bluuebunny/crossref_metadata_2025 huggingface dataset:
Measurement of angiotensin I converting enzyme inhibition in the heart.
Authors: Kinoshita A , Urata H , Bumpus F M , Husain A
Published: June 2012

Angiotensin (Ang) I converting enzyme (ACE) inhibitors represent a major advance in the treatment of congestive heart failure, and tissue, rather than circulating ACE, may be their major site of action. However, assessments of tissue ACE inhibition in treated patients has not always supported this contention. In these studies, ACE activity was measured in homogenates of sampled tissue by biochemical methods. In the present study, using a model system, we have examined the validity of these tissue-sampling methods. Functional ACE activity was determined by comparing positive inotropic responses to [Pro10]Ang I in either vehicle-pretreated or ACE inhibitor-pretreated papillary muscles. [Pro10]Ang I elicits a response, which is entirely dependent on ACE-mediated conversion to Ang II. The ACE inhibitors studied were captopril, enalaprilat, lisinopril, and quinaprilat. In a parallel study, papillary muscle ACE activity was also measured in homogenates using [125I]MK-351A (a radiolabeled ACE inhibitor) binding. The studies indicate that the tissue-sampling method significantly underestimated functional ACE inhibition in hamster papillary muscles (p < 0.001). Kinetic studies indicated that the half-time for the dissociation of [3H]enalaprilat and [3H]lisinopril from hamster ventricular ACE was 4.5 and 6.2 minutes, respectively. The dissociation of [3H]quinaprilat was biphasic (half-time, 47 and 90 minutes), indicating that the two active sites of somatic ACE differ in their ability to bind to this inhibitor. The rapid rate of ACE inhibitor dissociation suggests that, during the time taken to assay ACE activity biochemically, the enzyme becomes "disinhibited," leading to an underestimation of functional ACE inhibition. ACE inhibitor dissociation rates were partially predictive of the duration of functional ACE inhibition in papillary muscles; other factors that appeared to contribute were "tissue trapping" of the inhibitor and de novo synthesis of ACE in papillary muscles. Quantification of tissue ACE inhibition and its relation to drug efficacy must, therefore, involve a careful consideration of these factors to avoid artifacts in clinical decision making and in assessments of pathogenic mechanisms involved in congestive heart failure.
Mechanism of rapid desensitization of muscarinic K+ current in adult rat and guinea pig atrial cells.
Authors: Kim D
Published: June 2012

Acetylcholine (Ach) activates the muscarinic K+ current in atrial cells via the inhibitory GTP binding protein. After activation, the whole-cell K+ current decreases rapidly (rapid desensitization) to approximately half of the initial current within approximately 20 seconds. The mechanism of this rapid desensitization was investigated in adult rat and guinea pig atrial cells. Whole-cell voltage-clamp and patch-clamp techniques were used to study the K+ current. In voltage-clamped whole cells, ACh activated a K+ current that desensitized rapidly during the initial approximately 20 seconds followed by a slower decrease over several minutes. The rapid K+ current desensitization (a rapid decrease in channel open probability) was also observed at the single-channel level in cell-attached patches and was associated with a progressive shortening of the channel open time and prolongation of the closed time. These changes in channel current and kinetics were abolished by removal of the cytoplasm (by forming inside-out patches) and were partially inhibited by phosphatase inhibitors, suggesting an involvement of cytosolic phosphatase(s) in K+ current desensitization. In inside-out patches with ACh in the pipette and GTP in the bath, the open time of muscarinic K+ channels and channel open probability were increased by 1 mM Mg(2+)-ATP (but not by the nonhydrolyzable analogue, adenylylimidodiphosphate) and decreased by alkaline phosphatase. These results suggest that the rapid K+ current desensitization in adult rat or guinea pig atrial cells is produced by changes in the gating kinetics of the K+ channel, possibly mediated via membrane-associated protein kinase and cytosolic phosphatase(s).
Relative contributions of cardiopulmonary and sinoaortic baroreflexes in causing sympathetic activation in the human skeletal muscle circulation during orthostatic stress.
Authors: Jacobsen T N , Morgan B J , Scherrer U , Vissing S F , Lange R A , Johnson N , Ring W S , Rahko P S , Hanson P , Victor R G
Published: June 2012

The aim of this study was to reexamine the hypothesis that cardiopulmonary baroreflexes are more important than sinoaortic baroreflexes in causing vasoconstriction in the skeletal muscle circulation during orthostatic stress. We recorded muscle sympathetic nerve activity (MSNA) with microelectrodes in the peroneal nerve (and forearm blood flow with venous occlusion plethysmography) in normal subjects (innervated ventricles) and in heart transplant recipients (denervated ventricles) during graded lower body negative pressure (LBNP) performed alone and in combination with intravenous infusion of phenylephrine, which was titrated to eliminate the orthostatically induced fall in blood pressure and thus the unloading of both carotid and aortic baroreceptors. The principal new findings are as follows: (1) The increases in both MSNA and forearm vascular resistance during multiple levels of LBNP were not attenuated by heart transplantation, which causes ventricular but not sinoaortic deafferentation. (2) In heart transplant recipients, a small increase in MSNA during mild LBNP was dependent on a decrease in arterial pressure, but in normal subjects, a similar increase in MSNA occurred in the absence of any detectable decrease in the aortic pressure stimulus to the sinoaortic baroreceptors. (3) In normal subjects, the large increase in MSNA during a high level of LBNP was dependent on a decrease in arterial pressure and could be dissociated from the decrease in central venous pressure. Taken together, the findings strongly suggest that sinoaortic baroreflexes are much more important and ventricular baroreflexes are much less important than previously thought in causing reflex sympathetic activation and vasoconstriction in the human skeletal muscle circulation during orthostatic stress.
Alterations of K+ currents in isolated human ventricular myocytes from patients with terminal heart failure.
Authors: Beuckelmann D J , Näbauer M , Erdmann E
Published: June 2012

