bluuebunny/crossref_metadata_2025 huggingface dataset:
Ketanserin inhibits depolarization-activated outward potassium current in rat ventricular myocytes.
Authors: Zhang Z H , Boutjdir M , el-Sherif N
Published: June 2012

Ketanserin (KT), an antihypertensive agent, has been shown to prolong action potential duration (APD) and QT interval and to induce torsade de pointes in some patients. We previously suggested that the prolongation of APD could be due to KT inhibition of the fast component of the delayed rectifier current (IKr) in guinea-pig myocytes. However, in other tissue such as human atrium, Purkinje fibers, epicardial cells, and rat ventricular myocytes, the transient outward potassium current (Ito) is one of the major repolarizing currents. We investigated the possibility that KT could also increase APD by blocking Ito. Action potentials and membrane currents were recorded from rat ventricular myocytes known to have a large Ito by using whole-cell patch-clamp techniques. We found that KT (50 mumol/L) significantly prolonged APD at 50% repolarization by 218% (P < .05) and APD at 90% repolarization by 256% (P < .05) with no significant effect on other action potential parameters. Time-dependent Ito and sustained current (ISus) were measured in the presence of 400 nmol/L nisoldipine during depolarizing pulses to 40 mV from a holding potential of -100 mV every 10 seconds. KT resulted in a concentration- and time-dependent inhibition of charge area of Ito evaluated by integration with an EC50 of 8.3 mumol/L. The inhibitory effect of KT (10 mumol/L) was seen at voltages from 0 to 80 mV without any shift of the current-voltage relation of peak Ito. KT did not significantly change activation, inactivation, and reactivating curves of Ito. Kinetic analysis of Ito showed a biexponential fit of inactivation in 80.5% of total tracings studied at voltages between -30 and 80 mV (n = 149, R = .99 +/- .01). The inhibitory effect of KT was more prominent on charge areas of the slow component (Qs) than the fast component (Qf) of Ito (Qf = 33.2 +/- 6.2 s.pA and Qs = 235.5 +/- 7.4 s.pA for the control condition; 12.4 +/- 4.3 and 59.6 +/- 17 s.pA for KT at 40 mV; n = 4). The binding association (k) and dissociation (l) constants at 40 mV were 9.0 +/- 0.9x10(6) M-1.s-1 and 86.6 +/- 0.3 s-1, respectively. KT also blocked ISus in a dose-dependent manner with an EC50 of 11.2 mumol/L and had no significant effect on both the inward rectifier potassium current and the L-type calcium current.(ABSTRACT TRUNCATED AT 400 WORDS)
Nonadrenergic noncholinergic nerves regulate basal coronary flow via release of capsaicin-sensitive neuropeptides in the rat heart.
Authors: Yaoita H , Sato E , Kawaguchi M , Saito T , Maehara K , Maruyama Y
Published: June 2012

Nonadrenergic noncholinergic nerve fibers supposedly modulate basal coronary flow by releasing capsaicin-sensitive neuropeptides, but the physiological effects of this intrinsic action have not been clarified. We investigated the intrinsic function of nonadrenergic noncholinergic innervation in modulating basal coronary flow in rats. We administered capsaicin to 44 rats to deplete neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P and administered inert vehicle to 60 control rats. Four days later, we measured the coronary pressure-flow relation in the basal state and during maximal coronary vasodilation induced by intracoronary adenosine administration using Langendorff's method. Changes in basal coronary flow prompted by intracoronary infusion of CGRP or substance P and their antagonists were measured in 54 and 30 rats, respectively. Capsaicin-treated rats showed a 31.5 +/- 0.9% (mean +/- SEM) reduction (P < .01) of basal coronary flow in the range of perfusion pressures between 60 and 140 mm Hg compared with untreated control rats, but the maximal coronary flow after adenosine was similar between the two groups. Although basal coronary flow was reduced in capsaicin-treated hearts, left ventricular contractile force and myocardial oxygen consumption did not fall significantly. CGRP increased the coronary flow, but substance P did not. CGRP(8-37), a CGRP receptor antagonist, reduced basal coronary flow by 24.5 +/- 2.1% (P < .01), but FK888, a substance P antagonist, had little effect on it. Thus, capsaicin-sensitive neuropeptides in the rat heart modulate basal coronary flow, providing approximately 30% of it.(ABSTRACT TRUNCATED AT 250 WORDS)
Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis.
Authors: Miano J M , Cserjesi P , Ligon K L , Periasamy M , Olson E N
Published: June 2012

