
P
<.05). The induction of iNOS mRNA by LPS was preserved in hypoxia, and intracellular cGMP levels were similar at all levels of oxygen tension, indicating that iNOS induction and function were not altered by moderate or severe hypoxia. However, moderate hypoxia did alter the partitioning and oxidation of NO, favoring the appearance of NO in the “headspace” (defined as the gas overlying the cells) and NO
3
−
in the media. We conclude that although hypoxia can alter the partitioning and decomposition of NO, the induction of iNOS mRNA and the activity of its enzyme product are resistant to hypoxia (P
o
2
, >32 mm Hg).
Endothelial Cell Interactions With Synthetic Peptides From the Carboxyl-Terminal Heparin-Binding Domains of Fibronectin
Authors: Huebsch Joseph C. , McCarthy James B. , Diglio Clement A. , Mooradian Daniel L.
Published: June 2012

Abstract
Fibronectin (FN) plays an important role in endothelial cell adhesion, spreading, and motility. Within FN, a number of functional domains have been identified, including the 33/66-kD carboxyl-terminal heparin-binding fragments, which support the adhesion of vascular endothelial cells. A number of synthetic peptides representing amino acid sequences within the 33/66-kD fragments have been shown to promote the adhesion, spreading, and migration of a variety of cell types. Our working hypothesis is that one or more of these sequences may also mediate vascular endothelial cell adhesion, spreading, and migration to the 33/66-kD fragments. In support of this hypothesis, we have demonstrated that endothelial cells from various sources adhered in a concentration-dependent manner to surfaces coated with FN, the 33/66-kD fragments, and synthetic peptides derived from the 33/66-kD fragments of FN. FN and the 33/66-kD fragments also promoted endothelial cell spreading and migration. Although each of the six synthetic peptides tested supported endothelial cell adhesion, only one of these peptides within the carboxyl-terminal heparin-binding domain (FN-C/H-V) promoted endothelial cell spreading and migration. Cell spreading on FN-C/H-V, as well as on FN and the 33/66-kD fragments, was associated with the formation of a well-developed actin cytoskeleton and the formation of focal contacts. FN-C/H-V (but not scrambled FN-C/H-V) inhibited cell spreading on FN and the 33/66-kD fragments in a concentration-dependent manner. FN-C/H-V had a modest effect on the adhesion of a clonal population of rat heart endothelial cells (RHE-1A) to the 33/66-kD fragments of FN and no effect on RHE-1A cell adhesion to FN. These findings suggest that peptide FN-C/H-V is unique among this group of peptides derived from the 33/66-kD heparin-binding fragments of FN in its ability to promote the adhesion, spreading, and migration of vascular endothelial cells and further suggest that the sequence defined by this peptide plays an important role in vascular endothelial cell interactions with the 33/66-kD fragments of FN.
Stenosis Enhances Role of Platelets in Growth of Regional Thrombus and Intimal Wall Thickening in Rat Carotid Arteries
Authors: Igawa Takehiro , Nagamura Yoshie , Ozeki Yasushi , Itoh Hideki , Unemi Fumiko
Published: June 2012

Abstract
The authors present the results of a study in which stenosis was induced, resulting in either thrombus or intimal wall thickening, in rat carotid arteries. At ≥75% stenosis in mildly denuded arteries, an acute and occlusive thrombus formation was induced, but the thrombus was significantly reduced in thrombocytopenia. Thrombus formation near the site of stenosis decreased with decreasing degree of stenosis, whereas the percent formation in the distal region (percent total thrombus) increased. Numerous mural platelet microthrombi were noted at the distal region of the stenosed arteries. After chronic 50% stenosis of the carotid artery for 2 weeks, significant intimal thickening was observed, without any occlusive thrombus formation. The combination with mild denudation was critical in eliciting the effect of stenosis. The magnitude of intimal growth in the stenosed artery was marked by day 6 and plateaued thereafter, whereas it was slight in nonstenosed arteries. The 5-bromodeoxyuridine index of the cells of the medial layer at day 3 was significantly increased by the stenosis, and the effect was reversed in thrombocytopenia. Complete reendothelialization of the intimal surface was observed by 7 to 10 days after surgery in the stenosed arteries. These findings suggest that the introduction of stenosis in these arteries enhances the interaction of platelets with the damaged arterial walls under abnormal fluid shear and that this enhancement leads to acute and occlusive thrombus formation associated with more marked stenosis as well as to sustained increase of intimal wall thickness in less marked stenosis.
Gene Expression of Natriuretic Peptide Receptors in Myocardial Cells
Authors: Lin Xiongbin , Hänze Jörg , Heese Frauke , Sodmann Richard , Lang Rudolf E.
Published: June 2012

