bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular Ca
2+
, Intercellular Electrical Coupling, and Mechanical Activity in Ischemic Rabbit Papillary Muscle
Authors: Dekker Lukas R.C. , Fiolet Jan W.T. , VanBavel Ed , Coronel Ruben , Opthof Tobias , Spaan Jos A.E. , Janse Michiel J.
Published: June 2012

During myocardial ischemia, electrical uncoupling and contracture herald irreversible damage. In the present study, we tested the hypothesis that an increase of intracellular Ca
2+
is an important factor initiating these events. Therefore, we simultaneously determined tissue resistance, mechanical activity, pH
o
, and intracellular Ca
2+
(with the fluorescent indicator indo 1, Molecular Probes, Inc) in arterially perfused rabbit papillary muscles. Sustained ischemia was induced in three experimental groups: (1) control, (2) preparations preconditioned with two 5-minute periods of ischemia followed by reperfusion, and (3) preparations pretreated with 1 mmol/L iodoacetate to block anaerobic metabolism and minimize acidification during ischemia. In a fourth experimental group, intracellular Ca
2+
was increased under nonischemic conditions by perfusing with 0.1 mmol/L ionomycin and 0.1 μmol/L gramicidin. Ca
2+
transients and contractions rapidly disappeared after the induction of ischemia. In the control group, diastolic Ca
2+
began to rise after 12.6±1.3 minutes of ischemia; uncoupling, after 14.5±1.2 minutes of ischemia; and contracture, after 12.6±1.5 minutes of ischemia (mean±SEM). Preconditioning significantly postponed Ca
2+
rise, uncoupling, and contracture (21.5±4.0, 24.0±4.1, and 23.0±5.3 minutes of ischemia, respectively). Pretreatment with iodoacetate significantly advanced these events (1.9±0.7, 3.6±0.9, and 1.9±0.2 minutes of ischemia, respectively). In all groups, the onset of uncoupling always followed the start of Ca
2+
rise, whereas the start of contracture was not different from the rise in Ca
2+
. Perfusion with ionomycin and gramicidin permitted estimation of a threshold [Ca
2+
] for electrical uncoupling of 685±85 nmol/L. In conclusion, the rise in intracellular Ca
2+
is the main trigger for cellular uncoupling during ischemia. Contracture is closely associated with the increase of intracellular Ca
2+
during ischemia.
Hemodynamic Effects of
l
- and
d
-
S
-Nitrosocysteine in the Rat
Authors: Davisson Robin L. , Travis Mark D. , Bates James N. , Lewis Stephen J.
Published: June 2012

The vasorelaxant effects of the endothelium-derived relaxing factor
S
-nitrosocysteine (SNC) may not be simply due to its decomposition to NO. The biological actions of SNC may also involve the transnitrosation of amino acids in the blood and in plasma membranes. The possibility that the SNC moiety possesses biological activity prompted us to examine whether the hemodynamic effects of this
S
-nitrosothiol involves the activation of stereoselective
S
-nitrosothiol receptors within the cardiovascular system. We examined (1) the hemodynamic effects produced by intravenous injections of the
l
and
d
isomers of SNC (
l
- and
d
-SNC, respectively; 100 to 800 nmol/kg), the
l
and
d
isomers of the parent thiols (
l
- and
d
-cysteine, respectively; 100 to 800 nmol/kg), the oxidized thiol
l
-cystine (100 to 800 nmol/kg), and the NO donor sodium nitroprusside (SNP, 1 to 36 μg/kg) in conscious freely moving rats, (2) the baroreceptor reflex–mediated changes in heart rate elicited in response to the falls in arterial pressure produced by
l
- and
d
-SNC and SNP in conscious rats, and (3) the relative decomposition of
l
- and
d
-SNC to NO upon addition to heparinized rat blood or upon direct application to cultured porcine aortic smooth muscle (PASM) cells. We now report that (1)
l
-SNC is a more potent hypotensive and vasodilator agent within the mesenteric bed and sympathetically intact and sympathetically denervated hindlimb beds of conscious rats than is
d
-SNC, (2)
l
- and
d
-SNC markedly inhibit baroreceptor reflex–mediated tachycardia in conscious rats and
d
-SNC is considerably more effective than
l
-SNC, (3) the intravenous injections of
l
- and
d
-cysteine or
l
-cystine do not affect arterial blood pressure or vascular resistances, and (4)
l
- and
d
-SNC decompose equally to NO upon application to rat blood or cultured PASM cells. These results suggest that the hemodynamic effects of endogenous SNC may involve its interaction with stereoselective
S
-nitrosothiol recognition sites within the vasculature and the baroreflex arc. These findings provide tentative evidence that membrane-bound
S
-nitrosothiol receptors may exist within the cardiovascular system.
Thrombin Potently Stimulates Cytokine Production in Human Vascular Smooth Muscle Cells but Not in Mononuclear Phagocytes
Authors: Kranzhöfer Roger , Clinton Steven K. , Ishii Kenji , Coughlin Shaun R. , Fenton John W. , Libby Peter
Published: June 2012