Prolongation of the action potential has been postulated to be a major reason for the altered diastolic relaxation of the heart in patients with severe heart failure. To investigate the electrophysiological basis for this action potential prolongation in terminal heart failure, K+ currents were recorded in single ventricular myocytes isolated from 16 explanted hearts of patients undergoing transplantation. Results from diseased hearts were compared with ventricular cells isolated from six undiseased donor hearts. Action potential duration was significantly prolonged in cells from patients with heart failure. A delayed rectifier K+ current was hardly detectable in most cells, and if it could be recorded, it was very small in both diseased and undiseased cells. When currents were normalized for cell surface area, the average current density of the inward rectifier K+ current was significantly reduced in diseased cells when compared with normal control cells (hyperpolarization at -100 mV, -15.9 +/- 2.2 vs -9.0 +/- 1.2 microA/cm2; P < .01). In addition, a large transient outward K+ current could be recorded in human myocytes. The average current density of the time-dependent component of this transient outward K+ current was significantly reduced in heart failure (depolarization at +40 mV, 9.1 +/- 1.0 vs 5.8 +/- 0.64 microA/cm2; P < .01). Action potential prolongation in severe heart failure may partially be explained by a reduction in current densities of the inward rectifier K+ current and of the transient outward K+ current. These alterations may thereby have a significant effect on cardiac relaxation.
ATP-dependent closure and reactivation of inward rectifier K+ channels in endothelial cells.
Authors: Olesen S P , Bundgaard M
Published: June 2012

This report presents the results of a patch-clamp study examining the role of intracellular ATP in the regulation of endothelial inward rectifier K+ channels. Administration of ATP to the cytosolic surface of inside-out patches reversibly activated the K+ channel within seconds. ATP (1 mM) increased the mean open probability by a factor of 3.5, primarily by increasing the number of openings. Administration of the nonhydrolyzable ATP analogue ATP-gamma-S failed to modulate channel activity. Inhibition of ATP synthesis by administration of cyanide plus iodoacetate resulted in channel closure within 1 to 6 minutes. In experiments in which ATP was coadministered with the metabolic blockers, the channel activity continued unchanged for up to 30 minutes, but when ATP was removed, the activity rapidly decayed. We propose that normal functioning of the inward rectifier K+ channel is ATP dependent. Phosphorylation of the channel molecule is probably essential for maintaining activity.
Repeated dipyridamole administration enhances collateral-dependent flow and regional function during exercise. A role for adenosine.
Authors: Symons J D , Firoozmand E , Longhurst J C
Published: June 2012

Two main hypotheses concerning the mechanisms responsible for coronary collateral growth suggest the involvement of chemical or mechanical factors. Since we recently demonstrated that the development of the coronary collateral circulation is not closely related to the extent or duration of myocardial ischemia, we hypothesized that chronic repeated vasodilation and increased myocardial blood flow using dipyridamole would enhance collateral development in miniswine with an ameroid-occluded left circumflex coronary artery (LCx). Two days after surgical instrumentation, the animals received dipyridamole (n = 9), diltiazem as an adenosine-independent vasodilator (n = 8), or control vehicle (n = 7) 90 minutes per day, 5 days per week for 8 weeks. At 5 and 8 weeks, transmural blood flow and systolic wall thickening were measured during infusion of dipyridamole, diltiazem, or vehicle. Transmural blood flow increased similarly in the LCx and nonoccluded regions at 30 and 60 minutes during infusion of either vasodilator. Thus, we believe that similar mechanical stimulation resulted from dipyridamole and diltiazem infusion. There was no change in blood flow during administration of the vehicle. Systolic wall thickening in the collateral-dependent region was not altered by infusion of dipyridamole, diltiazem, or vehicle. Therefore, both vasodilators increased blood flow without eliciting ischemia. After 8 weeks of repeated treatment with each pharmacological agent, at least 24 hours after the last drug infusion, near maximal physiological capacity of the coronary collateral vessels was assessed during treadmill running (approximately 240 beats per minute). Transmural myocardial blood flow ratios, expressed as flow in the LCx divided by flow in the nonoccluded region of the left ventricle, were similar at rest for animals treated with dipyridamole (0.90 +/- 0.03), diltiazem (0.97 +/- 0.05), and control vehicle (0.89 +/- 0.02). However, collateral-dependent myocardial blood flow during exercise was greater (P < .05) in the dipyridamole-treated animals (0.78 +/- 0.04) than in either diltiazem-treated (0.63 +/- 0.09) or vehicle-treated (0.62 +/- 0.02) animals. LCx systolic wall thickening at rest was similar in animals treated with dipyridamole (44.4 +/- 6.3%), diltiazem (42.2 +/- 3.0%), and control vehicle (38.1 +/- 2.8%). During exercise, however, myocardial function in the collateral-dependent region was greater (P < .05) in the dipyridamole-treated (39.2 +/- 5.2%) compared with diltiazem-treated (23.9 +/- 4.0%) and vehicle-treated (26.9 +/- 2.9%) animals.(ABSTRACT TRUNCATED AT 400 WORDS)
Development of the cardiac coronary vascular endothelium, studied with antiendothelial antibodies, in chicken-quail chimeras.
Authors: Poelmann R E , Gittenberger-de Groot A C , Mentink M M , Bökenkamp R , Hogers B
Published: June 2012

