bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidant Stress Enhances Adenylyl Cyclase Activation
Authors: Tan C.M. , Xenoyannis S. , Feldman R.D.
Published: June 2012

Abstract
The generation of oxygen-derived free radicals has been implicated in the disordered vascular regulation of inflammation and reperfusion. In the vasculature, oxygen-derived free radicals are vasodilatory. The mechanisms underlying this effect remain unclear. To examine the cellular processes involved, we studied the effects of hydrogen peroxide (H
2
O
2
) on adenylyl cyclase activity in A10 cells, a murine vascular smooth muscle cell line. Pretreatment with H
2
O
2
caused a dose-dependent enhancement of forskolin-stimulated adenylyl cyclase activity (ED
50
, 44 μmol/L to a maximum of 166% of control activity; n=4). This enhancement was attenuated by iron chelation with deferoxamine and by the intracellular hydroxyl scavenger dimethylthiourea and mimicked by preincubation with purine/xanthine oxidase either alone or in the presence of superoxide dismutase. The effects of H
2
O
2
were completely blocked by the tyrosine kinase inhibitors genistein and tyrphostin A9 but not by its inactive analogue tyrphostin A1 (H
2
O
2
alone, 149±13%; H
2
O
2
+tyrphostin A9, 100±9%; H
2
O
2
+tyrphostin A1, 171±21%; n=4). H
2
O
2
comparably enhanced adenylyl cyclase activity stimulated by isoproterenol (166±17% of control, n=5) and sodium fluoride (177±18% of control, n=5). Thus oxygen-derived free radicals enhance adenylyl cyclase activation, probably via tyrosine kinase–mediated effects on the catalytic subunit of adenylyl cyclase. Sensitization of adenylyl cyclase activation may be an important mechanism by which free radicals modulate hormone-mediated vasodilation.
Effect of Mechanical Forces on Growth and Matrix Protein Synthesis in the In Vitro Pulmonary Artery
Authors: Kolpakov Valeri , Rekhter Mark D. , Gordon David , Wang Wei Hua , Kulik Thomas J.
Published: June 2012

Abstract
The effect of mechanical stimuli on pulmonary artery growth and matrix tissue synthesis (and how individual cell types in the vessel wall respond to such stimuli) is incompletely characterized. Rabbit pulmonary arteries were placed in tissue culture medium and subjected to varying magnitudes of stretch or hydrostatic pressure (separately) for 4 days. The rate of protein synthesis in smooth muscle cells (by quantitative autoradiography) was positively related to the magnitude of stretch, as were the percentage of procollagen type I–positive cells and the rate of cell replication. In adventitial fibroblasts, stretch increased the rate of replication but not of protein synthesis. Hydrostatic pressure had little or no effect on the variables measured in either smooth muscle cells or fibroblasts. Stretch also increased the rate of elastin and collagen synthesis in the whole pulmonary artery segment, and after 4 days of stretch, the contents of actin and elastin were increased. Removal of the endothelium did not affect stretch-induced protein, collagen, or elastin synthesis but augmented stretch-induced smooth muscle replication. These data suggest that in the intact pulmonary artery, stretch, but not pressure, can stimulate hypertrophy and hyperplasia in smooth muscle cells and hyperplasia in fibroblasts. Matrix protein synthesis and accumulation are also increased by stretch. Neither stretch-mediated growth nor matrix protein synthesis required endothelium in this model.
Endogenous and Exogenous Nitric Oxide Inhibits Norepinephrine Release From Rat Heart Sympathetic Nerves
Authors: Schwarz Petra , Diem Ricarda , Dun Nae J. , Förstermann Ulrich
Published: June 2012

Abstract
This study was designed to elucidate whether nitric oxide (NO) controls norepinephrine (NE) release from sympathetic nerves of the rat heart. Hearts were perfused in the Langendorff mode with Tyrode’s solution. The right sympathetic nerve was stimulated with trains of 1 or 3 Hz and NE release was measured. The NO synthase (NOS) inhibitor
N
G
-nitro-
l
-arginine (L-NNA) enhanced the evoked NE release in a concentration-dependent manner. This facilitation was independent of the increase in perfusion pressure and was stereospecifically reversed by
l
-arginine but not
d
-arginine. Another NOS inhibitor,
N
G
-methyl-
l
-arginine, produced a similar increase in NE release. The NO-donor compound
S
-nitroso-
N
-acetyl-
d,l
-penicillamine, added in the presence of L-NNA, restored the suppression of NE release in a concentration-dependent fashion. A similar suppression was achieved with 3-morpholinosydnonimine. These results demonstrated that NE release is under the inhibitory control of endogenous NO. Western blots demonstrated the presence of neuronal NOS I and endothelial NOS III in the hearts. Perfusion of the hearts with a low concentration of the detergent CHAPS produced functional damage of the endothelium, as evidenced by an increase in perfusion pressure and a conversion of the acetylcholine-induced coronary vasodilation to a constriction. However, CHAPS treatment did not produce a facilitation of NE release (as did the NOS inhibitors), and L-NNA still increased NE release in CHAPS-treated hearts. Double-labeling immunofluorescence histochemistry showed NOS I immunoreactivity in stellate ganglion cells and in neurons of the heart, some of which also stained positive for tyrosine hydroxylase. These findings suggest that NO, predominantly generated in neurons, controls NE release in the rat heart.
Divergent Mechanisms of ATP-Sensitive K
+
Channel–Induced Vasodilation in Renal Afferent and Efferent Arterioles
Authors: Reslerova Martina , Loutzenhiser Rodger
Published: June 2012