We cloned a portion of the mouse smooth muscle myosin heavy chain (SM-MHC) cDNA and analyzed its mRNA expression in adult tissues, several cell lines, and developing mouse embryos to determine the suitability of the SM-MHC promoter as a tool for identifying smooth muscle-specific transcription factors and to define the spatial and temporal pattern of smooth muscle differentiation during mouse development. RNase protection assays showed SM-MHC mRNA in adult aorta, intestine, lung, stomach, and uterus, with little or no signal in brain, heart, kidney, liver, skeletal muscle, spleen, and testes. From an analysis of 14 different cell lines, including endothelial cells, fibroblasts, and rhabdomyosarcomas, we failed to detect any SM-MHC mRNA; all of the cell lines induced to differentiate also showed no detectable SM-MHC. In situ hybridization of staged mouse embryos first revealed SM-MHC transcripts in the early developing aorta at 10.5 days post coitum (dpc). No hybridization signal was demonstrated beyond the aorta and its arches until 12.5 to 13.5 dpc, when SM-MHC mRNA appeared in smooth muscle cells (SMCs) of the developing gut and lungs as well as peripheral blood vessels. By 17.5 dpc, SM-MHC transcripts had accumulated in esophagus, bladder, and ureters. Except for blood vessels, no SM-MHC transcripts were ever observed in developing brain, heart, or skeletal muscle. These results indicate that smooth muscle myogenesis begins by 10.5 days of embryonic development in the mouse and establish SM-MHC as a highly specific marker for the SMC lineage. The SM-MHC promoter should therefore serve as a useful model for defining the mechanisms that govern SMC transcription during development and disease.
Dissociation of vasoconstrictor-stimulated basic fibroblast growth factor expression from hypertrophic growth in cultured vascular smooth muscle cells. Relevant roles of protein kinase C.
Authors: Ali S , Becker M W , Davis M G , Dorn G W
Published: June 2012

Thromboxane A2 (TXA2) and angiotensin II (Ang II) stimulate vascular smooth muscle hypertrophy by upregulating endogenous synthesis of basic fibroblast growth factor (bFGF). Because mitogenic phorbol esters can also stimulate bFGF formation, we investigated the role of protein kinase C (PKC) in vascular smooth muscle cell (VSMC) bFGF formation and hypertrophy. Preliminary characterization of PKC isoform expression in VSMC by use of polymerase chain reaction identified PKC alpha, delta, epsilon, and zeta. Western analysis confirmed the presence of these isoforms in cultured VSMC lines and demonstrated downregulation of PKC alpha, delta, and epsilon by phorbol 12-myristate 13-acetate (PMA) but not TXA2 or Ang II. PKC activation with 100 nmol/L PMA stimulated VSMC mitogenesis measured as incorporation of [3H]leucine and [3H]thymidine and increased cell number. Like TXA2 and Ang II, PMA increased endogenous VSMC bFGF in a time-dependent manner, whereas an inactive phorbol ester had no such effect. Addition of an antisense oligodeoxynucleotide against bFGF prevented PMA-stimulated bFGF expression and inhibited PMA-stimulated growth, suggesting that bFGF synthesis is necessary for VSMC growth stimulated by PMA. To clarify the role of PKC in vasoconstrictor-stimulated VSMC production of bFGF and hypertrophy, PKC was down-regulated by prolonged exposure to PMA or was inhibited with calphostin C or staurosporine before the addition of TXA2 or Ang II. PKC inhibition prevented TXA2-stimulated and Ang II--stimulated VSMC hypertrophy without attenuating the observed increase in bFGF expression. Furthermore, PKC inhibition with calphostin C inhibited VSMC mitogenesis stimulated by exogenous bFGF.(ABSTRACT TRUNCATED AT 250 WORDS)
K+ currents and K+ channel mRNA in cultured atrial cardiac myocytes (AT-1 cells).
Authors: Yang T , Wathen M S , Felipe A , Tamkun M M , Snyders D J , Roden D M
Published: June 2012