Thrombosis frequently occurs during atherogenesis and in response to vascular injury. Accumulating evidence supports a role for inflammation in the same situation. The present study therefore sought links between thrombosis and inflammation by determining whether thrombin, which is present in active form at sites of thrombosis, can elicit inflammatory functions of human monocytes and vascular smooth muscle cells (SMCs), two major constituents of advanced atheroma. Human α-thrombin (EC
50
, ≈500 pmol/L) potently induced interleukin (IL)-6 release from SMCs. The tethered-ligand thrombin receptor appeared to mediate this effect. Furthermore, α-thrombin also rapidly increased levels of mRNA encoding IL-6 and monocyte chemotactic protein-1 (MCP-1) in SMCs. In contrast, only α-thrombin concentrations of ≥100 nmol/L could stimulate release of IL-6 or tumor necrosis factor-α (TNFα) in peripheral blood monocytes or monocyte-derived macrophages. Lipid loading of macrophages did not augment thrombin responsiveness. Likewise, only α-thrombin concentrations of ≥100 nmol/L increased levels of IL-6, IL-1β, MCP-1, or TNFα mRNA in monocytes. Differential responses of SMCs and monocytes to thrombin extended to early agonist-mediated increases in [Ca
2+
]
i
. SMCs and endothelial cells, but not monocytes, contained abundant mRNA encoding the thrombin receptor and displayed cell surface thrombin receptor expression detected with a novel monoclonal antibody. Thus, the level of thrombin receptors appeared to account for the differential thrombin susceptibility of SMCs and monocytes. These data suggest that SMCs may be more sensitive than monocytes/macrophages to thrombin activation in human atheroma. Cytokines produced by thrombin-activated SMCs may contribute to ongoing inflammation in atheroma complicated by thrombosis or subjected to angioplasty.
PD 81,723, an Allosteric Enhancer of the A
1
Adenosine Receptor, Lowers the Threshold for Ischemic Preconditioning in Dogs
Authors: Mizumura Tsuneo , Auchampach John A. , Linden Joel , Bruns Robert F. , Gross Garrett J.
Published: June 2012

PD 81,723 (PD) acts allosterically to increase agonist binding to A
1
adenosine receptors and to enhance functional A
1
receptor–mediated responses in the heart and other tissues. To determine if PD lowers the threshold for ischemic preconditioning (PC), pentobarbital-anesthetized dogs were subjected to 60 minutes of left anterior descending coronary artery (LAD) occlusion and 3 hours of reperfusion. Ischemic PC was produced by either 2.5 or 5 minutes of LAD occlusion 10 minutes before the 60-minute occlusion. PD (100 μg/kg total dose, 5 to 50 μmol/L in coronary arterial blood) or vehicle was infused intracoronarily for 17.5 minutes before the 60-minute occlusion period in non-PC dogs or in dogs preconditioned with 2.5 minutes of ischemia. Myocardial infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Compared with the control group (26.3±3.6%, mean±SEM), infarct size was not significantly affected by 2.5 minutes of PC alone (23.4±4.2%) or by PD alone (26.5±1.7%) but was decreased by PD+PC (14.6±1.7%,
P
<.05) or by a longer period (5 minutes) of PC alone (12.5±3.3%). The intravenous administration of the selective antagonist of A
1
adenosine receptors, 8-cyclopentyl-1,3-dipropylxanthine (1 mg/kg), or the ATP-sensitive K
+
channel blocker, glibenclamide (0.3 mg/kg), for 15 minutes before PD+PC blocked the protection (23.6±2.3% or 25.9±3.3%, respectively). None of the compounds studied affected systemic hemodynamics, collateral blood flow, or AAR. To determine which subtypes of canine adenosine receptors were affected by 10 μmol/L PD, radioligand binding studies were conducted using membranes derived from COS-7 cells expressing recombinant canine receptors and agonist radioligands. PD enhanced the binding of [
125
I]
N
6
-4-amino-3-iodobenzyladenosine (
125
I-ABA) to A
1
receptors by increasing the t
1/2
for dissociation by 2.18-fold, but PD had no effect on the dissociation kinetics of
125
I-ABA from A
3
receptors or [
125
I]-[2-(4-amino-3-iodo-phenyl)ethylamino]adenosine from A
2A
receptors. Glibenclamide at concentrations up to 10 μmol/L had no effect on the binding of radioligands to recombinant canine A
1
, A
2A
, or A
3
receptors. These data suggest that PD reduces the amount of time required for ischemia to produce preconditioning by enhancing adenosine binding to its A
1
receptor. Glibenclamide prevents the protection afforded by A
1
receptor activation by a mechanism not involving adenosine receptor blockade.
Heterogeneity of Expression of E- and P-Selectins In Vivo
Authors: Eppihimer Michael J. , Wolitzky Barry , Anderson Donald C. , Labow Mark A. , Granger D. Neil
Published: June 2012

A novel technique involving radiolabeled monoclonal antibodies was used to characterize and compare the expression of E- and P-selectin on unstimulated, histamine-challenged, and endotoxin-challenged endothelial cells in various tissues of the mouse. Under unstimulated conditions, E-selectin was absent in all organs, but significant expression of P-selectin was observed in several organs. Histamine induced a rapid time-dependent upregulation of P-selectin, with the largest responses observed in mesentery and lung. Significant fold elevations in P-selectin expression occurred as early as 5 minutes after the histamine injection and remained elevated up to 1 hour. Histamine-induced P-selectin upregulation was inhibited by the H
1
receptor antagonist diphenhydramine, whereas the H
2
receptor antagonist cimetidine had no effect. Endotoxin (lipopolysaccharide [LPS]) also induced a time-dependent expression of P-selectin that reached a maximum between 4 and 8 hours after endotoxin administration. LPS-induced upregulation of P-selectin was greatest in heart and stomach, which exhibited insignificant constitutive expression of P-selectin. LPS also induced a time-dependent upregulation of E-selectin, with maximal expression occurring 3 to 5 hours after intraperitoneal administration. The lung and small intestine exhibited the largest responses to LPS challenge. Histamine administration did not affect E-selectin expression in any tissue. E- and P-selectin–deficient mice were used to test the specificity of monoclonal antibody binding in unstimulated, histamine-challenged, and LPS-stimulated tissues. Vascular binding of the radiolabeled E-selectin and P-selectin monoclonal antibodies was not observed in the respective deficient mice. These findings suggest that P-selectin is constitutively expressed on vascular endothelium in some tissues of the mouse and that there are significant regional differences in the magnitude and time course of histamine- and endotoxin-induced P-selectin expression. In contrast, E-selectin appears to be absent on unstimulated vascular endothelium but is upregulated within 3 hours after the administration of endotoxin in most tissues.
Identification of Distinct Luminal Domains for Macromolecules, Erythrocytes, and Leukocytes Within Mammalian Capillaries
Authors: Vink Hans , Duling Brian R.
Published: June 2012