Abstract
Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are cardiac hormones that serve to unload the heart through their effects on the kidney and vasculature. Whether the heart itself represents a site of action for these peptides is currently the subject of debate. Although functional studies indicate that ANP has some effects on isolated myocytes, several studies have been unable to detect binding of the hormone to these cells. The present study demonstrates that the genes for all three natriuretic peptide receptor (NPR) subtypes, NPR-A, NPR-B, and NPR-C, are expressed in the rat heart. For microlocalization of the receptor mRNAs in myocytes and nonmyocytic cells, a combination of cell isolation and reverse transcription–polymerase chain reaction (RT-PCR) was used. Cardiac myocytes were isolated by enzymatic dissociation of rat ventricular tissue, purified by density gradient centrifugation, and collected as single cells under microscopic control. Analysis by RT-PCR revealed the presence of transcripts for NPR-A as well as NPR-B and NPR-C. However, cGMP generation in purified myocytes was stimulated only by ANP and BNP, which specifically bind to NPR-A, whereas C-type natriuretic peptide (CNP, an NPR-B agonist) was ineffective. Therefore, rat ventricular myocytes appear to produce predominantly NPR-A. The expression of NPR-B may be low or even absent. The mRNAs for all three NPRs were also found in cultures of fibroblasts from the rat heart. In contrast to the myocytes, large increases in cGMP were observed in response not only to ANP but also to CNP. Cardiac fibroblasts may therefore synthesize functionally relevant amounts of both NPR-A and NPR-B. The expression of NPR-C in these cells is suggested by binding studies demonstrating that >70% of the maximum binding of [
125
I]ANP can be displaced by the NPR-C agonist C-ANP 4-23. These observations may help to resolve whether the heart muscle cells themselves are targets for natriuretic peptides. The finding that in addition to myocytes, cardiac fibroblasts are capable of expressing the NPR genes raises the interesting possibility that natriuretic peptides are involved in the structural remodeling of the heart.
Lymphocytic Na
+
-H
+
Exchange Increases After an Oral Glucose Challenge
Authors: Tepel Martin , Schlotmann Ralf , Barenbrock Michael , Kisters Klaus , Klaus Torsten , Spieker Claus , Walter Michael , Meyer Christian , Bretzel Reinhard G. , Zidek Walter
Published: June 2012

Abstract
The effects of oral glucose challenge on plasma glucose concentration, plasma insulin concentration, arterial blood pressure, cytosolic pH (pH
i
), cytosolic free Na
+
concentration ([Na
+
]
i
), and cellular Na
+
-H
+
exchange activity were investigated in 16 healthy subjects. The pH
i
, [Na
+
]
i
, and Na
+
-H
+
exchange activity were measured in intact lymphocytes by using the fluorescent dye technique. The oral glucose challenge significantly increased plasma glucose, plasma insulin, and the lymphocytic Na
+
-H
+
exchange activity, measured as change of pH
i
per second (control [0 hours], 5.20±0.53×10
−3
dpH
i
/s; 1 hour after glucose administration, 8.28±1.07×10
−3
dpH
i
/s; 2 hours after glucose administration, 8.15±1.18×10
−3
dpH
i
/s;
P
=.002). The lymphocytic Na
+
-H
+
exchange was significantly correlated with plasma glucose concentration (
r
=.357,
P
=.041). During steady state euglycemic hyperinsulinemic clamp, the Na
+
-H
+
exchange activity was not significantly changed compared with baseline values. The study shows that changes of blood glucose levels can induce an acute increase in Na
+
-H
+
exchange activity. Systolic blood pressure and Na
+
-H
+
exchange activity were significantly (
P
<.001) but weakly correlated during an oral glucose challenge.
Reduction of Intimal Hyperplasia by Naroparcil, a 4-Methylumbelliferyl β-
d
-Xyloside Analogue, After Arterial Injury in the Hypercholesterolemic Rabbit
Authors: Steg Philippe Gabriel , Ziol Marianne , Tahlil Ouafae , Robert Claude , Masson Philippe , Pruneau Didier , Bruneval Patrick , Bélichard Pierre
Published: June 2012