Abstract
K
+
channel openers (PCOs), such as pinacidil, elicit vasodilation primarily by hyperpolarization-induced inhibition of L-type Ca
2+
channel activation. The physiological role of other mechanisms suggested to contribute to PCO-induced vasodilation is not well established. In the renal microcirculation, L-type Ca
2+
channels play a prominent role in vasoconstriction of the afferent arteriole (AA) but are absent or physiologically silent in the efferent arteriole (EA). Thus, L-type Ca
2+
channel–dependent and –independent mechanisms can readily be distinguished in this model. In the present study, we found that pinacidil potently inhibited Bay K 8644–induced AA vasoconstriction. Pinacidil also preferentially inhibited angiotensin II–induced AA vasoconstriction (approximately ninefold greater potency than EA). These results are consistent with an AA effect of pinacidil on L-type Ca
2+
channel activation. Unexpectedly, 10 μmol/L pinacidil inhibited AA and EA responses to similar extents (84±10% and 71±9%, respectively). In both AAs and EAs, glibenclamide restored normal reactivity, indicating an involvement of the ATP-sensitive K
+
channels. In the EA, however, pretreatment with diltiazem did not alter the effects of pinacidil. Nevertheless, 45 mmol/L KCl reversed the EA actions of pinacidil, indicating an essential requirement for a normal K
+
gradient. These findings suggest that the EA actions of pinacidil involve alterations in membrane potential but not changes in L-type Ca
2+
channel activity. Overall, our findings do support the premise that L-type Ca
2+
channel modulation is involved in PCO-induced vasodilation in the renal microcirculation. The EA actions of pinacidil, however, suggest important additional vasodilatory mechanisms that also involve ATP-sensitive K
+
channel–induced hyperpolarization but are independent of L-type Ca
2+
channel modulation.
α
v
β
3
Integrin Expression in Normal and Atherosclerotic Artery
Authors: Hoshiga Masaaki , Alpers Charles E. , Smith Laura L. , Giachelli Cecilia M. , Schwartz Stephen M.
Published: June 2012

Abstract
Recent evidence suggests that α
v
β
3
integrin is a critical molecule in several processes involved in atherosclerosis progression and in restenosis, eg, smooth muscle cell (SMC) migration and angiogenesis. While several ligands for this integrin are known to be present in atherosclerotic plaque, little is known about the presence of α
v
β
3
integrin at this site. In the present study, we have examined α
v
β
3
expression in normal and atherosclerotic arteries. Thirty-six coronary artery segments from the recipient hearts of 24 patients undergoing heart transplantation were classified into two groups: nonatherosclerotic diffuse intimal thickening (DIT) and atherosclerotic plaques. Serial frozen sections were examined immunohistochemically with four different monoclonal antibodies directed against human α
v
β
3
complex or the β
3
subunit and with cell markers for SMCs, macrophages, and endothelial cells. The endothelium along the lumen of both DIT and plaque arteries showed high expression of α
v
β
3
. The media of both DIT and plaque arteries showed less intense but extensive expression of α
v
β
3
. Immunoprecipitation and reverse-transcribed polymerase chain reaction (RT-PCR) analyses performed on extracts from the aortic media confirmed the presence of α
v
β
3
in the media. In the intima of both DIT and plaque arteries, α
v
β
3
expression generally colocalized with SMCs but rarely with macrophages. The microvessels in the adventitia as well as in the plaque showed prominent expression of α
v
β
3
, in contrast to low expression in similar-sized microvessels of the skin. These results suggest that α
v
β
3
is present both in the normal artery and in sites of SMC accumulation and angiogenesis in atherosclerotic plaques.
Fractal
15
O-Labeled Water Washout From the Heart
Authors: Bassingthwaighte James B. , Beard Daniel A.
Published: June 2012

Abstract
To characterize the washout of water from the heart, we used a flow-limited (not diffusion- or permeability-limited) marker for blood-tissue exchange, namely, tracer-labeled water. Experiments were performed by injecting
15
O-labeled water into the inflow to isolated blood-perfused rabbit hearts and by recording the tracer content in the heart and in the outflow simultaneously for up to 5 minutes. The data exhibit a particular combination of power law forms: (1) The downslopes of the residue and outflow curves were both power law functions, with the residue diminishing as
t
−α
and the outflow as
t
−α−1
, where α is interpreted to be the dimensionless exponent of a fractal power law relation characterizing the self-similarity inherent in each curve. (2) The fractional escape rate, given by the outflow curve divided by the residue curve, diminished almost exactly as
t
−1
. In 18 sets of curves, α averaged 2.21±0.27. These results lead to an improved method for extrapolating the downslopes of indicator dilution curves to estimate their areas and therefore the blood flows. The evidence also points strongly to the conclusions that myocardial water washout is a fractal process and that stirred tank models are inappropriate for the heart.
The Development of the Atrioventricular Junction in the Human Heart
Authors: Wessels A. , Markman M.W.M. , Vermeulen J.L.M. , Anderson R.H. , Moorman A.F.M. , Lamers W.H.
Published: June 2012

Abstract
The histogenesis of the separation between atrial and ventricular myocardium at the atrioventricular junction in the developing human heart has been investigated immunohistochemically by using monoclonal antibodies specific for atrioventricular cushion tissue, mesenchymal cells, atrial and ventricular myocardium, and myocardium of the primary ring. It was found that the insulation between the muscle masses of atrium and ventricle is established by the fusion of the tissues of the atrioventricular sulcus (located at the epicardial side of the junctional myocardium) with those of the atrioventricular cushions (located at the endocardial side of the junctional myocardium). This process takes place at the ventricular margin of the myocardium of the atrioventricular canal. The separation of atrial and ventricular myocardium starts at ≈7 weeks of development in the anteromedial portion of the right atrioventricular junction and is largely completed around the 12th week of development. The only remaining myocardial continuity between atrial and ventricular myocardium is the atrioventricular axis of conduction. Our findings show that the nonmuscular part of the developing leaflets of the atrioventricular valves derives from the atrioventricular cushions and that the tissues of the atrioventricular groove do not contribute to the development of these leaflets.
High and Low Molecular Weight Fibroblast Growth Factor-2 Increase Proliferation of Neonatal Rat Cardiac Myocytes but Have Differential Effects on Binucleation and Nuclear Morphology
Authors: Pasumarthi Kishore B.S. , Kardami Elissavet , Cattini Peter A.
Published: June 2012