A thick endothelial surface coat consisting of the glycocalyx and associated plasma proteins has been hypothesized to reduce functional capillary volume available for flowing plasma macromolecules and blood cells. The purpose of this study was to compare anatomic and functional capillary diameters available for macromolecules, RBCs, and WBCs in hamster cremaster muscle capillaries. Bright-field and fluorescence microscopy provided similar estimates (mean±SE) of the anatomic capillary diameter: 5.1±0.1 μm (bright field, 39 capillaries in 10 animals) and 5.1±0.2 μm (membrane dye PKH26, 18 capillaries in 2 animals). Estimates of functional diameters were obtained by measuring the width of RBCs and WBCs and the intracapillary distribution of systemically injected fluorescein isothiocyanate (FITC)–dextran 70. WBCs (5.1±0.2 μm) fully occupied the anatomic capillary cross section. In contrast, the widths of RBCs (3.9±0.2 μm, 21 capillaries in 8 animals) and FITC-dextran (4.3±0.2 μm, 21 capillaries in 8 animals) were significantly smaller than the anatomic capillary diameter. Continuous (1- to 5-minute) excitation of fluorochromes in the capillary lumen (light-dye treatment) increased the width of RBCs passing the treated site from 3.6±0.3 to 4.4±0.3 μm (6 capillaries in 4 animals) and the width of the FITC-dextran column from 4.1±0.2 to 4.6±0.3 μm (10 capillaries in 7 animals). Furthermore, light-dye treatment increased capillary tube hematocrit by 60% in 40-μm-long capillary segments compared with untreated sites in the same capillaries. It is concluded that the wall of skeletal muscle capillaries is decorated with a 0.4- to 0.5-μm-thick endothelial surface coat, which may represent the true active interface between blood and the capillary wall.
Oleic Acid–Induced Mitogenic Signaling in Vascular Smooth Muscle Cells
Authors: Lu Gang , Morinelli Thomas A. , Meier Kathryn E. , Rosenzweig Steven A. , Egan Brent M.
Published: June 2012

As an initial step in testing the hypothesis that high oleic acid concentrations contribute to vascular remodeling in obese hypertensive patients by activating protein kinase C (PKC), the effects of oleic acid on primary cultures of rat aortic smooth muscle cells (RASMCs) were studied. Oleic acid, an 18-carbon
cis
-monounsaturated fatty acid (18:1 [
cis
]), from 25 to 200 μmol/L significantly increased [
3
H]thymidine uptake in RASMCs with an EC
50
of 41.0 μmol/L and a maximal response of 196±15% of control (
P
<.01). Oleic acid from 25 to 200 μmol/L caused a concentration-dependent increase in the number of RASMCs in culture at 6 days, reaching a maximum of 210±13% of control at 100 μmol/L (
P
<.001). PKC inhibition with 4 μmol/L bisindolylmaleimide I and PKC depletion (α, μ, ι, and ζ) with 24-hour exposure to 200 nmol/L phorbol 12-myristate 13-acetate in RASMCs eliminated the mitogenic effects of oleic acid but did not reduce responses to 10% FBS. Stimulation of intact cells with oleic acid induced a peak increase of cytosolic PKC activity, reaching 328±8% of control (
P
<.001), but did not enhance PKC activity in the membrane fraction (105±4%,
P
=NS). The oleic acid–induced increase of PKC activity in cell lysates was similar in the presence and absence of Ca
2+
, phosphatidylserine, and diolein (maximum response, 360±4% versus 342±9% of control,
P
=NS). Unlike phorbol 12-myristate 13-acetate, oleic acid over 24 hours did not downregulate any of the four PKC isoforms detected in RASMCs. Oleic acid treatment activated mitogen-activated protein (MAP) kinase. PKC depletion in RASMCs eliminated the rise in thymidine uptake, activation of PKC, and activation of MAP kinase in response to oleic acid. In contrast to oleic acid, 50 to 200 μmol/L stearic (18:0) and elaidic (18:1 [
trans
]) acids, which are less effective activators of PKC than oleic acid, did not enhance thymidine uptake. These data suggest that oleic acid induces proliferation of RASMCs by activating PKC, particularly one or more of the Ca
2+
-independent isoforms, and raise the possibility that the higher oleic acid concentrations observed in obese hypertensive patients may contribute to vascular remodeling.
Titin Develops Restoring Force in Rat Cardiac Myocytes
Authors: Helmes Michiel , Trombita´s Ka´roly , Granzier Henk
Published: June 2012

When relaxed after contraction, isolated cardiac myocytes quickly relengthen back to their slack length. The molecular basis of the force that underlies passive relengthening, known as restoring force, is not well understood. In a previous study of titin's elasticity in cardiac myocytes, we proposed that titin/connectin develops restoring force, in addition to passive force. This study tested whether titin indeed contributes to the restoring force in cardiac myocytes. Skinned rat cardiac myocytes in suspension were shortened by ≈20%, using Ca
2+
-independent shortening, followed by relaxation. Cells were observed to relengthen until they reached their original slack sarcomere length. However, the ability to relengthen was abolished after cells had been treated for 12 minutes with trypsin (0.25 μg/mL, 20°C). Gel electrophoresis showed that this treatment had degraded titin without clearly affecting other proteins, and immunoelectron microscopy revealed that the elastic segment of titin in the I band was missing from the sarcomere. Restoring force was also directly measured, before and after trypsin treatment. Restoring force of control cells was −61±20 μg (per cell) at a sarcomere length of 1.70 μm. Comparison of our results with those of activated trabeculae indicated that a large fraction of restoring force of cardiac muscle originates from within the myocyte. Restoring force of myocytes was found to be depressed after titin had been degraded with trypsin. We conclude that cardiac titin indeed develops restoring force in shortened cardiac myocytes, in addition to passive force in stretched cells, and that titin functions as a bidirectional spring. Our work suggests that at the level of the whole heart, part of the actomyosin-based active force that is developed during systole is harnessed by titin, allowing for elastic diastolic recoil and aiding in ventricular filling.
Fluid Flow Rapidly Activates G Proteins in Human Endothelial Cells
Authors: Gudi Siva R.P. , Clark Craig B. , Frangos John A.
Published: June 2012