The endothelium of the coronary vascular system has been described in the literature as originating from different sources, varying from aortic endothelium for the main coronary stems, endocardium for the intramyocardial network, and sinus venosus lining for the venous part of the coronary system. Using an antibody against quail endothelial cells (alpha-MB1), we investigated the development of the coronary vascular system in the quail (Hamburger and Hamilton stages 15 to 35) and in a series of 36 quail-chicken chimeras. In the chimeras, pieces of quail epicardial primordium and/or liver tissue were transplanted into the pericardial cavity of a chicken host. The results showed that the coronary vascular endothelial distribution closely followed the formation of the epicardial covering of the heart. However, pure epicardial primordium transplants did not lead to endothelial cell formation, whereas a liver graft with or without an epicardial contribution did have this capacity. The first endothelial cells were seen to reach the heart at the sinus venosus region, subsequently spreading through the inner curvature to the atrioventricular sulcus and the outflow tract and, last of all, over the ventricular surfaces. At these sites, the precursor cells and small vessels were seen to invade the sinus venosus wall, the ventricular and atrial myocardium, and the mesenchymal border of the aortic orifice. Connections with the endocardium of the heart tube were only observed in the right ventricular outflow region. Initially, the connections with the aortic endothelium were multiple, but later in development only two of these connections persisted to form the proximal part of the two main coronary arteries. Connections to the pulmonary orifice were never observed. Our transplantation data showed that the entire coronary endothelial vasculature originated from an extracardiac source. Moreover, using the developing subepicardial layer as a matrix, we showed that the endothelial cells reached the heart from the liver region. Ingrowth into the various cardiac segments was also observed. Implications for the relation to specific congenital cardiac malformations are discussed.
Peptidergic innervation of human epicardial coronary arteries.
Authors: Gulbenkian S , Saetrum Opgaard O , Ekman R , Costa Andrade N , Wharton J , Polak J M , Queiroz e Melo J , Edvinsson L
Published: June 2012

The peptidergic innervation of proximal (internal diameter, > 0.8 mm) and distal (internal diameter, < 0.8 mm) regions of human epicardial coronary arteries was investigated by means of immunohistochemical, chromatographic, radioimmunological, and in vitro pharmacological techniques. The use of an antiserum to the general neuronal marker protein gene product 9.5 revealed that the proximal part of epicardial arteries possessed a relatively sparse supply of nerve fibers forming a loose network in the adventitia. The perivascular network increased in density as the vessels were followed distally. In both proximal and distal regions, the majority of nerve fibers possessed neuropeptide Y and tyrosine hydroxylase immunoreactivity. Calcitonin gene-related peptide (CGRP)- and substance P-immunoreactive nerve fibers were very sparse in the proximal region of the arteries and increased in number distally. Only a few scattered vasoactive intestinal peptide (VIP)-immunoreactive nerve fibers were detected in both arterial regions. The use of high-performance liquid chromatography and radioimmunoassay revealed that the immunoreactive material present in coronary artery extracts closely resembled synthetic peptides. An in vitro pharmacological method demonstrated that neuropeptide Y elicited no detectable response in either proximal or distal arterial segments. In contrast, CGRP, substance P, and VIP all produced a concentration-dependent relaxation of both arterial regions. CGRP and substance P were stronger and more potent than VIP. CGRP and substance P induced a more potent response in distal compared with proximal regions of the arteries. These results suggest that the peptidergic nerves supplying human large epicardial coronary arteries may be predominantly involved in mediating vasodilation.
Expression of basic fibroblast growth factor and its receptor by smooth muscle cells and endothelium in injured rat arteries. An en face study.
Authors: Lindner V , Reidy M A
Published: June 2012

Release of endogenous basic fibroblast growth factor (bFGF) has been shown to initiate smooth muscle cell (SMC) proliferation following balloon catheter denudation in rat arteries. The mechanisms that contribute to the continued replication of the cells that subsequently form the neointima are not well understood. We have examined expression of bFGF and fibroblast growth factor receptor 1 (FGFR-1) in luminal SMCs as well as endothelium at various times after injury, which allowed us to study both replicating as well as quiescent cells. Using in situ hybridization on en face preparations, we were able to detect mRNA in luminal cells that was not observed by analysis of artery cross sections. We demonstrate that mRNA for bFGF was found in replicating SMCs and endothelial cells. bFGF mRNA was not detectable in either cell type at quiescence despite nuclear staining for bFGF. Expression of FGFR-1 mRNA was observed in replicating endothelial and SMCs at similar times after injury. These data provide evidence that in injured arteries the ligand/receptor system of bFGF and FGFR-1 may be involved in the continued proliferative response of SMCs leading to neointima formation. Furthermore, our results suggest a role for bFGF in reestablishing the endothelial lining in denuded vessels.
Potentiation by hypercholesterolemia of the induction of aortic intramural synthesis of plasminogen activator inhibitor type 1 by endothelial injury.
Authors: Sawa H , Sobel B E , Fujii S
Published: June 2012