Abstract
Basic fibroblast growth factor (FGF-2) plays a vital role in the growth and differentiation of cardiac myocytes. It exists in high and low molecular weight forms because of the use of alternative initiation codons in the same mRNA. Higher levels of high molecular weight forms (molecular mass of 22 and 21.5 kD) are present in the rat heart during the neonatal stage, whereas the low molecular weight form (molecular mass of 18 kD) is predominant in the adult heart, suggesting different roles in development. Rat FGF-2 cDNAs that can preferentially express high or low molecular weight forms were introduced into neonatal rat ventricular myocyte cultures. Significant and comparable increases in overall cardiac myocyte DNA synthesis and proliferation were seen with 22/21.5- and 18-kD FGF-2 expression. A significantly higher mitotic index was seen in the vicinity of cardiac myocytes overexpressing high or low molecular weight forms of FGF-2 compared with nonoverexpressing cells. This increase was inhibited in the presence of neutralizing antibodies to FGF-2, pointing to a proximity-dependent paracrine effect of 22/21.5- and 18-kD FGF-2 on mitosis. By contrast, overexpression of high but not low molecular weight FGF-2 was associated with a significant increase in binucleation (≈36% of cardiac myocytes overexpressing 22/21.5-kD FGF-2 were binucleated compared with 9% of cardiac myocytes overexpressing 18-kD FGF-2), which was not affected by neutralizing antibodies to FGF-2. These results suggest that 22/21.5-kD FGF-2 and 18-kD FGF-2 have similar paracrine effects on proliferation but that 22-21.5-kD FGF-2 exerts a distinct intracrine effect on binucleation.
Developmental Changes in Ionic Channel Activity in the Embryonic Murine Heart
Authors: Davies M.P. , An R.H. , Doevendans P. , Kubalak S. , Chien K.R. , Kass R.S.
Published: June 2012

Abstract
We have isolated murine embryonic atrial and ventricular cells derived from timed-pregnant females at different periods and used patch-clamp procedures to investigate age- and chamber-specific expression of ionic channels in the developing fetal mouse. Our data indicate that L-type Ca
2+
channels play a dominant role in excitation during early murine cardiac embryogenesis and that Na
+
channel expression increases dramatically just before birth. K
+
channel expression is particularly sensitive to changes during development. Neither atrial nor ventricular cells express a slowly activating component of delayed rectification (I
Ks
) until just before birth, and inwardly rectifying channel activity, associated with determination of cellular resting potential, is not markedly apparent until late stages of embryogenesis. Instead, we find robust expression of the ATP-regulated K
+
channel at early and late stages of embryonic development, which may indicate a novel functional role for this channel during morphogenesis of the heart. These results have important implications for the physiology and development of the murine cardiac conduction system and will also serve as a baseline for future studies designed to investigate developmental changes of ion channel expression in the myocardium of both wild-type and genetically modified mice.
Relation Between the Sarcolemmal Ca
2+
Current and Ca
2+
Sparks and Local Control Theories for Cardiac Excitation-Contraction Coupling
Authors: Santana L.F. , Cheng H. , Gómez A.M. , Cannell M.B. , Lederer W.J.
Published: June 2012

Abstract
Ca
2+
sparks, the elementary events underlying excitation-contraction (E-C) coupling, occur when sarcoplasmic reticulum (SR) Ca
2+
release channels open. They are activated locally by Ca
2+
influx through sarcolemmal (SL) Ca
2+
channels. By measuring the probability of spark occurrence under conditions in which their probability of occurrence is low, we address two important questions raised by our recent work: (1) When a Ca
2+
spark is triggered, how many SL Ca
2+
channels (at a minimum) contribute to its activation? (2) What is the relation between the subcellular local [Ca
2+
]
i
produced by the opening of SL Ca
2+
channels and the consequent SR Ca
2+
release? By comparing the voltage dependence of Ca
2+
sparks in rat ventricular myocytes with the Ca
2+
current, we show that the opening of a single SL Ca
2+
channel can trigger a Ca
2+
spark. Furthermore, we deduce that the probability of SR Ca
2+
release depends of the square of the local [Ca
2+
]
i
produced by SL Ca
2+
channel openings. These results are discussed with respect to the properties of Ca
2+
-induced Ca
2+
release (CICR) and the local control theory of excitation-contraction coupling.
Distribution and Physiological Roles of ATP-Sensitive K
+
Channels in the Vertebrobasilar System of the Rabbit
Authors: Nagao Tetsuhiko , Ibayashi Setsuro , Sadoshima Seizo , Fujii Koji , Fujii Kenichiro , Ohya Yusuke , Fujishima Masatoshi
Published: June 2012

Abstract
The effect of an opener (levcromakalim) and a blocker (glibenclamide) of ATP-sensitive K
+
(K
ATP
) channels was investigated in the vertebrobasilar system of the rabbit. Arterial tension and membrane potential were measured by the isometric tension recording method and the microelectrode technique, respectively. Glibenclamide (10
−6
mol/L) depolarized the membrane and potentiated the contraction to histamine in vertebral arteries. The sensitivity to the relaxant effects of levcromakalim was in the following descending order: vertebral>proximal basilar>distal basilar>superior cerebellar arteries. Vertebral arteries were ≈50 times more sensitive to levcromakalim than were superior cerebellar arteries. The relaxation to levcromakalim was abolished by glibenclamide (10
−6
mol/L). Glibenclamide attenuated vasorelaxation to adenosine in proximal arteries (vertebral and proximal basilar) but not in superior cerebellar arteries. Levcromakalim (7×10
−8
mol/L) and adenosine (10
−5
mol/L) induced glibenclamide-sensitive membrane hyperpolarization in vertebral arteries but not in distal basilar arteries. These results suggest that K
ATP
channels contribute to the determination of resting membrane potential and resting tone in vertebral arteries. Furthermore, there is a marked heterogeneity in the sensitivity to an opener of K
ATP
channels, and the heterogeneity has a functional link to the mechanism underlying vasorelaxation to adenosine in the vertebrobasilar system of the rabbit.
Multiple Domains Contribute to the Distinct Inactivation Properties of Human Heart and Skeletal Muscle Na
+
Channels
Authors: Makita Naomasa , Bennett Paul B. , George Alfred L.
Published: June 2012

