bluuebunny/crossref_metadata_2025 huggingface dataset:
Partial Inhibition of Ca
2+
Current by Methoxyverapamil (D600) Reveals Spatial Nonuniformities in [Ca
2+
]
i
During Excitation-Contraction Coupling in Cardiac Myocytes
Authors: Cheng H. , Cannell M. B. , Lederer W. J.
Published: June 2012

Abstract
The laser scanning confocal microscope was used in conjunction with the Ca
2+
indicator fluo 3 to examine the spatiotemporal properties of free Ca
2+
([Ca
2+
]
i
) transients in isolated rat cardiac myocytes. We show that localized increases in [Ca
2+
]
i
(Ca
2+
sparks) can be triggered by membrane depolarization in cardiac myocytes when the sarcolemmal Ca
2+
current amplitude is reduced by methoxyverapamil (D600). These depolarization-evoked Ca
2+
sparks are similar in amplitude and spatiotemporal properties to spontaneous Ca
2+
sparks previously observed at rest. These observations support the idea that Ca
2+
sparks are the result of the activation of functional elementary units of sarcoplasmic reticulum (SR) Ca
2+
release. The synchronous activation of a large number of Ca
2+
sparks can explain the increased amplitude and slower time course of the electrically evoked [Ca
2+
]
i
transient as well as the presence of spatial nonuniformities in [Ca
2+
]
i
during its rise. The data shown here suggest a model for excitation-contraction coupling in which the amplitude of the [Ca
2+
]
i
transient is regulated by variations in the probability of recruitment of elementary SR Ca
2+
release units as well as the amount of Ca
2+
released by each unit. Since the activation of each release unit will depend on the local amplitude of the Ca
2+
current, this model can explain the regulation of the amplitude of the [Ca
2+
]
i
transient by the Ca
2+
current. In addition, these data indicate that caution should be applied to the interpretation of signals obtained with nonlinear Ca
2+
indicators during the rising phase of the [Ca
2+
]
i
transient, when the nonuniformities in [Ca
2+
]
i
are largest.
Inositol Phosphate Release and Metabolism in Rat Left Atria
Authors: Woodcock Elizabeth A. , Suss Mark B. , Anderson Karen E.
Published: June 2012

Abstract
The phosphatidylinositol (PtdIns) turnover pathway in intact heart tissue differs from that in most cell types in that products of the inositol 1,4,5-trisphosphate [Ins(1,4,5)P
3
] kinase pathway are not detected in
3
H-labeling studies. In contrast, Ins(1,4,5)P
3
kinase products are detected in isolated neonatal cardiomyocytes. To understand the basis for the observed properties of the cardiac pathway, a detailed study of inositol phosphate (InsP) release has been undertaken by using isolated adult rat left atria. Addition of norepinephrine to
3
H-labeled atria caused a slow increase in
3
H-labeled Ins(1,4,5)P
3
and a more rapid increase in
3
H-labeled Ins(1,4)P
2
, its immediate dephosphorylation product. The mass of Ins(1,4,5)P
3
was high in unstimulated atria (13.5±1.1 pmol/mg tissue, mean±SEM, n=4) and did not change with stimulation. Measurements of the specific activities of Ins(1,4,5)P
3
and PtdIns(4,5)P
2
provided an estimate of the turnover rate of Ins(1,4,5)P
3
that was 20- to 40-fold lower than the rate of accumulation of
3
H label in InsP
1
and InsP
2
. In agreement with this, specific activities of InsP
1
and InsP
2
were higher than the specific activity of InsP
3
in both control and stimulated atria. Neomycin (5 mmol/L) did not inhibit the accumulation of
3
H-labeled InsP
1
and InsP
2
in left atria, even though it reduced the accumulation of
3
H label in Ins(1,4,5)P
3
, providing evidence that InsP
1
and InsP
2
do not derive primarily from Ins(1,4,5)P
3
. Stimulation with norepinephrine for 20 minutes resulted in a parallel decrease in
3
H-labeled Ins(1,4,5)P
3
and in Ins(1,4,5)P
3
mass, demonstrating that atria do not contain two different pools of Ins(1,4,5)P
3
. In contrast with findings in atria, the mass content of Ins(1,4,5)P
3
was low in unstimulated cardiomyocytes and increased with norepinephrine stimulation. Furthermore, neomycin (5 mmol/L) inhibited the accumulation of
3
H label in all of the InsPs, demonstrating that they are derived from Ins(1,4,5)P
3
. The data obtained when using left atria cannot be explained by models proposing either receptor-stimulated breakdown of Ins(1,4,5)P
3
or the presence of more than one pool of Ins(1,4,5)P
3
with different specific activities and are most readily explained by a model in which stimulation of receptors causes primarily the hydrolysis of PtdIns(4)P to Ins(1,4)P
2
. The high and unchanging content of Ins(1,4,5)P
3
may be related, in some way, to its poor Ca
2+
-mobilizing activity in heart tissue.
Circulating Leukocyte Counts, Activation, and Degranulation in Dahl Hypertensive Rats
Authors: Shen K. , DeLano F. A. , Zweifach B. W. , Schmid-Schönbein G. W.
Published: June 2012