Accumulation of plasminogen activator inhibitor type 1 (PAI-1) in the arterial wall may accelerate atherogenesis by inhibiting fibrinolysis, diminishing proteolysis of extracellular matrix proteins, or modifying migration of vascular smooth muscle cells. Increased intramural expression of the PAI-1 gene is induced by thrombosis. To determine whether it occurs also in response to a sustained mechanical insult to endothelium, hypercholesterolemia, or both, rabbits were subjected to sustained aortic injury induced by implantation of indwelling polyethylene tubing, to hyperlipidemia induced by cholesterol and peanut oil feeding over a period of 8 weeks, or both. Sustained vascular injury alone did not increase plasma PAI-1. However, hypercholesterolemia with or without mechanically induced vascular injury increased plasma PAI-1 twofold. The expression of PAI-1 mRNA in aorta (Northern blots) was significantly increased when vascular injury was combined with hyperlipidemia. In situ hybridization showed that the increase with mechanical injury alone occurred in endothelial cells covering the neointima (positive for factor VIII and thrombomodulin), in abnormally differentiated vascular smooth muscle cells (positive for embryonic myosin heavy chain), and in macrophages (positive for the RAM-11 anti-macrophage antibody). Qualitatively similar but much more marked increases in PAI-1 gene expression were seen when arterial injury was accompanied by hypercholesterolemia. Neither vitronectin, known to stabilize PAI-1, nor vitronectin mRNA increased in liver. However, immunocytochemistry and Western blots demonstrated marked aortic accumulation of vitronectin protein with hyperlipidemia, particularly in subendothelial fibrotic regions, accompanied by increased neointimal vitronectin mRNA as shown by in situ hybridization. These results suggest that increased synthesis and stabilization of vascular PAI-1 may potentiate accumulation of extracellular matrix, thereby accelerating atherosclerosis.
Selective induction of an embryonic fibronectin isoform in the rat aorta in vitro.
Authors: Hosoi M , Takasaki I , Pavlova-Rezakova A , Himeno H , Chobanian A V , Brecher P
Published: June 2012

The temporal changes in the expression of fibronectin and other extracellular matrix genes were studied in rat aortic rings incubated in vitro in a serum-free medium. Changes in all forms of fibronectin mRNA increased progressively during the 24-hour incubation period, although an increase in the alternatively spliced form of fibronectin designated EIIIA was most pronounced. Both collagen and elastin mRNA levels decreased markedly during the 24-hour interval, as did alpha-actin mRNA. The increase in the relative amount of the EIIIA isoform after a 24-hour incubation was also shown using ribonuclease protection assays. In situ hybridization showed the distribution of the induced fibronectin mRNA to be within all cell types, including endothelial cells, medial smooth muscle cells, and adventitial fibroblasts. Localization in the media was not uniform and was clearly identified mainly in clusters of cells distributed throughout the media. The early induction of fibronectin mRNA was inhibited by genistein, implicating tyrosine kinase activation as a causative factor in fibronectin expression. The in vitro changes reported may reflect a phenotypic change in vascular cell types that is both similar to and different from the changes reported in vivo under conditions in which vascular injury and repair occur.
Dynamic interaction between cardiac myosin isoforms modifies velocity of actomyosin sliding in vitro.
Authors: Sata M , Sugiura S , Yamashita H , Momomura S , Serizawa T
Published: June 2012

To study the functional significance of cardiac isomyosin heterogeneity, active sliding of actin-myosin was studied using two different types of in vitro motility assay systems: (1) a sliding actin filament assay, in which fluorescently labeled actin filaments were made to slide on a myosin layer attached to a glass coverslip, and (2) a myosin-coated bead assay, in which myosin-coated latex beads were made to slide on actin cables of an alga. Two different isomyosins were obtained from 3-week-old (V1) and hypothyroid (V3) rat hearts and were mixed to form solutions with various mixing ratios [V1/(V1 + V3)]. For these myosin mixtures, both ATPase activity and sliding velocity of actin-myosin were determined. As the relative content of V1 increased, both ATPase activity and velocity increased. However, in contrast to the linear relation between the mixing ratio and ATPase activity, the relation between the mixing ratio and sliding velocity was sigmoid, suggesting the existence of mechanical interaction between different isomyosins. To clarify the nature of this interaction, sliding velocity was measured for mixtures of V1 and p-N,N'-phenylene-dimaleimide-treated V1 myosin (pPDM-M). A convex relation was observed between the relative content of pPDM-M and velocity. Because pPDM-M is known to form a noncycling and weakly bound crossbridge with actin, it is expected to exert a constant internal load on V1, in contrast to the actively cycling V3. In conclusion, in actomyosin sliding, different isomyosins mechanically interact when they coexist. The interaction may be a dynamic one that cannot be explained by a simple load effect.
Protein kinase A-activated chloride channel is inhibited by the Ca(2+)-calmodulin complex in cardiac sarcoplasmic reticulum.
Authors: Kawano S , Hiraoka M
Published: June 2012