Abstract
Voltage-gated Na
+
channels are essential for the normal electrical excitability of neuronal and striated muscle membranes. Distinct isoforms of the Na
+
channel α-subunit have been identified by molecular cloning, and their functional attributes have been defined by heterologous expression coupled with electrophysiological recording. Two closely related Na
+
channel α-subunit isoforms, hH1 (human heart) and hSkM1 (human skeletal muscle), exhibit differences in their inactivation properties and in their response to the coexpressed β
1
-subunit. To localize regions that contribute to inactivation and to β
1
-subunit response, we have exploited these functional differences by studying chimeric channels composed of segments from both hH1 and hSkM1. Chimeras in which one or more of the cytoplasmic interdomain regions (ID1-2, ID2-3, and ID3-4) were exchanged between hH1 and hSkM1 exhibit inactivation properties identical with the background channel isoform, suggesting that these regions are not sufficient to cause gating differences. In contrast, inactivation properties of chimeras composed of approximately equal halves of the two channel isoforms were intermediate between hH1 and hSkM1. Furthermore, the response to the coexpressed β
1
-subunit was dependent on structures located in the carboxy-terminal half of the α-subunit, although domains D3, D4, and the carboxy terminal are not singularly responsible for this effect. These data indicate that inactivation differences between hH1 and hSkM1 are determined by multiple α-subunit domains.
EJ-Ras Inhibits Phospholipase C
γ1
but Not Actin Polymerization Induced by Platelet-Derived Growth Factor-BB via Phosphatidylinositol 3-Kinase
Authors: Heldman Alan W. , Kandzari David E. , Tucker Robert W. , Crawford Lawrence E. , Fearon Eric R. , Koblan Kenneth S. , Goldschmidt-Clermont Pascal J.
Published: June 2012

Abstract
Transformation of fibroblast-like cells (NIH 3T3) by a constitutively activated GTP-bound isoform of p21
ras
(EJ-Ras) produces morphogenic changes characterized by decreased attachment to the substratum, with retraction and rounding of the cell body. Transformed fibroblasts lose their “stressed” conformation and adopt a “relaxed” morphology. The specific molecular mechanisms responsible for these changes remain uncharacterized. We found that EJ-Ras transformation of NIH 3T3 cells decreased the cellular content of polymerized actin, particularly at the expense of actin stress fibers, but induced the accumulation of actin filaments in peripheral ruffling membranes. Polymerization of actin could be induced in EJ-Ras–transformed cells by exposure to platelet-derived growth factor (PDGF)-BB to an extent similar to that observed in wild-type NIH 3T3 cells. In EJ-Ras cells, actin polymerization was independent of phospholipase C
γ1
(PLCγ1) activity, because inositol tris-phosphate (IP
3
) production observed in control NIH 3T3 cells in response to PDGF-BB was absent. Although PDGF-BB did stimulate tyrosine phosphorylation of PLCγ1, the phospholipase was strongly inhibited by an inhibitory factor present in the cytoplasm of EJ-Ras–transformed cells. In addition, cytoplasmic extracts of EJ-Ras, but not of control cells, inhibited phosphatidylinositol 4,5-diphosphate (PIP
2
) hydrolysis catalyzed by a recombinant PLCγ1 in vitro. Although PIP
2
hydrolysis could not contribute to the reorganization of the actin cytoskeleton induced by PDGF-BB in EJ-Ras–transformed cells, phosphatidylinositol 3-kinase (PI3-K) was necessary for actin polymerization. Wortmannin, a specific PI3-K inhibitor, not only blocked actin polymerization in both control and EJ-Ras–transformed cells but actually led to rapid actin depolymerization when these cells were exposed to PDGF-BB. Thus, in EJ-Ras–transformed cells, cell morphogenic changes in response to PDGF-BB rely importantly on PI3-K and can occur in the complete absence of IP
3
production despite tyrosine phosphorylation of PLCγ1.
Identification of rab12 as a Secretory Granule Associated Small GTP-Binding Protein in Atrial Myocytes
Authors: Iida Hiroshi , Wang Luming , Nishii Kiyomasa , Ookuma Akiko , Shibata Yosaburo
Published: June 2012

Abstract
A subfamily of small GTP-binding proteins, rab, has been shown to be involved in regulation of vesicular traffic in eukaryotic cells. The goal of this study was to identify the rab proteins associated with atrial secretory granules. A [
32
P]GTP-overlay assay showed the presence of multiple small GTP-binding proteins on the atrial granules. By biochemical analysis, we have demonstrated that one of the small GTP-binding proteins associated with the atrial granules is a rab12 protein (rab12p), one of the rab proteins that are most closely related to a Sec4 protein of yeast. Association of rab12p with the atrial granules was confirmed by immunogold electron microscopy. Immunoprecipitation followed by immunoblot analysis with anti-rab12 antibody showed that in addition to atria, rab12p was expressed in multiple other organs and cell lines. These results suggest that rab12p may function in vesicular traffic in multiple diverse types of cells.
Developmental Changes in β-Adrenergic Modulation of L-Type Ca
2+
Channels in Embryonic Mouse Heart
Authors: An R.H. , Davies M.P. , Doevendans P.A. , Kubalak S.W. , Bangalore R. , Chien K.R. , Kass R.S.
Published: June 2012