Fluid shear stimulates endothelial cells, with the external hemodynamic forces transduced across the plasma membrane to modulate intracellular events. We report the first direct evidence that identifies specific GTP binding proteins (G proteins) activated within 1 second of flow onset, representing one of the earliest mechanochemical signal transduction events reported to date in shear-stimulated endothelium. A nonhydrolyzable GTP photoreactive analogue, azidoanilido [α-
32
P]GTP (AAGTP), allowed irreversible labeling of flow-stimulated G proteins, with two protein bands (42 kD and 31 kD) identified in human umbilical vein endothelial cells (HUVECs) subjected to laminar flow (10 dyne/cm
2
) in a parallel-plate flow chamber. Immunoprecipitation of labeled whole-cell lysates identified the specific G-protein subunits G
αq/α11
and G
αi3/αo
as being activated by flow. Endothelial cell membrane vesicles were sheared in a cone-and-plate viscometer, with the 42-kD protein band labeled by AAGTP, but the 31-kD protein absent, indicating that the 42-kD G protein is membrane associated and activated independently of intact cytoskeletal or cytosolic components. Our results describe one of the earliest flow-induced signaling events reported in HUVECs, providing insight into the primary mechanosensing and signal transduction mechanisms.
Pulse Pressure–Related Changes in Coronary Flow In Vivo Are Modulated by Nitric Oxide and Adenosine
Authors: Recchia Fabio A. , Senzaki Hideaki , Saeki Akio , Byrne Barry J. , Kass David A.
Published: June 2012

Acute increases in arterial pulsatile load imposed on the left ventricle can increase coronary flow without commensurate changes in myocardial oxygen consumption. One explanation is that augmenting pulsatile perfusion at the same mean pressure itself stimulates flow by releasing endothelium-mediated vasorelaxant factors such as NO. The present study tested this hypothesis and determined whether NO and adenosine modulate this response. In open-chest anesthetized dogs, the distal left anterior descending coronary artery (LAD) was whole-blood–perfused by a novel servopump system to control mean and pulsatile perfusion pressure within the isolated vascular bed. Central aortic pressure was measured, stored to computer memory, and then digitally modified (varying the pulse pressure [PP]) to generate a real-time servocommand that was still synchronous with ventricular contraction. Left heart workload was unchanged. LAD flow was measured before and after increasing the PP (to 60 to 100 mm Hg) from baselines of either 0 or 40 mm Hg. With normal basal coronary vascular tone, raising the PP increased flow (+9±2% at a PP of 100 mm Hg). This response was markedly amplified (+39±8%) when basal tone was first partially reduced by adenosine. Competitive inhibition of NO synthase by
N
ω
-monomethyl-
l
-arginine reduced acetylcholine and PP-dependent flow responses by 50%. Thus, enhanced pulsatile perfusion increases in vivo coronary flow in part by triggering NO release. The marked augmentation of the PP response with reduced basal coronary tone from adenosine suggests that this mechanism may play a role in improving myocardial perfusion during exercise.
Cardiac Efficiency Is Improved After Ischemia by Altering Both the Source and Fate of Protons
Authors: Liu Bin , Clanachan Alexander S. , Schulz Richard , Lopaschuk Gary D.
Published: June 2012

Cardiac efficiency is decreased in hearts after severe ischemia. We determined whether reducing the production of H
+
from glucose metabolism or inhibiting the clearance of H
+
via Na
+
-H
+
exchange could increase cardiac efficiency during reperfusion. This was achieved using dichloroacetate (DCA) to stimulate glucose oxidation and 5-(
N
,
N
-dimethyl)-amiloride (DMA) to inhibit Na
+
-H
+
exchange, respectively. Isolated working rat hearts were subjected to 30 minutes of global ischemia and 60 minutes of reperfusion. Glycolysis and oxidation rates of glucose, lactate, and palmitate were measured. Recovery of cardiac work, O
2
consumption (MV˙O
2
), and rates of acetyl-coenzyme A and ATP production during reperfusion were determined. After ischemia, cardiac work recovered to 35±5% of preischemic values in control hearts (n=23), although MV˙O
2
, tricarboxylic acid (TCA) cycle activity, and ATP production from glycolysis and oxidative metabolism rapidly recovered to preischemic levels. This decrease in cardiac efficiency was accompanied by a substantial production of H
+
from glucose metabolism. DCA caused a 2.2-fold increase in glucose oxidation, a 46±17% decrease in H
+
production, a 1.6-fold increase in cardiac efficiency, and a 2.0-fold increase in cardiac work during reperfusion (n=17). Inhibition of Na
+
-H
+
exchange with DMA did not alter TCA cycle activity and ATP production rates but did result in a 1.8-fold increase in cardiac efficiency and a 1.7-fold increase in cardiac work (n=12). These data show that cardiac efficiency and the contractile function after ischemia can be improved by either reducing the rate of H
+
production from glucose metabolism during reperfusion or inhibiting the clearance of H
+
via Na
+
-H
+
exchange. Our data suggest that an increased requirement for ATP to restore ischemia-reperfusion–induced alterations in ion homeostasis contributes to the decrease in cardiac efficiency and contractile function after ischemia.
A Balance Between Nitric Oxide and Oxidants Regulates Mast Cell–Dependent Neutrophil–Endothelial Cell Interactions
Authors: Niu Xiao-Fei , Ibbotson Geoffrey , Kubes Paul
Published: June 2012