Abstract
Previous evidence has shown that rats with spontaneous hypertension have on average about twice as many circulating leukocytes in comparison with their normotensive counterparts, the Wistar-Kyoto rats. Since such high levels of leukocytes may increase the risk for vascular complications for hypertensive animals, it is useful to ascertain whether a comparable derangement is present in other forms of hypertension. The present study deals with the properties of the circulating leukocytes in rats exhibiting another form of experimental hypertension; Dahl salt-sensitive (Dahl-S) hypertensive rats were compared with Dahl salt-resistant (Dahl-R) control rats. Measurements were performed to determine the following: circulating hematocrit levels, leukocyte counts, differential counts, number of activated leukocytes (by means of nitro blue tetrazolium [NBT] reduction), leukocyte adhesion in vitro and neutrophil CD-18 expression, alkaline phosphatase activity in individual neutrophils and in the plasma, and myeloperoxidase activity in neutrophils. The experimental cohort consisted of Dahl-S and Dahl-R rats maintained for a 6-week period on a 6% NaCl diet. The results show a highly significant elevation in the number of total leukocytes, neutrophil and monocyte counts, and NBT-positive neutrophils and monocytes in Dahl-S but not Dahl-R rats. There was a significant loss of alkaline phosphatase and myeloperoxidase activity in the neutrophils of the salt-treated Dahl-S rats but not in the neutrophils of the untreated Dahl-S or Dahl-R rats. No significant differences were found in neutrophil adhesion under in vitro test conditions between the two strains maintained on the salt diet. In salt-treated Dahl-S rats, there was a reduced expression of β
2
integrins (CD-18) in neutrophils, although a higher fraction of the circulating neutrophils exhibited above-threshold CD-18 expression. These results indicate an increased number of circulating neutrophils and monocytes in Dahl-S rats that are in an activated state and undergo spontaneous degranulation in the circulation without exhibiting elevated levels of membrane adhesive properties. Such circulating leukocyte kinetics may contribute to a range of vascular complications in the hypertensive rats.
Electrophysiological Effects of 4-Hydroxynonenal, an Aldehydic Product of Lipid Peroxidation, on Isolated Rat Ventricular Myocytes
Authors: Bhatnagar Aruni
Published: June 2012

Abstract
Aldehydic products of lipid peroxidation, such as 4-hydroxynonenal (4-HNE), have been implicated in the etiology of pathological changes under oxidative stress. To identify the mechanism by which 4-HNE alters cellular excitability, its effects on isolated rat ventricular myocytes were studied. Superfusion with 100 to 880 μmol/L 4-HNE led to a time- and concentration-dependent rigor shortening of myocytes. A reduction in [Ca
2+
]
o
and inhibition of transsarcolemmal Ca
2+
transport by 1 mmol/L La
3+
did not affect either the magnitude or the time course of 4-HNE–induced myocyte rigor. Superfusion of myocytes with 400 μmol/L 4-HNE led to an increase in the action potential duration, progressive depolarization of the resting membrane potential, and an increase in the input resistance (R
in
) of the myocyte (phase I), followed by a loss of electrical excitability. Continued superfusion with 4-HNE resulted in membrane hyperpolarization and a prominent decrease in the R
in
(phase II). The decrease in R
in
coincided with myocyte rigor. In whole-cell voltage-clamp experiments, superfusion with 4-HNE inhibited current through the inward rectifier K
+
channel (I
K1
). 4-HNE had no effect on either the magnitude or the rate of “rundown” of L-type Ca
2+
currents. Exposure to 4-HNE led to an increase in the magnitude of the fast inward Na
+
current (I
Na
). The voltage dependence of the steady state parameters for activation and inactivation of I
Na
shifted to more positive potentials, with a resultant increase in the window current. 4-HNE–induced myocyte rigor was accompanied by a large increase in time-independent currents that displayed linear dependence on the membrane potential and were inhibited by glibenclamide, suggesting activation of the ATP-sensitive K
+
channel. Steady state currents recorded in Cs
+
-containing Ringer’s solution with La
3+
and tetrodotoxin and Cs
+
-containing internal solution (leak currents) were not affected by 4-HNE. Superfusion with 4-HNE resulted in a significant decrease in the cellular concentration of nonprotein thiols and a severe decrease in [ATP]
i
. The energy charge of the myocytes fell from 0.9 to 0.3. These observations indicate that 4-HNE–induced membrane depolarization may be due to an inhibition of I
K1
. Changes in voltage dependence of I
Na
, inhibition of I
K1
, and membrane depolarization appear to contribute to the prolongation of the action potential, observed during phase I. Depletion of [ATP]
i
may be responsible for changes observed during phase II, ie, activation of the ATP-sensitive K
+
channels, membrane hyperpolarization, decrease in R
in
, and rigor shortening of the myocytes. These results suggest that stable products of lipid peroxidation, such as 4-HNE, are proarrhythmic and may contribute to the cytotoxic effects of oxidative stress.
Cloning and Functional Expression of an Inwardly Rectifying K
+
Channel From Human Atrium
Authors: Wible Barbara A. , De Biasi Mariella , Majumder Kumud , Taglialatela Maurizio , Brown Arthur M.
Published: June 2012

Abstract
The cardiac inward rectifier current (I
K1
) contributes to the shape and duration of the cardiac action potential and helps to set the resting membrane potential. Although several inwardly rectifying K
+
channels (IRKs) from different tissues have been cloned recently, the nature and number of K
+
channels contributing to the cardiac I
K1
are presently unknown. To address this issue in human heart, we have used the reverse-transcriptase–polymerase chain reaction (PCR) technique with human atrial total RNA as a template to identify two sequences expressed in heart that are homologous to previously cloned IRKs. One of the PCR products we obtained was virtually identical to IRK1 (cloned from a mouse macrophage cell line); the other, which we named hIRK, exhibited <70% identity to IRK1. A full-length clone encoding hIRK was isolated from a human atrial cDNA library and functionally expressed in
Xenopus
oocytes. This channel, like IRK1, exhibited strong inward rectification and was blocked by divalent cations. However, hIRK differed from IRK1 at the single-channel level: hIRK had a single-channel conductance of 36 pS compared with 21 pS for IRK1. We have identified single channels of 41, 35, 21, and 9 pS in recordings from dispersed human atrial myocytes. However, none of these atrial inward rectifiers exhibited single-channel properties exactly like those of cloned hIRK expressed in oocytes. Our findings suggest that the cardiac I
K1
in human atrial myocytes is composed of multiple inwardly rectifying channels distinguishable on the basis of single-channel conductance, each of which may be the product of a different gene.
Essential Role for Nitric Oxide in Neurogenic Inflammation in Rat Cutaneous Microcirculation
Authors: Kajekar Radhika , Moore Phillip K. , Brain Susan D.
Published: June 2012