Cardiac sarcoplasmic reticulum (SR) has several chloride (Cl-) channels, which may neutralize the charge across the SR membrane generated by Ca2+ movement. We recently reported a novel 116-picosiemen Cl- channel that is activated by protein kinase A-dependent phosphorylation in cardiac SR. This Cl- channel may serve as a target protein in the receptor-dependent regulation of cardiac excitation-contraction coupling. To understand further regulatory mechanisms, the effects of Ca2+ on the Cl- channel were studied using the planar lipid bilayer-vesicle fusion technique. In the presence of calmodulin (CaM, 0.1 mumol/L per microgram SR vesicles), Ca2+ (3 mumol/L to 1 mmol/L) added to the cis solution reduced the channel openings in a concentration-dependent fashion, whereas Ca2+ (1 nmol/L to 1 mmol/L) alone or CaM (0.1 to 1 mumol/L per microgram SR vesicles) with 1 nmol/L Ca2+ did not affect the channel activity. This inhibitory effect of Ca2+ in the presence of CaM was prevented by CaM inhibitors N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide and calmidazolium but not by CaM kinase II inhibitor KN62. These results suggest that the Ca(2+)-CaM complex itself, but not CaM kinase II, is involved in this channel inhibition. Thus, the cardiac SR 116-picosiemen Cl- channel is regulated not only by protein kinase A-dependent phosphorylation but also by the cytosolic Ca(2+)-CaM complex. This is a novel second messenger-mediated regulation of Cl- channels in cardiac SR membrane.
Mouse model of arterial injury.
Authors: Lindner V , Fingerle J , Reidy M A
Published: June 2012

In the present study, we established an injury model of the mouse carotid artery. Complete removal of the endothelium was achieved with a flexible wire. A platelet monolayer covered the denuded surface, and damage to underlying medial smooth muscle cells (SMCs) was detected. Injection of [3H]thymidine was used to determine the replication index for medial SMCs, which was found to be 1.6% at 2 days after denudation and 9.8% at 5 days. SMCs were observed in the intima by day 8 (replication index, 66%), and by 2 weeks the intimal lesion had a similar cell content as the media. In most animals, repair of the endothelial lining was complete 3 weeks after injury. The present model will allow us to use transgenic animals to address questions relevant to vascular biology and atherosclerosis.
Negative feedback regulation of endothelial cell function by nitric oxide.
Authors: Buga G M , Griscavage J M , Rogers N E , Ignarro L J
Published: June 2012

The objective of this study was to determine whether nitric oxide (NO) could function as a negative feedback modulator of endothelial cell function by inhibiting NO synthase in vascular endothelial cells. The rationale for this approach was a previous study from this laboratory, which revealed that NO inhibits neuronal NO synthase from rat cerebellum. In the present study, NO and NO-donor agents noncompetitively inhibited NO synthase derived from bovine aortic endothelial cells. Oxyhemoglobin blocked the inhibitory action of NO and by itself increased NO synthase activity. This finding suggests that NO acts as a negative feedback modulator of NO synthase. In intact aortic endothelial cells grown on microcarrier beads and perfused in a bioassay cascade system, pretreatment of cells with NO-donor agents caused a marked inhibition of endothelial NO biosynthesis in response to bradykinin and increased fluid shear or flow. When isolated bovine pulmonary arterial rings precontracted by phenylephrine were used, pretreatment of arterial rings with NO-donor agents diminished endothelium-dependent arterial relaxation involving the L-arginine-NO pathway without altering endothelium-independent relaxation to NO itself. On the basis of these studies, NO is suggested to play an important negative feedback regulatory role on endothelial NO synthase and, therefore, vascular endothelial cell function.
Use-dependent block and use-dependent unblock of the delayed rectifier K+ current by almokalant in rabbit ventricular myocytes.
Authors: Carmeliet E
Published: June 2012

A voltage-clamp analysis of the effect of almokalant on the delayed rectifier K+ current (IK) was made in rabbit ventricular myocytes. The two-suction pipette method was used, and appropriate voltage-clamp protocols were used to study more specifically use dependence, block development, and recovery from block. Almokalant interacted with the IK in two ways: it shifted the activation curve in the hyperpolarizing direction (stimulatory effect) and blocked the open IK channel in a use-dependent way (inhibitory effect). For 2-second voltage clamps to +20 mV, half-maximum block was obtained at 5 x 10(-8) mol/L, with a Hill coefficient of 1.76. Use-dependent block was related to an open-channel block that occurred at 0 mV with a time constant of 1.07 second and a rather slow recovery from block: at -50 mV, recovery time constant was approximately 10 seconds; at -75 mV, recovery was practically absent. The absence of an important recovery at negative membrane potentials is consistent with the hypothesis of the drug being trapped in the channel. A limited frequency-dependent block could be demonstrated. Use-dependent unblock was demonstrated by a rapid recovery from block during stimulation following complete washout of the drug. It is concluded that almokalant shifts the activation curve of IK in the hyperpolarizing direction, blocks the open channel, and is trapped by the closure of the activation gate.
Intracellular myoglobin loading worsens H2O2-induced, but not hypoxia/reoxygenation-induced, in vitro proximal tubular injury.
Authors: Zager R A
Published: June 2012