Nitric oxide (NO) synthesis inhibition causes neutrophil adhesion to endothelium via a mast cell– and oxidant-dependent mechanism. The objective of this study was to delineate the cascade of events in the mast cell– and oxidant-induced neutrophil-endothelium interactions after NO synthesis inhibition. Mast cells were isolated and purified from the rat peritoneal cavity and coadministered with neutrophils to wells of endothelium. This system was treated with an NO synthesis inhibitor (
N
G
-nitro-
l
-arginine methyl ester; L-NAME) for 60 minutes. L-NAME did not induce neutrophil-endothelium interactions in the absence of mast cells, but the addition of mast cells in a ratio as low as 1:50 mast cells to neutrophils was sufficient to induce a large increase in neutrophil adhesion to endothelium within 20 to 25 minutes.
l
-arginine, NO donors, and 8-bromo-cGMP reversed the L-NAME effect, whereas
N
G
-nitro-
d
-arginine methyl ester alone had no proadhesive effect. The adhesion was inhibited by an anti-CD18 or an anti–intracellular adhesion molecule-1 antibody and a platelet-activating factor-receptor antagonist. Inhibition of NO in isolated endothelial monolayers induced oxidant release (reduction of cytochrome C) into extracellular fluid. The endothelium-derived superoxide contributed to the mast cell–induced adhesion, inasmuch as the extracellular antioxidant superoxide dismutase reduced the neutrophil adhesion response as did disruption of endothelial function. There was some direct activation of mast cells with L-NAME (independent of endothelium) inasmuch as intracellular calcium and oxidative stress increased within mast cells after L-NAME treatment, and this translated into increased neutrophil adhesion to nonendothelial substrata. These data demonstrate that depletion of NO increases oxidative stress within mast cells and endothelium and together these events promote neutrophil adhesion within the vasculature.
An Improved Permeabilization Protocol for the Introduction of Peptides Into Cardiac Myocytes
Authors: Johnson John A. , Gray Mary O. , Karliner Joel S. , Chen Che-Hong , Mochly-Rosen Daria
Published: June 2012

We have developed an improved, less disruptive procedure for the transient permeabilization of neonatal cardiac myocytes using saponin. The method allows delivery of peptides to a high percentage of cells in culture without effects on long-term cell viability. Permeation was confirmed microscopically by cellular uptake of a fluorescently labeled peptide and biochemically by uptake of
125
I-labeled calmodulin and a 20-kD protein kinase Cε fragment into the cells. The intracellular molar concentration of the introduced peptide was ≈10% of that applied outside. We found no significant effects of permeabilization on spontaneous, phorbol ester–modulated, or norepinephrine-modulated contraction rates. Similarly, the expression of c-
fos
mRNA (measured 30 minutes after permeabilization) and the incorporation of [
14
C]phenylalanine following agonist stimulation (measured 3 days after permeabilization) were not altered by saponin permeabilization. Finally, permeabilization of cells in the presence of a protein kinase C pseudosubstrate peptide, but not a control peptide, inhibited phorbol ester–induced [
14
C]phenylalanine incorporation into proteins by 80%. Our results demonstrate a methodology for the introduction of peptides into neonatal cardiac myocytes that allows study of their actions without substantial compromises in cell integrity.
Role of Reactive Oxygen Intermediates in Cytomegalovirus Gene Expression and in the Response of Human Smooth Muscle Cells to Viral Infection
Authors: Speir Edith , Shibutani Tomoko , Yu Zu-Xi , Ferrans Victor , Epstein Stephen E.
Published: June 2012

Because cytomegalovirus (CMV) may contribute to restenosis and atherosclerosis and because smooth muscle cells (SMCs) are involved in these disease processes, we examined CMV-SMC interactions. Using confocal microscopy to identify a redox-sensitive fluorescent marker, we found that CMV infection of SMCs generates intracellular reactive oxygen intermediates (ROIs). CMV also activated nuclear factor κB (NFκB), a cellular transcription factor, as demonstrated by increased NFκB binding to DNA (electrophoretic mobility shift assay). Antioxidants inhibited activation, suggesting a role of ROIs in CMV-induced NFκB activation. By using antioxidants to assess the role of ROIs in modulating virally mediated effects, we also found that CMV-induced ROIs (1) are critical to the transactivation of the viral major immediate promoter (MIEP) by its immediate-early protein IE72 (determined by cotransfection of an IE72 expression vector and a reporter gene downstream from the MIEP) and (2) are necessary for IE72 expression (determined by immunocytochemistry) and viral replication (determined by viral titer assay on indicator cells) following CMV infection of SMCs. Because ROIs, through activation of NFκB, can also induce expression of cellular genes involved in immune and inflammatory responses, the ROI response to CMV infection may also represent a parallel survival mechanism that has evolved in the host cell to protect against viral infection. We conclude that CMV induces intracellular ROI generation within minutes after infection of SMCs and then uses these ROIs to facilitate its own gene expression and replication. Conversely, antioxidants inhibit CMV immediate-early gene expression and viral replication.
Metabolism of Ischemic Cardiac Muscle
Authors: KALTENBACH JOHN P. , JENNINGS ROBERT B.
Published: June 2012