Abstract
The possible modulatory role of nitric oxide (NO) in neurogenic edema formation in rat paw skin, induced by electrical stimulation of the saphenous nerve, was investigated by using two NO synthase inhibitors,
N
G
-nitro-
l
-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI). Both L-NAME (100 mg/kg IV,
P
<.05) and 7-NI (10 mg/kg IV,
P
<.05) caused an
l
-arginine (100 mg/kg IV,
P
<.01)–reversible inhibition of neurogenic edema as measured by
125
I-albumin accumulation, whereas D-NAME (inactive enantiomer of L-NAME) and 6-aminoindazole (structurally similar to 7-NI) were without inhibitory effect. L-NAME produced the predicted vasopressor effect (before, 115±18 mm Hg; 5 minutes after, 174±18 mm Hg; n=6;
P
<.05), whereas 7-NI showed no significant increase in blood pressure (before, 96±9 mm Hg; 5 minutes after, 102±10 mm Hg; n=6), and neither L-NAME nor 7-NI had any effect on basal or vasodilator calcitonin gene-related peptide (CGRP, 10 pmol per site)–stimulated local blood flow in rat skin, as measured by laser Doppler flowmetry. Furthermore, systemic and local 7-NI had no effect on edema formation induced by local administration of substance P (with or without CGRP) and histamine (with or without CGRP) in rat skin. Since 7-NI blocks edema produced by stimulation of the saphenous nerve, it is suggested that release of NO is involved in neurogenic edema formation, but the vasodilator action of NO is unimportant in this context. We suggest that NO is involved in the release of neuropeptides from sensory nerves.
Porcine Aortic Smooth Muscle Cells Secrete a Serine Protease for Insulin-like Growth Factor Binding Protein-2
Authors: Gockerman Amy , Clemmons David R.
Published: June 2012

Abstract
Porcine aortic smooth muscle cells secrete two forms of insulin-like growth factor (IGF) binding proteins (IGFBP-2 and -4), and both forms have been shown to modulate IGF-I actions in this cell type. Recently, we showed that IGFBP-4 inhibited IGF-I action and that the cells produced a protease that cleaved IGFBP-4 into non-IGF binding fragments. After the cleavage of IGFBP-4, the cellular DNA synthesis response to IGF-I was enhanced. This study reports that these cells also secrete a protease for IGFBP-2. Like the IGFBP-4 protease, this protease is also secreted constitutively, but unlike the IGFBP-4 protease, its secretion is enhanced if the cells are serum-deprived for 24 hours before the collection of conditioned medium. The protease cleaved IGFBP-2 into 25- and 16-kD fragments, which had reduced IGF-I binding activity. Protease activity was enhanced by coincubation with IGF-I or IGF-II, and IGF-II was more potent than IGF-I. The protease is a serine protease, since its activity can be inhibited by 3,4-dichloroisocoumarin and aprotinin. It is also inhibited by EDTA, and its activity can be restored with calcium but not zinc. The heparin-binding serpins, specifically, heparin cofactor II and antithrombin III, are inhibitory. Heparin alone also had activity, and the combination of antithrombin III plus heparin caused complete inhibition. The conditioned medium also contained proteolytic activities for IGFBP-4 and -5 but it did not cleave IGFBP-1 and -3. Chromatography of the conditioned medium on heparin-sepharose indicated that the IGFBP-2 protease bound very weakly to heparin, since it was eluted with 0.2 mol/L NaCl. This contrasts with the IGFBP-5 protease activity, which required 2.0 mol/L NaCl to elute most of the activity. α
1
-Antichymotrypsin inhibited the IGFBP-2 protease, but it had no effect on the IGFBP-5 protease. Exposure to other growth factors such as transforming growth factor-β, fibroblast growth factor, or platelet-derived growth factor did not alter protease activity. In summary, porcine aortic smooth muscle cells secrete a serine protease for IGFBP-2. This protease cleaves IGFBP-2 into two fragments that have reduced IGF binding, and its activity is enhanced by prior serum deprivation of the cells. The protease has the potential to significantly modify IGF actions in this cell type.
Effects of Second Messengers on the Permeability and Morphology of Eel Rete Capillaries
Authors: Rasio Eugenio A. , Bendayan Moı̈se , Goresky Carl A.
Published: June 2012

Abstract
The effects of second-messenger concentration changes on capillary diffusion capacity (permeability–surface area product [PS]) to cellular and paracellular tracers and on capillary ultrastructure were studied during countercurrent perfusion of the rete of the eel swim bladder. Cyclic nucleotide effects were investigated with isoproterenol, forskolin, and dibutyryl cAMP. Isoproterenol (5×10
−6
mol/L) did not modify water and solute permeability or capillary structure. Forskolin (10
−4
mol/L) immediately raised the concentrations of cAMP in the rete and produced interstitial edema but did not change permeability. The addition of dibutyryl cAMP (10
−6
mol/L) to the perfusate had rapid effects: it reduced the PS of [
3
H]water and oxygen and increased the PS of [
125
I]albumin, [
14
C]sucrose, and
22
Na. No structural changes were observed. Phosphoinositide effects were studied with 1,2-dioctanoyl-
sn
-glycerol (DG) and phorbol 12-myristate 13-acetate (PMA). DG (10
−5
mol/L) had no effect on the permeability of the rete to water and solutes, while inducing cell membrane vacuolization. PMA (10
−5
mol/L) progressively reduced the PS of [
3
H]water. In contrast, PS values of [
125
I]albumin, [
14
C]sucrose, and
22
Na rose gradually. Membrane vacuoles bulging into the lumen and in the cytoplasm were a common feature. The Ca
2+
effect was investigated with the Ca
2+
ionophore A23187. At 5×10
−6
mol/L, unsteady permeability changes and extensive cytolysis were observed. At 5×10
−7
mol/L, the PS of [
125
I]albumin, [
14
C]sucrose, and
22
Na rapidly increased. The PS values for water were not modified. No structural changes were identified. It is concluded that increments of second-messenger concentrations in the rete induce characteristic selective effects on the paracellular and transcellular pathways of transport and create significant but nonselective alterations of capillary structure.
Human Cardiac Troponin T: Cloning and Expression of New Isoforms in the Normal and Failing Heart
Authors: Mesnard Laurence , Logeart Damien , Taviaux Sylvie , Diriong Sylvie , Mercadier Jean-Jacques , Samson Françoise
Published: June 2012