Intracellular iron reportedly mediates many forms of tissue injury, including ischemic and myohemoglobinuric acute renal failure. This action may be explained by the ability of iron to catalyze the formation of the highly toxic hydroxyl radical (.OH) from H2O2 via the Fenton/Haber-Weiss reactions. To assess whether renal tubular myoglobin/iron loading, induced by a physiological mechanism (endocytosis), alters its susceptibility to O2 deprivation/reoxygenation- and H2O2-mediated injury, rats were infused with myoglobin or its vehicle (5% dextrose, control rats), and after 2 hours, proximal tubular segments (PTSs) were isolated for study. This infusion caused substantial myoglobin endocytic uptake (approximately 25 micrograms/mg PTS protein), and it doubled PTS catalytic iron content (assessed by bleomycin assay). Nevertheless, PTS viability (percent lactate dehydrogenase release) was minimally affected (4% to 6% increase), and an increased .OH burden (assessed by the salicylate trap method) did not appear to result. Deferoxamine addition, reported to protect against in vivo acute renal failure, paradoxically increased .OH levels (approximately 25%) in myoglobin-loaded, but not control, PTSs. Conversely, dimethylthiourea (an .OH scavenger) depressed .OH (by approximately 80%) in all PTSs. Myoglobin/iron loading modestly increased PTS vulnerability to exogenous H2O2 addition (P < .001). However, tubular susceptibility to hypoxia (15 and 30 minutes)/reoxygenation injury was not affected. .OH levels appeared to fall in response to both forms of injury, suggesting decreased .OH production and/or .OH scavenging. To assess whether myoglobin decreases .OH levels in the presence of Fenton reactants, myoglobin and six other test proteins were incubated with Fe2+/H2O2. Myoglobin decreased .OH levels by approximately 70%, a significantly greater decrement than was observed with the other proteins tested.(ABSTRACT TRUNCATED AT 250 WORDS)
Metabolism and excretion of nitric oxide in humans. An experimental and clinical study.
Authors: Wennmalm A , Benthin G , Edlund A , Jungersten L , Kieler-Jensen N , Lundin S , Westfelt U N , Petersson A S , Waagstein F
Published: June 2012

Despite the increasing insight in the clinical importance of nitric oxide (NO), formerly known as endothelium-derived relaxing factor (EDRF), there is limited information about the metabolism and elimination of this mediator in humans. We studied the degradation of NO in healthy subjects inhaling 25 ppm for 60 minutes and in patients with severe heart failure inhaling 20, 40, and 80 ppm in consecutive 10-minute periods. In other healthy subjects, the renal clearance of NO metabolite was measured. The metabolism ex vivo was evaluated by direct incubation of nitrite, the NO oxidation product, in blood from healthy humans. During inhalation of NO, the plasma levels of nitrate increased progressively, both in the healthy subjects (from 26 to 38 mumol/L, P < .001) and in the patients (from 72 to 90 mumol/L, P < .001). Methemoglobin (MetHb) also increased in the healthy subjects (from 7 to 13 mumol/L, P < .001) as well as in the patients (from 19 to 42 mumol/L, P < .01). No change in nitrosohemoglobin (HbNO) was detected, either in the healthy subjects or in the patients. In arterialized blood (O2 saturation, 94% to 99%), incubated nitrite was semiquantitatively converted to nitrate and MetHb. In venous blood (O2 saturation, 36% to 85%) moderate amounts of HbNO were also formed. Plasma and urinary clearance of nitrate in healthy subjects averaged 20 mL/min. We conclude that uptake into the red blood cells with subsequent conversion to nitrate and MetHb is a major metabolic pathway for endogenously formed NO. Nitrate may then enter the plasma to be eliminated via the kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of hypertrophy and atrophy in cultured adult heart cells.
Authors: Clark W A , Rudnick S J , LaPres J J , Andersen L C , LaPointe M C
Published: June 2012

Mechanical loading and alpha-adrenergic receptor stimulation have both been shown to induce hypertrophy in isolated neonatal heart cells. The present study examined the effects of adrenergic hormones and contractile activity on the hypertrophic response in isolated adult feline cardiomyocytes maintained for more than 14 days in insulin- and serum-supplemented medium. Measurements of the hypertrophic response included cell size, total protein content, myosin heavy chain content, and the time course of activation of increased protein synthesis. Reactivation of the "fetal" gene program was evaluated by secretion of atrial natriuretic factor (ANF) into the medium. Significant myocyte hypertrophy was induced in both quiescent myocytes treated with alpha 1-adrenergic agonists and in beating myocytes treated with beta-adrenergic agonists. However, there were both quantitative and qualitative differences in the response to each type of stimulation. alpha-Adrenergic agonists promoted an increase in cell size, protein content, and ANF secretion but not myofibrillar reorganization, which was observed only in beating myocytes. In contrast to results reported for neonatal heart cells, determinants of hypertrophy in beating myocytes exceeded those in nonbeating alpha 1-adrenergic agonist-treated heart cells in every parameter examined. In addition, in the case of both beating and alpha-adrenergic stimulation, there were marked time-dependent variations in rates of protein synthesis over the interval of 4 hours to 7 days of treatment with each type of stimulus. Differences were also encountered in correlations between rates of protein synthesis and protein accumulation over this interval. The effect of beating was particularly important both to the reorganization of myofibrillar structure and the metabolism of myosin heavy chain. In cultures in which beating was inhibited with the calcium channel antagonist nifedipine, the loss of myosin heavy chain was significantly greater than that of total protein.
Relative significance of endothelium and internal elastic lamina in regulating the entry of macromolecules into arteries in vivo.
Authors: Penn M S , Saidel G M , Chisolm G M
Published: June 2012