Abstract
In the adult mammalian myocardium, cellular Ca
2+
entry is regulated by the sympathetic nervous system. L-type Ca
2+
channel currents are markedly increased by β-adrenergic (β-A) agonists, which contribute to changes in pacing and contractile activity of the heart. In the developing mammalian heart, the regulation of Ca
2+
entry by this enzyme cascade has not been clearly established, because changes in receptor density and coupling to downstream elements of the signaling cascade are known to occur during embryogenesis. In this study, we systematically investigated the regulation of L-type Ca
2+
channel currents during development of the murine embryonic heart. We used conventional whole-cell and perforated-patch–clamp procedures to study modulation of L-type Ca
2+
channel currents and to assay functional activity of distinct steps in the β-A signaling cascade in murine embryonic myocytes at different stages of gestation. Our data indicate that L-type Ca
2+
channels in early-stage (day-11 to -13) myocytes are unresponsive to either isoproterenol or cAMP. L-type Ca
2+
channels in late-stage (day-17 to -19) murine myocytes, however, exhibit responses to isoproterenol and cAMP similar to responses in adult cells, providing evidence that the β-A cascade becomes functionally active during this period of embryonic development. We found that L-type Ca
2+
channel activity in early-stage cells is increased by cell dialysis with the catalytic subunit of cAMP-dependent protein kinase (cA-PK) and that dialysis of early-stage cells with the holoenzyme of cA-PK restores functional responses to forskolin and cAMP, but not to isoproterenol. Our results provide strong evidence that a key factor in the early-stage insensitivity of L-type Ca
2+
channels to cAMP is the absence, or low expression level, of the holoenzyme of cA-PK but that in addition, another element in the signaling cascade upstream from adenylate cyclase is expressed at a nonfunctional level or is uncoupled from the cascade and thus contributes to L-type Ca
2+
channel insensitivity to β-A agonists in early stages of the developing murine heart.
Migration of Arterial Wall Cells
Authors: Reidy Michael A. , Irvin Colleen , Lindner Volkhard
Published: June 2012

Abstract
The expression of plasminogen activators and inhibitors was examined in denuded arteries. Within 5 days, smooth muscle cells (SMCs) on the luminal surface expressed the mRNA for tissue-type plasminogen activator (TPA), urokinase-type plasminogen activator (UPA), the receptor for UPA (UPAR), and plasminogen activator inhibitor type-1 (PAI-1). Similar results were seen after 8 days. Six weeks later, only TPA mRNA was still expressed by SMCs on the luminal surface. En face casein zymograms revealed a net fibrinolytic activity in areas covered with luminal SMCs. Reverse zymography showed no antifibrinolytic activity in these zones. Quiescent endothelial cells did not express TPA, UPA, UPAR, or PAI-1 mRNA. Regenerating endothelium at the wound edge strongly expressed TPA, UPA, and UPAR, as well as PAI-1. UPA and UPAR expression was highly restricted to cells at the wound edge and was not present elsewhere. En face zymography showed no plasmin activity in endothelialized areas, and reverse zymography showed a net antifibrinolytic activity in endothelialized zones. These results suggest that plasminogen activator and inhibitor expression correlates with the migration of both SMCs and endothelial cells into an arterial wound.
Synergistic Modulation of ATP-Sensitive K
+
Currents by Protein Kinase C and Adenosine
Authors: Liu Yongge , Dong Gao Wei , O’Rourke Brian , Marban Eduardo
Published: June 2012

Abstract
Ischemic preconditioning has been shown to involve the activation of adenosine receptors, protein kinase C (PKC), and ATP-sensitive K
+
(K
ATP
) channels. We investigated the effects of PKC activation and adenosine on K
ATP
current (I
K,ATP
) and action potentials in isolated rabbit ventricular myocytes. Responses to pinacidil (100 to 400 μmol/L), an opener of K
ATP
channels, were markedly increased by preexposure to the PKC activator phorbol 12-myristate 13-acetate (PMA, 100 nmol/L). I
K,ATP
measured at 0 mV was increased by PMA pretreatment from 0.55±0.32 to 3.25±0.47 nA (n=6,
P
<.01). We next determined whether PKC activation abbreviates the time required to turn on I
K,ATP
during metabolic inhibition (MI). In control cells in which MI was induced by 2 mmol/L cyanide and 0 glucose, I
K,ATP
developed after an average of 15.1±2.4 minutes (n=8). Ten-minute pretreatment with PMA alone (PMA+MI) did not significantly alter this latency (11.9±2.0 minutes, n=8). Since adenosine receptor activation has been shown to play an important role in the preconditioning response, two groups of myocytes were studied with adenosine (10 μmol/L) included during MI. Without PMA, adenosine alone (MI+Ado) did not affect the latency to develop I
K,ATP
(12.3±1.5 minutes, n=8). However, if cells were pretreated with PMA and then subjected to MI in the presence of adenosine (PMA+MI+Ado), the latency was greatly shortened to 5.5±1.6 minutes (n=8;
P
<.02 versus MI, PMA+MI, and MI+Ado groups). This effect could not be reproduced by an inactive phorbol but was completely abolished by the adenosine receptor antagonist 8-(
p
-sulfophenyl)-theophylline. The opening of K
ATP
channels may be cardioprotective because of the abbreviation of action potential duration (APD) during ischemia. Therefore, we tested whether PKC activation could modify the time course of APD shortening during MI. Consistent with the ionic current measurements, PMA pretreatment significantly accelerated APD shortening, but only when adenosine (10 μmol/L) was included during MI. The effects were not attributable to accelerated ATP consumption: PMA pretreatment did not alter the time required to induce rigor during MI, whether or not adenosine was included. Our results indicate that PKC activation increases the I
K,ATP
induced by pinacidil or by MI. The latter effect requires concomitant adenosine receptor activation. The synergistic modulation of I
K,ATP
by PKC and adenosine provides an explicit basis for current paradigms of ischemic preconditioning.
Is a High Glycogen Content Beneficial or Detrimental to the Ischemic Rat Heart?
Authors: Cross Heather R. , Opie Lionel H. , Radda George K. , Clarke Kieran
Published: June 2012