Abstract
Troponin T, like many myofibrillar proteins, exists as multiple isoforms encoded by distinct genes or generated by splicing of the same primary RNA transcript. We have previously cloned the first human cardiac troponin T (cTnT) cDNA and showed the differential expression of cTnT in cardiac and skeletal muscle during ontogenic development. In this work we located the human cTnT gene by means of fluorescent in situ hybridization to 1q32 and, by sequencing thirteen cDNAs isolated from a human fetal heart cDNA library, identified three new isoforms resulting from specific combinations of three variable regions in human cTnT cDNA. The first variable region is a 30-bp box located at the 5′ end of the cDNA, which can be excised either totally or only from the first 3 bp onwards; the second is a codon which can be completely excised; and the third is a 9-bp box in the 3′ half of the cDNA, which can also be excised either totally or only from the first 3 bp. The existence of the corresponding RNAs in fetal and adult ventricles was confirmed by RNase protection studies. No accumulation of the fetal isoforms was found in failing ventricles compared with controls.
Cardiac Muscarinic Potassium Channel Activity Is Attenuated by Inhibitors of G
βγ
Authors: Nair Lawrence A. , Inglese James , Stoffel Robert , Koch Walter J. , Lefkowitz Robert J. , Kwatra Madan M. , Grant Augustus O.
Published: June 2012

Abstract
The cardiac muscarinic potassium channel (I
K.ACh
) is activated by a G protein upon receptor stimulation with acetylcholine. The G protein subunit responsible for activation (G
α
versus G
βγ
) has been disputed. We used G
βγ
inhibitors derived from the β-adrenergic kinase 1 (βARK1) to assess the relative importance of G
βγ
in I
K.ACh
activation. In rabbit atrial myocytes, I
K.ACh
had a conductance of 49±6.2 pS. In inside-out patches, the mean open time was 1.60±0.57 ms, mean time constant (τ
o
) was 1.59±0.53 ms, and mean closed time was 3.02±1.35 ms (n=38). βARK1 is a G
βγ
-sensitive enzyme that interacts with G
βγ
through a defined sequence near its carboxyl terminus. A 28-amino-acid peptide derived from the carboxyl terminus of βARK1 (peptide G) increased the closed time to 10.04 ms (
P
<.001) and decreased opening probability (N
P
o
) by 71% (
P
<.001). Fusion proteins containing the entire carboxyl terminus of βARK1, glutathione
S
-transferase βARK1ct and hexahistidine βARK1ct, decreased N
P
o
by 67% (
P
=.03) and 48% (
P
=.009), respectively. They also both significantly increased the closed time. None of the inhibitors affected mean open time or channel amplitude. A control peptide derived from a neighboring region of βARK1 had no significant effect on I
K.ACh
activity. These results provide further evidence for the role of G
βγ
in the activation of I
K.ACh
.
Endothelin-Mediated Positive Inotropic Effect Induced by Reactive Oxygen Species in Isolated Cardiac Muscle
Authors: De Keulenaer Gilles W. , Andries Luc J. , Sys Stanislas U. , Brutsaert Dirk L.
Published: June 2012

Abstract
Cardiac endothelium, both coronary and endocardial, produces a number of inotropic molecules. Changes in cardiac endothelial function by substances in the superfusing blood may thus participate in the control of muscle-pump performance of the heart. Reactive oxygen species (ROS) have been implicated in normal and pathological vascular physiology by influencing vascular endothelial function. Therefore, we examined the influence of ROS on endocardial endothelial modulation of myocardial performance. Right ventricular cat papillary muscles were briefly (15 s) exposed to electrolysis-generated ROS. Peak total isometric twitch tension and peak rate of tension development increased by 7.8±0.7% (
P
<.05) and 9.7±1.5% (
P
<.05), respectively (n=12). Isometric twitch duration was slightly increased (time from stimulus to half isometric relaxation, +2.7±0.6%;
P
<.05). ROS scavengers such as ascorbic acid (n=6), superoxide dismutase and catalase (n=8), or catalase alone (n=6), but not superoxide dismutase alone (n=6), blocked the inotropic effect. Interestingly, the positive inotropic effect was completely blocked by selectively damaging endocardial endothelium (Triton X-100, 0.5%, 1-s immersion, n=7) before ROS generation and by preincubating the muscles with the endothelin-A receptor antagonist BQ 123 (n=11). Preincubation with
N
G
-nitro-
l
-arginine methyl ester and indomethacin (n=5) or with atenolol (n=6) did not influence the inotropic effect. Confocal scanning laser microscopic observations of muscles stained with viability tracers (n=9) revealed that significantly more but not all endocardial endothelial cells were damaged in electrolysis-treated muscles than in control muscles (42±5% versus 14±4%,
P
<.05). Accordingly, brief exposure of isolated cardiac muscle to electrolysis-generated ROS damaged the endocardial surface in part and increased contractile performance by stimulating endothelin release from endocardial endothelium. Hence, ROS-induced endothelin release from endocardial endothelium may be involved in normal and/or disturbed regulation of cardiac function.
Role of Calcium-Activated Neutral Protease (Calpain) in Cell Death in Cultured Neonatal Rat Cardiomyocytes During Metabolic Inhibition
Authors: Atsma Douwe E. , Bastiaanse E.M. Lars , Jerzewski Anastazia , Van der Valk Lizet J.M. , Van der Laarse Arnoud
Published: June 2012