The aerobic metabolism of homogenates of normal and ischemic left ventricle was compared in dogs at various time intervals (15 min. to 24 hours) after high ligation of the circumflex branch of the left coronary artery. Glucose and fructose-1,6-diphosphate were used as substrates. The injured tissue was taken from the posterior papillary muscle (PP) and the non-ischemic tissue from the anterior superior septum (LV). Both oxygen consumption and organic phosphorus (Δ organic phosphorus) were slightly decreased in PP at 15 and 30 min. after ligation. Between 30 and 60 min., there was a sharp decrease in oxygen consumption and Δ organic phosphorus falling to 40 to 50 per cent of control values at 60 min. A further decrease was observed at 180 min., and at 24 hours there was a complete loss of oxidative metabolism in the PP. A striking increase in organic phosphorus metabolism and oxygen consumption was observed in the non-ischemic portion of the myocardium 60 and 180 min. after ligation.
The Effect of Tetraethylpyrophosphate (TEPP) on the Transmembrane Potentials of Pacemaker and Non-pacemaker Fibers of Isolated Rabbit Atrium
Authors: KLEINFELD MORRIS , MAGIN JOHN , STEIN EDWARD
Published: June 2012

When 7 x 10
-7
M tetraethylpyrophosphate (TEPP) was administered as constant perfusion, the results were: (1) A progressive slowing of atrial rate and subsequent block in conduction; (2) A decrease in slope of the prepotential of pacemaker fibers; (3) No significant change in membrane resting potential of sinoatrial and atrial fibers, even during block in conduction; (4) An accelerated repolarization of both types of fibers. Simultaneously with the changes in electrical activity TEPP consistently produced a progressive decrease in inotropism. Pyridine 2-aldoxime methiodide (PAM) in concentrations of 1.26-2.52 x 10
-4
M consistently reversed the effects of TEPP. The inotropism often exceeded the control. The data suggests that 1) the changes in transmembrane potential produced by TEPP are principally due to an acetylcholine action, 2) the decrease in contractility is more intimately related to a disturbance in metabolic energy, and 3) the reversibility of the TEPP effects by PAM is due to a reactivation of acetylcholinesterase, an action similar to that observed in vitro.
Ventricular Pressure-Volume Relationships and Oxygen Consumption in Fibrillation and Arrest
Authors: MONROE R. GRIER , FRENCH GORDON
Published: June 2012

Pressure-volume relationships and myocardial oxygen consumption (V
·
O
2
) were obtnined in an isolated heart during ventricular fibrillation, potassium-induced arrest, and isovolumetric contractions. The heart was found to be less distensible in fibrillation than in diastole or arrest. The V
·
O
2
increased from 4.6 to 14.6 cc./100 Gm. LV/min. as the fibrillating ventricle was distended. During arrest the V
·
O
2
remained constant despite ventricular distention. When the fibrillating ventricle was distended beyond a critical point coronary flow was compromised and the V
·
O
2
decreased. Beyond and only beyond this point could the V
·
O
2
be increased by the greater coronary flow induced by a higher perfusion pressure.
The Influence of Experimentally Induced Atherosclerosis on the Sulfate Content of the Aortic Wall
Authors: FORMAN DONALD T. , McCANN D. S. , MOSHER R. E. , BOYLE A. J.
Published: June 2012

By autoradiography it has been demonstrated that parenterally administered S
35
as sulfate is localized in the area of arterial plaque formation in rabbits with induced atherosclerosis. Dialysis of such tissue, after fat extraction to rid the tissue of inorganic sulfate, shows a much larger amount of sulfate remaining in the aortas of diseased animals as compared to controls suggesting that it is in combination with the mucopolysaccharides of the artery.
Experimental Atheromatosis: Acid Mucopolysaccharide Content of the Aorta
Authors: BOLLET ALFRED JAY , WANG CHUNI , ADLERSBERG DAVID
Published: June 2012

Histochemical and chemical studies of the acid mucopolysaccharide content of the aorta were made in rabbits fed cholesterol for up to seven months. Colloidal iron staining suggested an increase in acid mucopolysaccharide content of the aortas with mild atheromatous lesions but not when severe lesions were present. Chemical determination, however, revealed no increase in the concentration of acid mucopolysaccharide in early lesions, but did reveal a significant increase in the more severely atheromatous aortas of rabbits fed cholesterol for 6 months or more.
Abdominal Baro- and Chemosensitivity in Dogs
Authors: HEYMANS C. , DE SCHAEPDRYVER A. F. , DE VLEESCHHOUWER G. R.
Published: June 2012

Experiments performed in anesthetized dogs have shown that occlusion of the coeliac and mesenteric arteries may determine minor increases of systemic blood pressure, obviously of hemodynamic nature and not induced by baroreceptive vasomotor reflexes. Increase of pressure into the mesenteric arterial circulation does not decrease the systemic blood pressure. The aortic and carotid sinus baroreceptors are the major means of the reflex blood pressure homeostasis. Evidence for the existence of specific chemoreceptors in the abdominal vascular area could not be obtained.
Myocardial Necrosis in Experimental Occlusion of the Portal Vein
Authors: MEJIÍA RAUL H.
Published: June 2012

Fourteen dogs were subjected to temporary occlusion of the portal vein. Electrocardiographic changes suggestive of epicardial or transmural necrosis of the high lateral region of the left ventricle and of subendocardial ischemia, were found in 55 per cent of the dogs. These electrocardiographic changes were accompanied by a significant rise of plasma GOT. Myocardial sections showed areas of multifocal necrosis, extending from endocardium to epicardium, with predominance in the lateral and anterior wall and pillars of the left ventricle and interventricular septum.
Reflex Effects of Left Ventricular Distention
Authors: SALISBURY PETER F. , CROSS CECIL E. , RIEBEN P. ANDRE
Published: June 2012