A role for the internal elastic lamina (IEL), which separates the intima and media of an artery wall, as a restrictive barrier to macromolecular movement has been suggested in atherosclerotic lesion development or restenosis during angioplasty. The permeability coefficient of the IEL, however, has never been quantified in unperturbed vessels in vivo. Using a newly developed technique, we measured the concentration distributions in both intima and media of cationic (pI approximately 8.5) and anionic (pI approximately 6.3) isozymes of the 44-kD macromolecule horseradish peroxidase (HRP). Two mathematical models of arterial wall transport differing in their resolution of the intima were required to simulate the concentration distribution data and to estimate the parameters of interest. Optimal estimates of the permeability coefficients of the endothelium (PE) and IEL (PIEL) to HRP were determined by the best least-squares fit of the two models to experimental data. These estimates (anionic: PE = 0.050 +/- 0.021 microns/min, PIEL = 0.146 +/- 0.082 microns/min, n = 8; cationic: PE = 0.034 +/- 0.018 microns/min, PIEL = 0.110 +/- 0.047 microns/min, n = 8) indicate that the IEL is responsible for approximately 25% (anionic, 26 +/- 9%; cationic, 25 +/- 13%) of the resistance to HRP transport from the blood into the arterial media. Although both parameters were less for the cationic preparation, the differences were not significant, and the relative role of the IEL was similar for both molecules.(ABSTRACT TRUNCATED AT 250 WORDS)
Cellular uncoupling induced by accumulation of long-chain acylcarnitine during ischemia.
Authors: Yamada K A , McHowat J , Yan G X , Donahue K , Peirick J , Kléber A G , Corr P B
Published: June 2012

Long-chain acylcarnitines (LCACs) increase rapidly within minutes after the onset of ischemia in vivo or hypoxia in vitro and produce a time-dependent reversible reduction in gap junctional conductance in isolated myocyte pairs. The present study was performed to assess whether LCACs contribute to cellular uncoupling in response to ischemia in isolated blood-perfused rabbit papillary muscles by use of simultaneous measurements of transmembrane action potentials, extracellular electrograms, extracellular K+, and tissue LCACs and ATP. LCACs increased threefold in response to 20 minutes of no-flow ischemia from 127 +/- 5 to 397 +/- 113 pmol/mg protein (P < .01), concomitant with the onset of cellular uncoupling, extracellular K+ accumulation, and a marked reduction in conduction velocity and action potential duration. To assess whether inhibition of the accumulation of LCACs modified the electrophysiological alterations during ischemia, muscles were pretreated with either sodium 2-(5-(4-chlorophenyl)-pentyl)-oxirane-2-carboxylate (POCA, 10 mumol/L) or oxfenicine (100 mumol/L), inhibitors of carnitine acyltransferase I. Both POCA and oxfenicine completely prevented the increase in LCACs even with 40 minutes of ischemia (138 +/- 37 and 56 +/- 4 pmol/mg protein, respectively), associated with a marked delay in the onset and progression of cellular uncoupling and ischemic contracture. Although POCA and oxfenicine did not affect either the initial early rise in extracellular K+ or the initial fall in conduction velocity, both agents markedly delayed the secondary rise in extracellular K+ as well as the secondary fall in conduction velocity, independent of the level of tissue ATP. Thus, LCACs accumulate during myocardial ischemia and contribute substantially to the initiation of cell-to-cell uncoupling. Inhibition of carnitine acyltransferase I and prevention of the increase in LCACs markedly delays cellular uncoupling and development of ischemic contracture in response to ischemia.
Viral infection and the pathogenesis of dilated cardiomyopathy.
Authors: Martino T A , Liu P , Sole M J
Published: June 2012

Long-term follow-up studies of patients with suspected viral myocarditis reveal progression to dilated cardiomyopathy (DCM) in a significant number of cases. Thus, an underlying viral etiology has been hypothesized in the pathogenesis of ongoing heart disease that leads to DCM. Recent application of molecular biology in clinical diagnosis has strengthened this hypothesis. By use of probe hybridization and polymerase chain reaction, enteroviral RNA has been detected in the myocardium of patients at all stages of the disease process: myocarditis, chronic heart disease, and DCM. Experimental murine models of enterovirus-induced heart disease provide a framework for examining the pathogenic mechanisms. Viral cytotoxicity, immunological responses, viral RNA persistence, and spasm of the coronary microvasculature are all implicated in the ongoing disease process. Abnormal cardiac function and heart failure are attributed to the pathological changes that occur.
Expression of beta-arrestins and beta-adrenergic receptor kinases in the failing human heart.
Authors: Ungerer M , Parruti G , Böhm M , Puzicha M , DeBlasi A , Erdmann E , Lohse M J
Published: June 2012

The beta-adrenergic receptor system of the failing human heart is markedly desensitized. We have recently postulated that this desensitization may in part be caused by an increase in beta-adrenergic receptor kinase (beta ARK) expression. beta ARK is thought to effect desensitization by acting in concert with an inhibitor protein, called beta-arrestin. Two isoforms have been identified both for beta ARK and for beta-arrestin. In the present study, we have investigated the expression of the individual isoforms of beta-arrestin and of beta ARK in left ventricles from failing and control human hearts. mRNAs for all four proteins, beta-arrestin-1, beta-arrestin-2, beta ARK-1, and beta ARK-2, were identified in human heart. Quantitation by reverse-transcription polymerase chain reactions showed that in heart failure there were no changes of the mRNA levels for beta-arrestin-1 and beta-arrestin-2, a slight (< 50%) increase of the mRNA for beta ARK-2, and a threefold increase for beta ARK-1 mRNA. At the protein level, beta-arrestin-1 was readily detected by Western blotting in human heart. Its absolute values were approximately 350 fmol/mg cytosolic protein, and its expression was not changed in heart failure. beta-Arrestin-2 levels were too low to be detectable using the same methods. beta ARK levels as determined by enzymatic activity were approximately 20 fmol/mg cytosolic protein (beta ARK-1 plus beta ARK-2) and thus almost 20-fold lower than those of beta-arrestin. beta ARK levels were increased approximately twofold in heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)
Hyaluronate degradation affects ventricular function of the early postlooped embryonic rat heart in situ.
Authors: Baldwin H S , Lloyd T R , Solursh M
Published: June 2012