Abstract
Calcium-activated neutral protease (CANP), also known as calpain, has been implicated in the development of cell death in ischemic hearts. CANP is thought to be activated by the calcium overload that develops during ischemia. We studied the involvement of CANP in cell death in cultured neonatal rat cardiomyocytes during metabolic inhibition (5 mmol/L NaCN+10 mmol/L 2-deoxyglucose). First, we isolated CANP using ion exchange and affinity chromatography. Then the efficacy of the CANP inhibitors calpain I inhibitor, leupeptin, and E64 to inhibit isolated CANP activity was tested with the use of fluorescently labeled β-casein as a substrate. The IC
50
for the inhibitors was between 2.1 and 56 μmol/L. Uptake of the inhibitors by intact cells was assessed with the use of
99m
Tc-radiolabeled inhibitors. The calculated intracellular inhibitor concentrations were sufficiently high to yield substantial inhibition of intracellular CANP activity. Intracellular CANP activity was measured directly with the use of the cell-permeant fluorogenic CANP-specific substrate
N
-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl-coumarin. During metabolic inhibition, intracellular CANP activity was increased compared with control incubation. The time course of CANP activation was compatible with that of the rise in [Ca
2+
]
i
, as measured by fura 2 and digital imaging fluorescence microscopy. Calpain I inhibitor and leupeptin inhibited intracellular CANP activity both during metabolic inhibition and control incubation, whereas E64 did not. Despite their substantial inhibition of intracellular CANP activity, calpain I inhibitor and leupeptin did not attenuate cell death during metabolic inhibition. We therefore conclude that intracellular CANP in cardiomyocytes is activated during metabolic inhibition, but it does not play a major role in the development of cell death.
Adenosine-Recruitable Flow Reserve Is Absent During Myocardial Ischemia in Unanesthetized Dogs Studied in the Basal State
Authors: Canty John M. , Smith Thomas P.
Published: June 2012

Abstract
We conducted the present study to determine if pharmacologically recruitable flow reserve was present during ischemia in unanesthetized dogs studied in the absence of adrenergic activation. Microsphere measurements of regional perfusion at reduced coronary pressures in a distal circumflex region subjected to intracoronary adenosine infusion (0.265 mg/min IC) were compared with measurements in a proximal circumflex region where pressure was reduced but flow was being autoregulated normally. Ischemia began when coronary pressure was reduced below 40 mm Hg. At a coronary pressure of 28±1 (mean±SEM) mm Hg, subendocardial flow with autoregulation intact was 0.59±0.05 mL · min · g
−1
and similar to that in the region receiving adenosine, which averaged 0.61±0.05 mL · min
−1
· g
−1
(
P
=NS). Although adenosine increased subepicardial flow from 0.81±0.05 to 1.16±0.09 mL · min
−1
· g
−1
(
P
<.001), the magnitude of the increase was minimal when coronary pressure fell to a level that caused subepicardial flow during autoregulation to be reduced below resting values. These results demonstrate that the substantially lower autoregulatory break point found in unanesthetized dogs studied in the basal state reflects the ability of intrinsic autoregulatory mechanisms to match local vasodilator reserve to that recruitable pharmacologically. This contrasts with the presence of pharmacologically recruitable subendocardial flow reserve in anesthetized animals and is most likely related to a low level of circulating catecholamines and lack of sympathetic activation in unanesthetized animals studied under basal conditions.
Inhibition of Vascular Smooth Muscle Cell Growth Through Antisense Transcription of a Rat Insulin-Like Growth Factor I Receptor cDNA
Authors: Du Jie , Delafontaine Patrick
Published: June 2012

Abstract
Insulin-like growth factor I (IGF I) is an autocrine/paracrine growth factor that is produced in multiple tissues and is essential for normal developmental growth. Its effects are mediated by activation of a membrane-bound tyrosine kinase receptor, IGF IR. On the basis of the partial rat IGF IR α-chain cDNA sequence previously reported, we cloned cDNA encoding the full-length rat IGF IR. The deduced amino acid sequence predicts a 1370–amino acid receptor precursor, which includes signal sequence, a 707–amino acid α-chain, a 4-Arg cleavage site, and a 629–amino acid β-chain. Overall, similarity to human IGF IR is 89% and 98% at the nucleotide and amino acid levels, respectively. Antisense IGF IR expression constructs in vectors incorporating Epstein-Barr virus replicative signals and the cytomegalovirus promoter/enhancer or the inducible human metallothionein IIa promoter/enhancer were assembled and stably transfected into cultured rat aortic smooth muscle cells. Clone CA9 (constitutively expressing abundant antisense IGF IR transcripts), clones MA5 and MA7 (expressing antisense IGF IR transcripts inducibly), and clones ME8 and ME10 (expressing vector alone) were characterized. There was a 57% reduction in IGF IR mRNA levels in clone CA9 after confluence compared with clone ME10. This resulted in a 51% decrease in IGF I binding sites in clone CA9, without a change in binding affinity (
K
d
), and a 55% and 57% reduction in DNA synthesis rates, basally and in response to 10 ng/mL IGF I, respectively. Clones MA5/MA7 similarly showed a 54% reduction in IGF IR number after confluence following exposure to 100 μmol/L ZnSO
4
and a 44% and 58% reduction in DNA synthesis, basally and in response to 10 ng/mL IGF I, respectively. Growth curves indicated that proliferation of clone CA9 in the presence of 10% serum was reduced by 60% compared with clone ME10. Thus, cloning of cDNA encoding the full-length rat IGF IR indicates that this receptor is highly conserved. Antisense targeting of this receptor in vascular smooth muscle cells (VSMCs) demonstrates that a decrease in IGF IR density results in marked inhibition of VSMC proliferation. These findings indicate an important role for this ligand-receptor system in regulating VSMC growth. Specifically, they suggest that modulation of VSMC IGF IR density may be an important mechanism whereby growth of these cells is controlled.
Nongenomic Effects of Aldosterone on Intracellular Ca
2+
in Vascular Smooth Muscle Cells
Authors: Wehling Martin , Neylon Craig B. , Fullerton Meryl , Bobik Alex , Funder John W.
Published: June 2012