Abstract
A high glycogen level may be beneficial to the ischemic heart by providing glycolytic ATP or detrimental by increasing intracellular lactate and protons. To determine the effects of high glycogen on the ischemic myocardium, the glycogen content of Langendorff-perfused rat hearts was either depleted or elevated before 32 minutes of low-flow (0.5 mL/min) ischemia with Krebs-Henseleit buffer with or without 11 mmol/L glucose, followed by 32 minutes of reperfusion with buffer containing 11 mmol/L glucose.
31
P nuclear magnetic resonance spectra were acquired sequentially throughout. Further experiments involved early reperfusion or the addition of HOE 694, a Na
+
-H
+
exchange inhibitor, during reperfusion. When glucose was supplied throughout ischemia, no ischemic contracture occurred, and postischemic recovery of contractile function was highest, at 88% of preischemic function. In the absence of glucose, normal-glycogen hearts underwent ischemic contracture at 5 minutes, had an end-ischemic pH of 6.87, and recovered to 54%, whereas in high-glycogen hearts, contracture was delayed to 13 minutes, the end-ischemic pH was 6.61, and functional recovery decreased to 13%. Contracture onset coincided with the decrease in glycolysis, which occurred as glycogen became fully depleted. Functional recovery in the high-glycogen hearts increased to 89% when reperfused before contracture and to 56% when reperfused in the presence of HOE 694. Thus, during brief ischemia in the high-glycogen hearts, ischemic glycogen depletion and contracture were avoided, and the hearts were protected from injury. In contrast, during prolonged ischemia in the high-glycogen hearts, glycogen became fully depleted, and myocardial injury occurred; the injury was exacerbated by the lower ischemic pH in these hearts, leading to increased Na
+
-H
+
exchange during reperfusion. The contradictory findings of past studies concerning the effect of high glycogen on the ischemic myocardium may thus be due to differences in the extent of glycogen depletion during ischemia.
Aging Does Not Affect the Activation of the Myocyte Insulin-Like Growth Factor-1 Autocrine System After Infarction and Ventricular Failure in Fischer 344 Rats
Authors: Cheng Wei , Reiss Krzysztof , Li Peng , Chun Micky J. , Kajstura Jan , Olivetti Giorgio , Anversa Piero
Published: June 2012

Abstract
To determine whether the attenuation in the growth capacity of myocytes in the overloaded aging heart is associated with an impairment in the activation of insulin-like growth factor-1 (IGF-1) and its receptor (IGF-1R) in the stressed cells, large myocardial infarcts were produced in Fischer 344 rats at 4 and 16 months of age, and the animals were killed 6 hours, 3 days, and 7 days later. After the documentation of cardiac failure, the unaffected myocytes were enzymatically dissociated, and the expression of IGF-1 and IGF-1R was measured at these three time points after surgery. The level of expression of IGF-1R mRNA increased at 3 days and remained elevated at 7 days in both age groups. In addition, an increase in IGF-1R protein in these cells was found, with no apparent difference with age. This phenomenon was coupled with an upregulation of IGF-1 mRNA of comparable magnitude in the younger and older animals. In contrast, the increases in the dimensional properties of myocytes were delayed and of smaller magnitude in the older infarcted rats. Moreover, the expression of atrial natriuretic factor, used as a molecular marker of myocyte cellular hypertrophy, was greater at 3 days in 4-month-old rats and at 7 days in 16-month-old rats. Thus, aging may affect the hypertrophic response of myocytes after infarction but has no impact on the ability of the cells to enhance the expression of IGF-1 and IGF-1R, which may sustain only in part the growth reserve mechanisms of the pathological heart.
Regulation of Endothelial Production of C-Type Natriuretic Peptide in Coculture With Vascular Smooth Muscle Cells
Authors: Komatsu Yasato , Itoh Hiroshi , Suga Shin-ichi , Ogawa Yoshihiro , Hama Norio , Kishimoto Ichiro , Nakagawa Osamu , Igaki Toshio , Doi Kentaro , Yoshimasa Takaaki , Nakao Kazuwa
Published: June 2012

Abstract
Recently, we have demonstrated that C-type natriuretic peptide (CNP) is produced in vascular endothelial cells (ECs). In the present study, we investigated the interaction of ECs and vascular smooth muscle cells (SMCs) for endothelial production of CNP and its action on vascular growth, using the EC/SMC coculture system. The concentration of CNP-like immunoreactivity in the medium was increased 60-fold within 48 hours in the EC/SMC coculture with direct contact compared with that in EC alone. Northern blot analysis revealed the augmented expression of CNP mRNA in the EC/SMC coculture. The accumulation of intracellular cGMP in the coculture was concomitantly increased, and this response was blocked by anti-CNP monoclonal antibody and HS-142-1, a nonpeptide atrial natriuretic peptide receptor antagonist. The concentration of biologically active transforming growth factor-β (TGF-β) in the culture medium of the coculture with direct contact of ECs and SMCs was elevated to the level to stimulate endothelial production of CNP. Actually, the neutralizing antibody against TGF-β abrogated the cGMP accumulation in the coculture. These results show that endothelial production of CNP in the EC/SMC coculture is at least in part regulated by TGF-β. Furthermore, the conditioned medium from ECs stimulated by TGF-β was demonstrated to have a growth-inhibitory effect on SMCs, which was abolished by anti-CNP monoclonal antibody and HS-142-1. The treatment with anti-CNP monoclonal antibody and HS-142-1 also significantly increased the cell number of the EC/SMC coculture. The present study reveals the pathophysiological significance of endothelial CNP as a paracrine/autocrine vascular regulator for vascular growth in the interaction of ECs and SMCs.
Endothelin-Dependent Actions in Cultured AT-1 Cardiac Myocytes
Authors: Jiang Tianrong , Pak Elena , Zhang HongLu , Kline Richard P. , Steinberg Susan F.
Published: June 2012