In open-chest dogs, the peripheral circulation was carried on a heart-lung machine. The pulmonary artery was obstructed and the left atrium and the right ventricle were drained into the venous reservoir of the machine. A balloon in the bloodless left ventricle permitted its distention. Pressures were recorded in the left ventricle and the aortic arch or a femoral artery. After distention of the left ventricle, the left ventricular diastolic pressure rose, the systemic arterial pressure fell, and bradycardia occurred. Distention of the left ventricle also caused reflex dilation of systemic veins. These effects were reversible and were abolished by section of the vagi. They are attributed to receptors in the myocardium of the left ventricle. It is considered likely that these reflex effects of left ventricular distention contribute to the mechanism of cardiogenic shock.
Stroke Volume in the Dog During Graded Exercise
Authors: WANG YANG , MARSHALL ROBERT J. , SHEPHERD JOHN T.
Published: June 2012

Measurements of stroke volume were made in dogs during graded exercise on a horizontal treadmill up to the maximal exercise which they could maintain for 2 to 3 minutes. Eleven series of experiments were performed on 7 dogs. The cardiac output was measured by the indicator-dilution technic and the heart rate was measured from the aortic pressure tracing. At all grades of exercise, there was little or no change in stroke volume. Even with severe exercise, when the cardiac output had increased from 1.5 to 3.5 (average, 2.4) times the values obtained with the dog standing at rest, the stroke volume showed less than 5 per cent change over resting values in 5 experiments on 4 dogs, while in the remainder the increases were 8, 11, 11, 14, 15, and 19 per cent. Thus, the increase in cardiac output in the untrained exercising dog is almost directly proportional to the increase in heart rate. This is in contrast to other situations, such as the combination of exercise with infusion of catecholamines, where an increase in stroke volume may make an important contribution to the increase in cardiac output.
Effect of Insulin in the Induction and Regression of Atherosclerosis in the Chick
Authors: STAMLER JEREMIAH , PICK RUTH , KATZ LOUIS N.
Published: June 2012

Insulin administration to intact cockerels made atherosclerotic by the feeding of a cholesterol-oil-containing diet prevented regression of coronary atherosclerosis. Insulin administration during the induction of atherosclerosis had no effect on the development of lesions. In some experiments, insulin slightly inhibited estrogen-induced protection of the coronary arteries when the birds were on a high cholesterol-oil diet.
Effects of Anoxia on the Vascular Resistance of the Dog's Hind Limb
Authors: LITWIN JERZY , DIL ABDUL HABIB , AVIADO DOMINGO M.
Published: June 2012

In anesthetized dogs, the inhalation of 5 per cent oxygen caused vasoconstriction of perfused hind limb but vasodilatation occurred during reoxygenation. The vasoconstriction during anoxia amounted to a mean increase of vascular resistance of +12 per cent if blood from the same anoxic dog was used for perfusion, and to +35 per cent if blood from a nonanoxic donor was used. This vasoconstrictor response was almost entirely the outcome of anoxemic stimulation of chemoreceptors in the carotid and aortic bodies. The vasodilatation during reoxygenation persisted after chemoreceptor denervation, but was eliminated by either spinal cord transection or denervation of the limb. The nervous pathways responsible for this postanoxic vasodilatation were resistant to bretylium but were partially blocked by either atropine or hexamethonium.
Cardiovascular Adaptation to Partial Heart-Lung Bypass
Authors: GALLETTI PIERRE M. , BRECHER GERHARD A.
Published: June 2012

Partial heart-lung bypass was performed in open-chest dogs, using the gravity arterial in-fusion technique. Arterial infusion pressure, venous suction and pressures in the left ventricle, aortic arch, and airways were phasically recorded. The mean flow in the aortic arch and the blood content of the extracorporeal circuit were continuously recorded. According to the height of the bag oxygenator above the heart level, partial heart-lung bypass resulted in a shift of blood either from the extracorporeal circuit into the animal or vice versa. When the body blood volume was increased by the bypass procedure, the cardiac output decreased only slightly. When the body blood volume was diminished by the bypass procedure, the cardiac output dropped markedly. It was found that when the systemic arterial pressure was maintained at approximately normal levels, independent of cardiac output, the change in cardiac output was proportional to the change in the body blood volume.
Effect of Cholesterol Atherosclerosis, Hypertension and Cortisone on Aortic Oxygen Consumption in Rabbit
Authors: FISHER EDWIN R. , GELLER J. H.
Published: June 2012

In vitro estimation of aortic QO
2
in normal rabbits revealed the thoracic portion to be lower than that of the arch and abdominal segments. These latter were not significantly different. Renal hypertension, cortisone administration and their combination with or without cholesterol feeding resulted in significant elevations of the oxygen consumption of all segments. The thoracic portion under these conditions was lowest and that of the arch was greater than that observed in the abdominal aorta. A depression of QO
2
was observed in the aortas from normotensive rabbits subjected to cholesterol feeding with or without cortisone treatment.

Although these findings indicate that hypertension and cortisone may profoundly alter the metabolic activity of the aorta, they could not be correlated with the degrees of cholesterol atherosclerosis observed in the various experimental situations explored.
Study of the Relationship Between Plasma Volume and Transcapillary Protein Exchange Using I
131
-Labeled Albumin and I
125
-Labeled Globulin
Authors: WALKER W. GORDON , ROSS RICHARD S. , HAMMOND JOHN D. S.
Published: June 2012