Abstract
Genomic mechanisms of steroid action have been increasingly elucidated over the past four decades. In contrast, rapid steroid actions have been widely recognized only recently, and detailed analysis of the mechanisms involved are still lacking. The present article describes rapid effects of mineralocorticoid hormones on free intracellular calcium in vascular smooth muscle cells as determined by fura 2 spectrofluorometry in single cultured cells from rat aorta. These effects are almost immediate and reach a plateau after only 3 to 5 minutes and are characterized by high specificity for mineralocorticoids versus glucocorticoids. The potent mineralocorticoids aldosterone and fludrocortisone are agonists with estimated apparent EC
50
values of ≈0.1 to 0.5 nmol/L; deoxycorticosterone acetate is an agonist with an EC
50
of ≈5 nmol/L; and progesterone, cortisol, corticosterone, and estradiol have much lower potency (EC
50
values of ≈0.5 to 5 μmol/L). The effect of aldosterone is blocked by neomycin and short-term treatment with phorbol esters but augmented by staurosporine, indicating an involvement of phospholipase C and protein kinase C. The Ca
2+
effect appears to involve the release of intracellular Ca
2+
, as shown by the inhibitory effect of thapsigargin; intriguingly, a relatively small maximum effect (≈40 nmol/L increase) is consistently seen. This mechanism operates at physiological subnanomolar aldosterone concentrations and appears to be a likely candidate for rapid fine tuning of cardiovascular responsivity. It may also contribute to known clinical features of mineralocorticoid action that are difficult to explain by the traditional genomic mechanism alone.
Effects of Levosimendan, a Cardiotonic Agent Targeted to Troponin C, on Cardiac Function and on Phosphorylation and Ca
2+
Sensitivity of Cardiac Myofibrils and Sarcoplasmic Reticulum in Guinea Pig Heart
Authors: Edes Istvan , Kiss Eva , Kitada Yoshimi , Powers Frances M. , Papp Julius G. , Kranias Evangelia G. , Solaro R. John
Published: June 2012

Abstract
A new cardiotonic agent, (
R
)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-phenyl] hydrazono]propanedinitrile (Levosimendan), has been developed and screened for its ability to bind to cardiac troponin C. In perfused hearts, low concentrations of 0.03 or 0.1 μmol/L Levosimendan increased +dP/dt, but did not affect the speed of relaxation and produced only a slight increase in spontaneous heart rate in the hearts perfused with 0.1 μmol/L of the drug. In these same hearts, perfusion with 0.03 μmol/L Levosimendan did not alter the
32
P incorporation into troponin I or C protein, whereas a slight but significant increase was noted for phospholamban, with no detectable change in tissue cAMP levels. Administration of 0.1 or 0.3 μmol/L Levosimendan significantly increased myocardial cAMP levels as well as the phosphorylation of phospholamban, troponin I, and C protein. Levosimendan (0.03 to 10 μmol/L) reversibly increased force generated by detergent-extracted fiber bundles over a range of submaximally activating free Ca
2+
concentrations with no significant effect on maximum force or on Ca
2+
binding to myofilament troponin C. There was no direct effect of Levosimendan on Ca
2+
uptake by vesicles of sarcoplasmic reticulum (SR). In contrast, under conditions optimal for cAMP-dependent phosphorylation, Levosimendan slightly but significantly lowered the concentration of Ca
2+
, yielding half-maximal uptake rates by the SR vesicles. Our results indicate that at low concentrations Levosimendan acts preferably as a Ca
2+
sensitizer, whereas at higher concentrations its action as a phosphodiesterase inhibitor contributes to the positive inotropic effect.
Acetylcholine-Sensitive Intracellular Ca
2+
Store in Fresh Endothelial Cells and Evidence for Ryanodine Receptors
Authors: Wang Xiaodong , Lau Frankie , Li Li , Yoshikawa Akiyoshi , van Breemen Cornelis
Published: June 2012

Abstract
In a freshly isolated endothelial cell preparation from rabbit aorta, the regulation of the acetylcholine (ACh)–sensitive intracellular Ca
2+
store and the effects of the Ca
2+
-induced Ca
2+
release agonists ryanodine and caffeine were studied using fura 2 imaging fluorescence microscopy. ACh (10 μmol/L) caused a transient release of Ca
2+
from an intracellular store, presumably via an inositol tris-phosphate–sensitive mechanism. This ACh response could be repeated in the presence of extracellular Ca
2+
but was obtained only once in Ca
2+
-free bathing solution, which shows that a depleted intracellular Ca
2+
store can be rapidly refilled from the extracellular space. Refilling can be prevented by the endoplasmic reticulum Ca
2+
-ATPase inhibitor cyclopiazonic acid (10 μmol/L), implying that Ca
2+
enters the cytoplasm before accumulation in the endoplasmic reticulum. Ionomycin (10 μmol/L) caused a large Ca
2+
release even after the ACh-releasable store had been emptied, indicating the existence of other ACh-insensitive stores, perhaps including the mitochondria. In one third of the cells studied, ACh induced oscillations in [Ca
2+
]
i
that were dependent on extracellular Ca
2+
. Also investigated were the effects of caffeine and ryanodine. In this cell preparation neither caffeine nor ryanodine induced a Ca
2+
transient but instead slowly increased [Ca
2+
]
i
. It was observed that both caffeine and ryanodine were able to slowly deplete the ACh-sensitive store. These results indicate the presence of functional ryanodine receptors in native endothelial cells and demonstrate overlap between the caffeine and agonist-sensitive Ca
2+
stores. We also found that caffeine was able to directly inhibit the process of ACh-induced Ca
2+
release. It is hypothesized that endothelial endoplasmic reticulum contains both inositol tris-phosphate receptors and ryanodine receptors but that the former class are more densely distributed.
Apoptosis of Rat Vascular Smooth Muscle Cells Is Regulated by p53-Dependent and -Independent Pathways
Authors: Bennett M. R. , Evan G. I. , Schwartz S. M.
Published: June 2012