Abstract
4-Methylumbelliferyl β-D-xylosides (β-
d
-xylosides) inhibit proteoglycan synthesis, and this is associated with reduced proliferation and extracellular matrix production by vascular smooth muscle cells. This study evaluated whether treatment with naroparcil, a β-
d
-xyloside analogue, reduced intimal hyperplasia after arterial injury in the hypercholesterolemic rabbit. Forty-two rabbits were assigned to three groups that received either a 1% cholesterol-enriched diet (group 1, n=15) or the same diet with added 100 mg · kg
−1
naroparcil (group 2, n=15) or 300 mg · kg
−1
naroparcil (group 3, n=12). All animals underwent iliac artery endothelial abrasion at day 14 and were killed at day 56. At the time of death, the angiographic minimal luminal diameter was significantly larger in both treated groups. Morphometric analysis showed a larger luminal area in treated rabbits (groups 2 and 3) compared with control rabbits (group 1) (0.75±0.54 and 0.85±0.61 mm
2
versus 0.32±0.25 mm
2
, respectively;
P
<.05), with a decreased intimal thickness in groups 2 and 3 (average reduction of 37% and 39%, respectively, compared with group 1;
P
<.05) but without changes in medial area. Total vessel area was comparable among all groups. In the media, immunohistochemistry suggested reduced infiltration by macrophages and a larger fractional area of smooth muscle cells. There were no differences in plasma or arterial wall cholesterol content between groups. Plasma levels of glycosaminoglycans and dermatan sulfate content were increased only in group 3. In this model, oral treatment with naroparcil appears to preserve the arterial lumen and reduce intimal thickness after arterial injury.
Differential Regulation of Circulating Mg
2+
in the Rat by β
1
- and β
2
-Adrenergic Receptor Stimulation
Authors: Keenan D. , Romani A. , Scarpa A.
Published: June 2012

Abstract
Extracellular Mg
2+
homeostasis was studied in vivo in the anesthetized rat. Animals were infused with isoproterenol (ISO) for 10 minutes, and serum Mg
2+
was measured after the infusion and then 10 and 20 minutes later. A dose-dependent increase in circulating Mg
2+
was observed in animals infused with ISO at a rate of 0.1 μg · kg
−1
· min
−1
or higher. The time course of the effect demonstrated that circulating Mg
2+
continued to increase 20 minutes after the end of the ISO infusion. A predicted maximal increase in serum Mg
2+
concentration of 19.3% was derived with a predicted EC
50
of 0.08 μg · kg
−1
· min
−1
. The maximal percent increase corresponded to a net increase of 6.7 μmol/300 g body wt. Because infusion of ISO resulted in changes in hemodynamic parameters, most notably a drop in blood pressure, a group of animals was infused with nitroprusside to mimic the hypotensive response via a nonadrenergic mechanism. Under these conditions, there was a transient increase in circulating Mg
2+
that was largely inhibited by propranolol, indicating that hypotension per se was not responsible for the mobilization of Mg
2+
. Infusion of salbutamol, but not prenalterol, also induced an increase in circulating Mg
2+
. Pretreatment with butoxamine, ICI-118551, or propranolol prevented the ISO-induced increase in serum Mg
2+
. Pretreatment with atenolol minimally affected the ISO-induced changes in circulating Mg
2+
. Pretreatment with CGP-20271A actually enhanced the ISO-induced increase in circulating Mg
2+
. This evidence demonstrates the existence of a pool of Mg
2+
that is mobilized into the circulation in response to selective β
2
-adrenergic stimulation.
Involvement of Multiple
cis
Elements in Basal- and α-Adrenergic Agonist–Inducible Atrial Natriuretic Factor Transcription
Authors: Sprenkle Amy B. , Murray Susan F. , Glembotski Christopher C.
Published: June 2012