Abstract
The consequences of endothelin receptor activation were examined in atrial tumor myocytes derived from transgenic mice (AT-1 cells). Endothelin-1 (endothelin) stimulates phosphoinositide hydrolysis in a dose-dependent manner. Endothelin also induces the rapid and transient translocation of protein kinase C (PKC)-ε immunoreactivity from the soluble to the particulate cell fraction. The subcellular distributions of PKCα and PKCζ (also expressed by AT-1 cells) are not influenced by endothelin. Using quantitative fluorescence microscopy with fura 2, we examined the effects of endothelin on intracellular calcium. In electrically driven myocytes, endothelin induces a rapid and transient increase in the amplitude of the calcium transient. This is blocked by both phorbol 12-myristate 13-acetate (PMA) pretreatment to downregulate PKC and the PKC inhibitor chelerythrine, arguing that PKCε plays a critical role in endothelin receptor–dependent increases in intracellular calcium. Endothelin also stimulates mitogen-activated protein kinase (MAPK). MAPK activation is markedly attenuated by pretreatment with PMA or pertussis toxin (PTX, to inactivate susceptible G protein α subunits); it is completely prevented by combined pretreatment with PMA and PTX. In contrast, it is not attenuated by chelation of intracellular calcium with BAPTA. These findings indicate that the pathway for endothelin receptor stimulation of MAPK involves PKCε and PTX-sensitive G protein(s). Thus, these studies identify a functional role for PKCε as a mediator of endothelin receptor–dependent increases in cytosolic calcium and MAPK activity in AT-1 cells. Accordingly, the AT-1 cell system should provide a uniquely useful model to identify the intracellular targets for PKCε and investigate their function in the regulation of intracellular calcium homeostasis and the induction of the growth response in cardiac myocytes.
Salicylate or Aspirin Inhibits the Induction of the Inducible Nitric Oxide Synthase in Rat Cardiac Fibroblasts
Authors: Saeid Farivar R. , Chobanian Aram V. , Brecher Peter
Published: June 2012

Abstract
To determine if fibroblasts are a source of NO in inflammatory myocardial diseases, we have studied the effect of cytokines on the inducible NO synthase (iNOS) in neonatal cardiac fibroblasts and tested whether nonsteroidal anti-inflammatory drugs can diminish the induction of iNOS. In primary cultures, interferon gamma (IFN), interleukin-1β (IL-1), or tumor necrosis factor-α (TNF) separately did not stimulate nitrite production, whereas IFN combined with IL-1 or TNF synergistically induced iNOS, both at the level of steady state mRNA and nitrite accumulation. Steady state mRNA levels for iNOS were obvious as early as 3 hours after the addition of IFN+TNF and remained elevated for at least 72 hours. Sodium salicylate inhibited cytokine-induced nitrite accumulation in a time- and dose-dependent manner (IC
50
, 750 μmol/L). The inhibition was reversible and occurred when salicylate was added either before or after cytokine induction. Aspirin (1 mmol/L) also inhibited nitrite production, whereas indomethacin (25 μmol/L) or acetaminophen (100 μmol/L) did not. TNF, either alone or combined with IFN, significantly stimulated prostaglandin E
2
, which was inhibited by either salicylate (4 mmol/L) or indomethacin (25 μmol/L). Salicylate, when given either before or after IFN+TNF, reduced mRNA levels of iNOS induced by cytokines. Salicylate did not affect iNOS enzymatic activity when added to the cytosolic lysate, although it was able to reduce enzymatic activity to 32% of induced levels when given to intact cells. These studies implicate cardiac fibroblasts as a source of NO in inflammatory cardiac diseases and suggest a possible therapeutic role for salicylate and aspirin in diminishing the steady state levels of iNOS mRNA.
Developmental Remodeling of the Internal Elastic Lamina of Rabbit Arteries
Authors: Wong Lisa C. Y. , Langille B. Lowell
Published: June 2012

Abstract
We examined remodeling of the internal elastic lamina (IEL) of rabbit arteries from 3 to 23 weeks of age. The IELs were fenestrated at all ages; however, the sizes of the fenestrae increased dramatically during postnatal development. Mean areas occupied by the individual fenestrae of the carotid artery IEL increased from 11.3±0.7 μm
2
in 3-week-old rabbits to 61.2±5.5 μm
2
in adult rabbits. The estimated number of fenestrae per vessel also increased greatly, from 2.68×10
5
to 9.27×10
5
; however, the increased number of fenestrae did not keep pace with growth of the artery, since fenestrae per square millimeter decreased by 26%. Large increases in the size of fenestrae were also observed in the renal and iliac arteries, although greater decreases in fenestrae per square millimeter occurred with age (70% in iliac arteries). Morphological assessments suggested that enlarging fenestrae frequently fuse with neighbors. By contrast with other arteries, the IEL of the abdominal aorta was not a continuous fenestrated sheet in young animals, perhaps reflecting the extensive remodeling that this vessel undergoes in the postnatal period. We decreased common carotid blood flow by 70% in 5 rabbits at 10 weeks of age by ligating the ipsilateral external carotid artery, and we approximately doubled blood flow in 5 others at the same age, by contralateral common carotid ligation. At 15 weeks of age, fenestrae in the artery carrying increased flow were 39% larger than fenestrae in the control artery, whereas fenestrae were 53.5% smaller after 70% decreases in flow (
P
<.05). We conclude that flow-dependent enlargement of fenestrae contributes to developmental remodeling of the IEL. Remodeling of the IEL may also have important implications for transport of materials and cell-cell communication between the intima and media.
Multiple Mechanisms of Na
+
Channel– Linked Long-QT Syndrome
Authors: Dumaine Robert , Wang Qing , Keating Mark T. , Hartmann Hali A. , Schwartz Peter J. , Brown Arthur M. , Kirsch Glenn E.
Published: June 2012