Abstract
Apoptosis of vascular smooth muscle cells has recently been described in culture and also in remodeling of the artery after birth. However, the genes that regulate apoptosis in smooth muscle cells are mostly unknown. We studied the regulation of apoptosis in rat smooth muscle cells stably infected with retrovirus constructs containing c-
myc
, adenovirus E1A,
bcl
-
2
, and a temperature-sensitive mutant of the tumor suppressor gene p53. Apoptosis was verified by electron microscopy and quantified by time-lapse videomicroscopy. Death was induced by c-
myc
and E1A when cells were deprived of serum survival factors.
bcl
-
2
suppressed apoptosis of cells infected with c-
myc
and E1A and also normal smooth muscle cells. Overexpression of wild-type p53 induced apoptosis of cells infected with E1A and c-
myc
but not normal cells. In contrast, expression of mutant p53, which blocks wild-type p53 function, suppressed apoptosis of cells infected with E1A or c-
myc
but not normal cells. Both adenovirus E1A and c-
myc
increased the expression of endogenous p53 protein but not p53 mRNA. Although
bcl
-
2
suppressed apoptosis induced by E1A and c-
myc
, upregulation of p53 protein induced by these agents was unaffected. We conclude that apoptosis of vascular smooth muscle cells is regulated by p53-dependent and -independent pathways. Death induced by c-
myc
and E1A is mediated by, and dependent on, p53. However, the suppression of apoptosis by
bcl
-
2
is not mediated by changes in p53 expression, and the low level of apoptosis seen in normal VSMCs upon removal of survival factors is independent of p53.
Pulmonary Microvascular Permeability
Authors: Townsley Mary I. , Fu Zhenxing , Mathieu-Costello Odile , West John B.
Published: June 2012

Abstract
The pressure threshold for injury of pulmonary capillaries is ≈50 to 55 cm H
2
O in the canine lung, as measured by changes in the filtration coefficient (
K
f,c
). Since the pulmonary endothelial basement membrane has been observed to thicken in patients with heart failure and pulmonary venous hypertension, we hypothesized that both baseline permeability and the threshold for high-vascular-pressure injury would be altered as a result. Dogs (n=12) were chronically paced at 245 beats per minute for ≈4 weeks, then were paced at 225 beats per minute for an additional 3 weeks. Lung lobes from anesthetized paced dogs and additional control dogs (n=14) were then isolated, ventilated, and perfused with blood. Although vascular resistance was increased nearly threefold and vascular compliance reduced by 50% in the paced group,
K
f,c
referenced to 1 g blood-free dry weight was no different from control. Despite this lack of difference at normal pulmonary vascular pressures, several significant results were obtained. First, in the paced group there was a significant increase in the threshold for high-vascular-pressure injury:
K
f,c
measured at pulmonary vascular pressures commonly seen in heart failure (20 to 50 cm H
2
O) were significantly less in this group compared with control. Model predictions showed that in vivo, this difference in
K
f,c
would result in a 50% reduction in the amount of water and protein cleared across the pulmonary capillary endothelial barrier in the paced group. Next, challenge with high vascular pressure resulted in less accumulation of residual blood in this group compared with control. Finally, in separate groups of animals, morphometric analysis of the alveolocapillary barrier showed that after either 4 or 7 to 8 weeks of pacing, endothelial, interstitial, and epithelial thicknesses were increased compared with control. Together, these results suggest that vascular remodeling confers a modest but important increase in the resistance of these lungs to high-vascular-pressure injury and the development of alveolar edema.
Glibenclamide, an ATP-Sensitive K
+
Channel Blocker, Inhibits Cardiac cAMP-Activated Cl
−
Conductance
Authors: Tominaga Makoto , Horie Minoru , Sasayama Shigetake , Okada Yasunobu
Published: June 2012

Abstract
Stimulation of the β-adrenoceptor activates a time-independent Cl
−
conductance that is known to be regulated via phosphorylation by cAMP-dependent protein kinase in guinea pig ventricular myocytes. Since epithelial cystic fibrosis transmembrane conductance regulator Cl
−
channels are known to be sensitive to an antidiabetic sulfonylurea, glibenclamide, we tested whether the drug modulates cardiac cAMP-activated Cl
−
conductance. Bath application of isoproterenol (1 μmol/L, n=11) or forskolin (1 μmol/L, n=17) or the intracellular application of cAMP (1 mmol/L, n=9) activated whole-cell Cl
−
currents recorded from single myocytes at 36°C. External glibenclamide (≥10 μmol/L, n=26) inhibited the Cl
−
current induced by either of the stimulants in a concentration-dependent manner. The half-maximal inhibition concentration (IC
50
) of glibenclamide and the Hill coefficient were 24.5 to 37.9 μmol/L and 1.6 to 2.2, respectively. During current-clamp experiments, forskolin was found to shorten the action potential significantly (250±45 to 201±52 milliseconds,
P
<.05) in 7 of 11 cells tested. Glibenclamide antagonized the forskolin-induced shortening (to 243±54 milliseconds, n=7,
P
<.05). Intracellular administration of sodium orthovanadate (0.5 to ≈1 mmol/L, n=6) brought about persistent activation of Cl
−
current after brief bath application of forskolin. This Cl
−
current was not affected by H-89 (100 μmol/L, n=3), a specific inhibitor of cAMP-dependent protein kinase, and was suppressed by glibenclamide similarly, with an IC
50
of 29.7 μmol/L. Thus, it is concluded that glibenclamide inhibits cardiac cAMP-activated Cl
−
channels at some step(s) downstream from the phosphorylation/dephosphorylation process.
β-Adrenergic Stimulation Induces Acetylcholine to Activate ATP-Sensitive K
+
Current in Cat Atrial Myocytes
Authors: Wang Yong Gao , Lipsius Stephen L.
Published: June 2012