A mean transcapillary exchange rate for albumin and gamma globulin has been obtained from plasma disappearance curves of I
131
-labeled albumin and I
125
-labeled gamma globulin. A much closer correlation exists between transcapillary exchange rate for albumin and the plasma volume than between the rate and serum albumin concentrations. Also, for albumin this rate varies with plasma volume and circulating protein in such a manner as to suggest that the capillary permeability changes with changing plasma volume. The relation between vascular and extravascular albumin is consistent with this interpretation, the extravascular mass of albumin appearing to approach zero as vascular albumin approaches the vicinity of 0.35 Gm/Kg. In experiments in which I
131
-labeled albumin and I
125
-labeled globulin were employed to obtain simultaneous data on albumin and gamma globulin, a good correlation has been obtained between the simultaneously measured albumin and globulin transcapillary exchange rates. The ratio of the fractional capillary exchange rates of these 2 substances is very nearly the same as the ratio of their diffusion coefficients, a relation to be expected if diffusion is the mechanism of transcapillary exchange.
Regulation of Ventricular Contraction
Authors: SARNOFF S. J. , BROCKMAN S. K. , GILMORE J. P. , LINDEN R. J. , MITCHELL J. H.
Published: June 2012

At constant heart rates, efferent stimulation of the vagus nerve and of the left stellate ganglion revealed the following: 1. Vagal stimulation exerts a profound depressant effect on the strength of the atrial contraction and can thereby influence ventricular filling and ventricular stroke work; it elevates mean atrial pressure at any given level of ventricular stroke work. This occurs under experimental conditions wherein the vagal stimulation used does not produce an alteration in the performance characteristics of the ventricle. The effects of vagal stimulation are blocked by atropine. 2. Stellate ganglion stimulation or norepinephrine infusion augments the strength of atrial contraction and thus the atrial contribution to ventricular filling. The augmented atrial contraction takes place in a shorter period of time. 3. Stellate ganglion stimulation or norepinephrine infusion increases the external stroke work and power produced by the ventricle from any given mean atrial pressure and from any given end-diastolic pressure or fiber length.4. There is a family of curves representing the relation between end-diastolic fiber length and stroke work, as well as a family of curves representing the relation between mean atrial or end-diastolic pressure and stroke work.5. When taken together with the well-known sympathetic and parasympathetic effects on heart rate, the above data are believed to comprise a reasonably comprehensive description of the means available to the central nervous system for directly inducing acute changes in the activity of the heart.
Studies on Starling's Law of the Heart
Authors: BRAUNWALD EUGENE , FRYE ROBERT L. , ROSS JOHN , Lewis Robert
Published: June 2012

The relationship between left ventricular end-diastolic pressure and circumference was studied in 27 open-chest dogs, utilizing a mercury-resistance gage to measure left ventricular end-diastolic circumference. In 6 experiments aortic pressure, cardiac output and heart rate were varied independently. Tachycardia above a rate critical for each heart elevated left ventricular eud-diastolic pressure for any given end-diastolic circumference. Hypothermia at a constant heart rate had a similar effect. The altered left ventricular end-diastolic pressure-end-diastolic circumference relationships resulting from tachycardia and hypothermia are believed to be related to the incomplete ventricular relaxation which occurs as the duration of diastole is encroached upon. Acute, spontaneous heart failure was accompanied by an augmented left ventricular end-diastolic circumference for any given end-diastolic pressure, an effect which may be considered to reflect an increase in myocardial extensibility. The relationship between left ventricular end-diastolic pressure and end-diastolic circumference was not modified either by changes of aortic pressure or of cardiac output. There was no constant relationship between the left ventricular end-diastolic circumference and the tension-time index. Indeed, it was possible to manipulate aortic pressure and cardiac output so that these 2 parameters moved in opposite directions. These observations are not consonant with the view that myocardial oxygen consumption is primarily dependent on end-diastolic fiber length.
Electrolyte Content of Rat Heart Atria and Ventricles
Authors: BARCLAY J. A. , HAMLEY E. J. , HOUGHTON HELGA
Published: June 2012

Extracellular tissue spaces are compared using inulin, sucrose, chloride and sodium in hearts perfused with Ringer-Locke solution by Langendorff's method. Also chloride and sodium spaces are given for unperfused, fresh hearts. From the inulin spaces the intracellular chloride, sodium, potassium and water concentrations in both perfused and unperfused heart chambers have been calculated. Data showing the specificity of the atrium and ventricles of the heart are presented.
Evidence That Outwardly Rectifying Cl
−
Channels Underlie Volume-Regulated Cl
−
Currents in Heart
Authors: Duan Dayue , Hume Joseph R. , Nattel Stanley
Published: June 2012

Swelling-induced Cl
−
current (I
Cl.swell
) is present in most cardiac tissues, but the unitary channel underlying I
Cl.swell
is unknown. We used the cell-attached patch-clamp technique to assess the properties of single channels underlying I
Cl.swell
and the basally active Cl
−
current (I
Cl.b
) in rabbit atrial myocytes. Under isotonic conditions, single outwardly rectifying Cl
−
channels (ORCCs) with a slope conductance of 28±1 pS at the reversal potential were observed in 21 (5.7%) of 367 patches. Unconditional kinetic analysis revealed at least three open and four closed-channel states. Hypotonic superfusion-induced swelling resulted in the appearance of active channels in 41 (15.5%) of 265 patches without channel activity under isotonic conditions and caused a second active channel to appear in 3 of 14 patches showing a single channel under isotonic conditions. Overall, channels were seen in 54 of 336 patches under hypotonic conditions (16.1%,
P
<.001 versus isotonic conditions). The current-voltage relations, reversal potential–[Cl
−
]
o
relations, open probability, and kinetics of swelling-induced channels were indistinguishable from those of ORCCs under isotonic conditions. Unitary ORCCs, I
Cl.b
, and I
Cl.swell
were strongly and similarly inhibited by tamoxifen. Swelling-induced increases in macroscopic Cl
−
current were attributable to an increase in the number of active ORCCs with no significant effects on single-channel amplitude or open probability. Estimated macroscopic currents based on cell surface area, patch dimensions, single-channel ORCC current amplitude, open probability, and density were consistent with measured values of I
Cl.b
and I
Cl.swell
. We conclude that ORCCs underlie volume-regulated basal and swelling-induced Cl
−
currents in isolated rabbit atrial myocytes.