Abstract
In the present study,
cis
elements in the 5′-flanking sequence (FS) of the rat atrial natriuretic factor (ANF) gene involved in regulating basal and α
1
-adrenergic–inducible transcription were investigated. Truncation analyses using ANF-luciferase reporter constructs transfected into primary neonatal rat cardiac myocytes showed that an A/T-rich serum response element (SRE) at −114 bp of the ANF 5′-FS, which bound serum response factor (SRF), was required for basal and inducible transcription. In constructs composed of 134 bp of rat ANF 5′-FS driving luciferase (ANF-134Luc), mutations in the SRE at −114 bp disrupted SRF binding and ANF promoter activity. However, the same mutations in ANF-638Luc had little effect, suggesting a collaborating role for more distal sequences, such as the other SRE in ANF-638 at −406 bp. In ANF-638Luc, mutations in the SRE at −406 bp that disrupted SRF binding to that site decreased ANF reporter activity by only 25%; however, mutating both of the SREs completely blocked α
1
-adrenergic–inducible activity. Mutation analyses showed that an ••• (SP-1)–like site at −69 bp, shown previously to confer inducibility in reporters with 134 bp of ANF 5′-FS, was not required in ANF-638Luc. However, double mutants in the SP-1–like region and either SRE completely blocked α
1
-adrenergic–inducible ANF promoter activity. These findings emphasize that no single element is responsible for α
1
-adrenergic agonist–regulated ANF transcription but that the SREs at −114 and −406 bp and the SP-1–like sequence at −69 bp mediate the effect in collaboration.
Anti-minK Antisense Decreases the Amplitude of the Rapidly Activating Cardiac Delayed Rectifier K
+
Current
Authors: Yang Tao , Kupershmidt Sabina , Roden Dan M.
Published: June 2012

Abstract
The rapidly and slowly activating delayed rectifier K
+
currents (I
Kr
and I
Ks
, respectively), which have different physiological properties, have been identified in cardiac cells from several species, including humans. Although expression of the minimal K
+
channel protein (minK) cDNA in some systems results in a current resembling I
Ks
, the role of this gene product in channel function remains controversial. In atrial tumor myocytes (AT-1 cells), no I
Ks
is recorded, but minK mRNA is detected, raising the possibility that expression of the minK gene serves an as-yet-unidentified function. In these experiments, AT-1 cells were exposed to antisense oligonucleotides targeting the 5′ translation start site of the minK cDNA cloned from an AT-1 library. Cell size, I
Kr
, and L-type and T-type Ca
2+
currents were measured 24 to 48 hours after exposure and compared with data in cells exposed to the corresponding sense oligonucleotide or grown in medium only. Antisense oligonucleotide significantly reduced I
Kr
compared with sense and medium-only control cells in 0 of 2 experiments (n=3 to 6 cells per treatment in each experiment) at 50 nmol/L, 1 of 2 at 250 nmol/L, 6 of 6 at 1000 nmol/L, and 2 of 2 at 10 000 nmol/L. At 1000 nmol/L, maximum tail current in antisense-exposed cells was 2.5±0.1 pA/pF (mean±SEM, n=28, 6 separate experiments), 6.6±0.4 pA/pF in sense-exposed cells (n=27), 5.4±0.6 pA/pF in medium-only cells (n=21), and 5.8±0.7 pA/pF in cells exposed to a random oligonucleotide (n=9). I
Kr
activation, rectification, deactivation, and sensitivity to the blocker dofetilide were unaffected. Two different antisense oligonucleotides produced the same effect, and there was no effect of antisense treatment on cell size or on L- or T-type Ca
2+
currents. These data indicate that in AT-1 cells, expression of the minK gene plays a role in determining I
Kr
amplitude.