Abstract
Our previous work on atrial myocytes suggested that the effect of acetylcholine (ACh) to increase K
+
conductance can be potentiated by prior loading of the sarcoplasmic reticulum (SR) with Ca
2+
. The present study, therefore, sought to determine whether prior exposure to isoproterenol (ISO) potentiates ACh-induced increases in K
+
conductance and the underlying mechanisms. A nystatin–perforated patch whole-cell configuration was used to record from cat atrial myocytes. Voltage-clamp ramps (40 mV/s) were used to assess total membrane conductance. The experimental protocol consisted of two consecutive 30-second ACh exposures (ACh
1
and ACh
2
) separated by a 6-minute recovery period in ACh-free solution. In general, experimental interventions, such as exposure to ISO, were imposed during the period between ACh
1
and ACh
2
to determine their effects on the response to ACh
2
in relation to ACh
1
. Under control conditions, K
+
conductances induced by ACh
1
and ACh
2
were not different from one another with or without activation of L-type Ca
2+
current (I
Ca,L
) during the recovery period. When 1 μmol/L ISO plus I
Ca,L
activation was imposed during the recovery period, ACh
2
induced a significantly larger increase in K
+
conductance than ACh
1
. The ACh
2
-induced K
+
current potentiated by ISO was time independent and selectively blocked by 10 μmol/L glibenclamide and therefore identified as ATP-sensitive K
+
current (I
K,ATP
). The effect of ISO to induce ACh
2
-activated I
K,ATP
was mimicked by 1 μmol/L forskolin or 200 μmol/L 8-(4-chlorophenylthio)-cAMP, but not by 0.5 μmol/L BAY K 8644, and was selectively abolished by (1) 5 μmol/L thapsigargin or 1 μmol/L ryanodine, agents that prevent accumulation of SR Ca
2+
, (2) inhibition of L-type Ca
2+
current (I
Ca,L
) by 1 μmol/L nisoldipine or zero external Ca
2+
, (3) 50 μmol/L Rp-cAMPs, an inhibitor of cAMP-dependent protein kinase A, or (4) 2 μmol/L propranolol. Atropine (1 μmol/L) abolished all ACh-induced currents. Moreover, ACh
2
-activated I
K,ATP
was selectively blocked by 0.2 μmol/L pirenzepine, an M
1
muscarinic receptor antagonist, or 0.1 μmol/L calphostin C, a selective inhibitor of protein kinase C. AFDX116 (100 μmol/L), an M
2
muscarinic receptor antagonist, blocked the conventional ACh-activated K
+
current and revealed ACh
2
-activated I
K,ATP
. These results indicate that prior exposure to ISO potentiates ACh-induced increases in K
+
current via ACh-activated I
K,ATP
. ISO acts via β-adrenergic receptors and cAMP to enhance both Ca
2+
influx via I
Ca,L
and SR Ca
2+
uptake. By loading SR Ca
2+
, ISO facilitates ACh-induced stimulation of the M
1
receptor/phosphoinositol signaling pathway to activate I
K,ATP
. These findings suggest that prior β-adrenergic stimulation accentuates subsequent cholinergic inhibition of atrial function via ACh-induced activation of ATP-sensitive K
+
channels.
Osteopontin and β
3
Integrin Are Coordinately Expressed in Regenerating Endothelium In Vivo and Stimulate Arg-Gly-Asp–Dependent Endothelial Migration In Vitro
Authors: Liaw Lucy , Lindner Volkhard , Schwartz Stephen M. , Chambers Ann F. , Giachelli Cecilia M.
Published: June 2012

Abstract
Osteopontin is an Arg-Gly-Asp (RGD)–containing acidic glycoprotein postulated to mediate cellular adhesion and migration in a growing number of normal and pathological conditions through interaction with integrin molecules. In this report, we have investigated the potential contributions of osteopontin and one of its receptors, the α
v
β
3
integrin, to endothelial regenerative processes by using both in vivo and in vitro models. In vivo, uninjured rat arterial endothelium had undetectable levels of osteopontin and β
3
-integrin mRNA by in situ hybridization. After balloon catheter denudation, osteopontin mRNA levels correlated temporally and spatially with active endothelial proliferation and migration, with the highest levels observed at the wound edge between 8 hours and 2 weeks after injury, declining to uninjured levels at 6 weeks, when regeneration was complete. Osteopontin protein levels, as determined by immunocytochemistry, paralleled the time course of mRNA expression. Likewise, β
3
-integrin mRNA and protein levels were substantially elevated in regenerating endothelial cells but were not detectable in uninjured or healed endothelium. In vitro, rat smooth muscle cell–derived and bacterial expressed mouse recombinant osteopontins both stimulated the adhesion and directed migration of bovine aortic endothelial cells through interactions with the α
v
β
3
receptor. Structural mutants of osteopontin confirmed the importance of the RGD domain for both adhesion and migration of endothelial cells through α
v
β
3
. These data suggest important roles for osteopontin and β
3
integrin in regenerating endothelium.
Inhibitory Effects of Insulin on Cytosolic Ca
2+
Level and Contraction in the Rat Aorta
Authors: Han Shu-Zhong , Ouchi Yasuyoshi , Karaki Hideaki , Orimo Hajime
Published: June 2012

Abstract
To determine the mechanism of the inhibitory effect of insulin on vascular tone, contraction was measured simultaneously with endothelial and smooth muscle cytosolic Ca
2+
level ([Ca
2+
]
i
) in the isolated rat aorta. Insulin (200 mU/mL) increased endothelial [Ca
2+
]
i
and decreased resting muscle tone. The removal of endothelium abolished the effects of insulin. In the aorta precontracted with norepinephrine, insulin (3 to 120 mU/mL) induced concentration-dependent inhibition of contraction. The relaxant effect followed the increase in endothelial [Ca
2+
]
i
and decrease in smooth muscle [Ca
2+
]
i
. The relaxant effect was attenuated by removal of endothelium or by the addition of 10
−5
mol/L
N
G
-monomethyl-
l
-arginine but not by 10
−5
mol/L indomethacin. In the absence of endothelium, the relaxant effect of insulin followed the decrease in smooth muscle [Ca
2+
]
i
. These results suggest that insulin inhibits vascular contraction by dual mechanisms in the isolated rat aorta: (1) Insulin acts on vascular endothelium by increasing endothelial [Ca
2+
]
i
and releasing NO, which decreases smooth muscle [Ca
2+
]
i
and the Ca
2+
sensitivity of the contractile elements. (2) Insulin also directly acts on smooth muscle and decreases smooth muscle [Ca
2+
]
i
.