Abstract
Inheritable long-QT syndrome (LQTS) is a disease in which delayed ventricular repolarization leads to cardiac arrhythmias and the possibility of sudden death. In the chromosome 3–linked disease, one mutation of the cardiac Na
+
channel gene results in a deletion of residues 1505 to 1507 (ΔKPQ), and two mutations result in substitutions (N1325S and R1644H). We compared all three mutant-channel phenotypes by heterologous expression in
Xenopus
oocytes. Each produced a late phase of inactivation-resistant, mexiletine- and tetrodotoxin-sensitive whole-cell currents, but the underlying mechanisms were different at the single-channel level. N1325S and R1644H showed dispersed reopenings after the initial transient, whereas ΔKPQ showed both dispersed reopenings and long-lasting bursts. Thus, two distinct biophysical defects underlie the in vitro phenotype of persistent current in Na
+
channel–linked LQTS, and the additive effects of both are responsible for making the ΔKPQ phenotype the most severe.
Unitary Cl
−
Channels Activated by Cytoplasmic Ca
2+
in Canine Ventricular Myocytes
Authors: Collier Mei Lin , Levesque Paul C. , Kenyon James L. , Hume Joseph R.
Published: June 2012

Abstract
Recent whole-cell studies have shown that Ca
2+
-activated Cl
−
currents contribute to the Ca
2+
-dependent 4-aminopyridine–insensitive component of the transient outward current and to the arrhythmogenic transient inward current in rabbit and canine cardiac cells. These Cl
−
-sensitive currents are activated by Ca
2+
release from the sarcoplasmic reticulum and are inhibited by anion transport blockers; however, the unitary single channels responsible have yet to be identified. We used inside-out patches from canine ventricular myocytes and conditions under which the only likely permeant ion is Cl
−
to identify 4-aminopyridine–resistant unitary Ca
2+
-activated Cl
−
channels. Ca
2+
applied to the cytoplasmic surface of membrane patches activated small-conductance (1.0 to 1.3 pS) channels. These channels were Cl
−
selective, with rectification properties that could be described by the Goldman-Hodgkin-Katz current equation. Channel activity exhibited time independence when cytoplasmic Ca
2+
was held constant and was blocked by the anion transport blockers, DIDS and niflumic acid. Ca
2+
(ranging from pCa ≥6 to pCa 3) applied to the cytoplasmic surface of inside-out patches increased, in a dose-dependent manner, NP
o
, where N is the number of channels opened and P
o
is open probability. At negative membrane potentials (−60 to −130 mV), an estimate of the dependence of NP
o
on cytoplasmic Ca
2+
yielded an apparent
K
d
of 150.2 μmol/L. At pCa 3, an average channel density of ≈3 μm
−2
was estimated. Calculations based on these estimates of cytoplasmic Ca
2+
sensitivity and channel current amplitude and density suggest that these small-conductance Cl
−
channels contribute significant whole-cell membrane current in response to changes in intracellular Ca
2+
within the physiological range. We suggest that these small-conductance Ca
2+
-activated Cl
−
channels underlie the transient Ca
2+
-activated 4-aminopyridine–insensitive current, which contributes to phase-1 repolarization, and under conditions of Ca
2+
overload, these channels may generate transient inward currents, contributing to the development of triggered cardiac arrhythmias.
α
1
-Adrenergic Inhibition of the β-Adrenergically Activated Cl
−
Current in Guinea Pig Ventricular Myocytes
Authors: Oleksa Lisa M. , Hool Livia C. , Harvey Robert D.
Published: June 2012

Abstract
α-Adrenergic receptor stimulation regulates the activity of a number of different cardiac ion channels, including those underlying one or more distinct Cl
−
conductances. The whole-cell patch-clamp technique was used in the present study to investigate the effects of α-adrenergic stimulation on the β-adrenergically regulated Cl
−
current in guinea pig ventricular myocytes. Neither α
1
-adrenergic receptor stimulation with methoxamine (25 to 500 μmol/L) nor direct activation of endogenous protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu, 100 nmol/L) evoked a Cl
−
current. On the contrary, the Cl
−
current activated by 30 nmol/L isoproterenol was inhibited by methoxamine, with an EC
50
of 6.7±2.6 μmol/L, and this response was blocked by prazosin, an α
1
-adrenergic receptor antagonist. Prazosin also decreased the EC
50
for current activation by norepinephrine from 53±7.1 to 18±3.8 nmol/L, demonstrating that the ability of this endogenous neurotransmitter to activate the Cl
−
current through β-adrenergic receptor stimulation is limited by its intrinsic ability to also activate α-adrenergic receptors. Methoxamine did not inhibit the Cl
−
current evoked by either direct activation of adenylate cyclase with forskolin or inhibition of phosphodiesterase activity with 3-isobutyl-1-methylxanthine, indicating that α-adrenergic stimulation inhibits β-adrenergic responses at a point upstream of adenylate cyclase activation. Methoxamine also did not inhibit the Cl
−
current activated by histamine, suggesting that α-adrenergic stimulation specifically inhibits β-adrenergic receptor–mediated responses. The inhibitory effect of methoxamine was not mimicked by PDBu, and it persisted in the presence of bisindolylmaleimide, a selective PKC inhibitor. However, methoxamine inhibition of the isoproterenol-activated Cl
−
current was sensitive to pertussis toxin. These results suggest that α-adrenergic receptor stimulation inhibits the β-adrenergically activated Cl
−
current, demonstrating a novel mechanism by which α-adrenergic receptors may regulate ion channel activity in the heart.