Hyaluronic acid is the major glycosaminoglycan of the early cardiac extracellular matrix or "cardiac jelly," yet little is known about its role in the ontogeny of early ventricular performance. To investigate the in situ effect of hyaluronate degradation on ventricular function, whole rat embryos were cultured in rat serum alone (control embryos) or rat serum plus 20 TRU/mL of Streptomyces hyaluronidase (treatment embryos) from gestational day 9.5 (before formation of the heart tube) through initial looping of the heart. Cardiac function was measured before looping (24 hours in culture) and immediately after looping (36 hours in culture) by video motion analysis of the external wall motion of the bulbus cordis and primitive ventricle. Degradation of hyaluronic acid in the treated embryos was confirmed by Alcian blue staining at pH 2.5. Significant increases in heart rate, circumferential shortening fraction, maximum velocity of circumferential contraction, and maximum velocity of circumferential relaxation were observed with looping in both control and treatment embryos. Although there was minimal difference in ventricular performance between control and treatment embryos before looping, there was a significant increase in all parameters of ventricular performance in the hyaluronidase-treated embryos immediately after looping of the heart. Endocardial cushions were absent in hyaluronidase-treated embryos, and an additional group of embryos cultured in the presence of Streptomyces hyaluronidase for 48 to 72 hours failed to develop endocardial cushions. These experiments are the first to (1) document a quantifiable increase in ventricular performance during early cardiac looping and (2) demonstrate that hyaluronate degradation results in abnormal endocardial cushion formation and altered ventricular performance of the postlooped heart.(ABSTRACT TRUNCATED AT 250 WORDS)
Interleukin-2-induced lung injury. The role of complement.
Authors: Rabinovici R , Sofronski M D , Borboroglu P , Spirig A M , Hillegas L M , Levine J , Vernick J , Scesney S M , Feuerstein N , Feuerstein G
Published: June 2012

Pulmonary edema and sepsis-like syndrome are grave complications of interleukin-2 (IL-2) therapy. Recent animal studies have suggested IL-2-induced microvascular injury as the underlying mechanism. Since complement factors have been shown to mediate increased vascular permeability in diverse conditions that lead to pulmonary injury and recombinant human IL-2 is known to activate the complement system in patients undergoing IL-2 therapy, we hypothesized that complement factors play a pivotal role in the development of increased vascular permeability after IL-2 treatment. To test this hypothesis, we evaluated the capacity of recombinant soluble human complement receptor type 1 (sCR1, BRL 55730), a new highly specific complement inhibitor, to attenuate IL-2-induced lung injury in the rat. Recombinant human IL-2 (intravenously for 60 minutes) at 10(6) U per rat (n = 4) elevated lung water content (37 +/- 6%, P < .05), myeloperoxidase activity (162 +/- 49%, P < .05), and serum thromboxane B2 (30 +/- 1 pg/100 microL, P < .01) and had no effect on serum tumor necrosis factor-alpha sCR-1 at 30 mg/kg (n = 5), but not at 10 mg/kg (n = 6), attenuated the elevation of lung water content (18 +/- 2%, P < .05) and myeloperoxidase activity (42 +/- 9%, P < .05) but failed to alter serum thromboxane B2 response to IL-2. These data suggest the involvement of complement in the pathogenesis of IL-2-induced pulmonary microvascular injury and point to the potential therapeutic capacity of complement inhibitors in combating this toxic effect of IL-2 therapy.
Modulation of ischemia/reperfusion-induced microvascular dysfunction by nitric oxide.
Authors: Kurose I , Wolf R , Grisham M B , Granger D N
Published: June 2012

Leukocyte-endothelial cell adhesion and an altered metabolism of endothelial cell-derived nitric oxide (NO) have been implicated in the microvascular dysfunction associated with ischemia/reperfusion (I/R). The objective of this study was to determine whether NO donors can attenuate the reperfusion-induced increase in venular albumin leakage via an effect on leukocyte-endothelial cell adhesion. Leukocyte adherence and emigration as well as albumin extravasation were monitored in single postcapillary venules in rat mesentery subjected to 20 minutes of ischemia followed by 30 minutes of reperfusion. This I/R protocol elicits significant leukocyte adherence and emigration as well as a profound albumin leakage response. Superfusion of the mesenteric microcirculation with the NO donors sodium nitroprusside, spermine-NO, and SIN1 significantly reduced the I/R-induced leukocyte adherence/emigration and albumin leakage in postcapillary venules, whereas neither spermine nor the NO synthase inhibitor NG-nitro-L-arginine methyl ester affected the I/R-induced responses. Platelet-leukocyte aggregation and mast cell degranulation were also observed in the postischemic mesentery, and the responses were also attenuated by the NO donors. Plasma nitrate/nitrite levels in the superior mesenteric vein were significantly reduced by I/R. The results of this study indicate that I/R-induced microvascular dysfunction (albumin leakage) is attenuated by NO and that the protective effect of NO donors may be related to their ability to reduce leukocyte-endothelial cell and leukocyte-platelet interactions and/or mast cell degranulation.