Atrial tumor myocytes derived from transgenic mice (AT-1 cells) maintain a well-differentiated cardiac biochemical and histological phenotype. In addition, they beat spontaneously in culture and exhibit long action potentials whose repolarization resembles that observed in native mammalian myocytes. In this study, we identified the major depolarization-activated outward currents in AT-1 cells; also, the presence of mRNAs that encode outwardly conducting ion channels was determined by cloning from an AT-1 cDNA library or by Northern hybridization. Among K+ channel isoforms, Kv2.1, minK, and Kv1.4 were readily detected in tumors and at 1 day in culture. Their abundance remained relatively stable (twofold or less change) after 14 days. The major outward current in AT-1 cells is a delayed rectifier that displays prominent inward rectification, activates rapidly (eg, 182 +/- 27 milliseconds [mean +/- SEM] at + 20 mV, n = 12), exhibits biexponential deactivation kinetics, and is extremely sensitive to the methanesulfonanilide dofetilide (IC50, 12 nmol/L). These characteristics identify this current as IKr, a delayed rectifier observed only in cardiac cells. IKr in AT-1 cells displayed slow inactivation: dofetilide-sensitive deactivating tails were greater after 1-second than after 5-second pulses. When IKr was blocked by > or = 0.5 mumol/L dofetilide, time-independent current was usually recorded (50 of 65 experiments); rapidly inactivating (6 of 65) or slowly inactivating (9 of 65) outward currents were occasionally observed. We conclude that AT-1 cells express mRNAs encoding cardiac K+ channels and display a cardiac electrophysiological phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)
Cardiac Electrophysiological Actions of the Histamine H
1
-Receptor Antagonists Astemizole and Terfenadine Compared With Chlorpheniramine and Pyrilamine
Authors: Salata Joseph J. , Jurkiewicz Nancy K. , Wallace Audrey A. , Stupienski Raymond F. , Guinosso Peter J. , Lynch Joseph J.
Published: June 2012

Abstract
We compared the cardiac electrophysiological actions of two types of H
1
-receptor antagonists—the piperidines, astemizole and terfenadine, and the nonpiperidines, chlorpheniramine and pyrilamine—in vitro in guinea pig ventricular myocytes and in vivo in chloralose-anesthetized dogs. Astemizole and terfenadine significantly increased action potential duration of guinea pig myocytes. This concentration-dependent prolongation of action potential duration was reverse frequency dependent and led to development of early afterdepolarizations, which occurred more frequently at higher concentrations and slower pacing frequencies. Astemizole and terfenadine potently blocked the rapidly activating component of the delayed rectifier, I
Kr
, with IC
50
values of 1.5 and 50 nmol/L, respectively. At 10 μmol/L, terfenadine but not astemizole blocked the slowly activating component of the delayed rectifier, I
Ks
(58.4±3.1%), and the inward rectifier, I
K1
(20.5±3.4%). Chlorpheniramine and pyrilamine blocked I
Kr
relatively weakly (IC
50
=1.6 and 1.1 μmol/L, respectively) and I
Ks
and I
K1
less than 20% at 10 μmol/L. Astemizole and terfenadine (1.0 to 3.0 mg/kg IV) significantly prolonged the QTc interval and ventricular effective refractory period in vivo. Chlorpheniramine and pyrilamine (≤3.0 mg/kg) did not significantly affect these parameters. Block of repolarizing K
+
currents, particularly I
Kr
, by astemizole and terfenadine produces reverse rate–dependent prolongation of action potential duration and development of early afterdepolarizations, delays ventricular repolarization, and may underlie the development of torsade de pointes ventricular arrhythmias observed with the use and abuse of these agents.
Rate of Tension Development in Cardiac Muscle Varies With Level of Activator Calcium
Authors: Wolff Matthew R. , McDonald Kerry S. , Moss Richard L.
Published: June 2012

Abstract
In skeletal muscle, the rate of transition from weakly bound to force-generating crossbridge states increases as calcium concentration is increased. To examine possible calcium sensitivity of this transition in cardiac muscle, we determined the kinetics of isometric tension development during steady activation in detergent-permeabilized rat ventricular trabeculae (n=7) over a range of calcium concentrations. Force-generating crossbridges in activated trabeculae were disrupted by a brief, rapid release and restretch equivalent to 20% muscle length (15°C), which resulted in a subsequent phase of tension redevelopment that was well fit by a monoexponential function (rate constant,

k
tr

). Sarcomere length was monitored by laser diffraction and held constant during tension redevelopment by an iterative adaptive feedback control system. The

k
tr

increased from 3.6±0.8 s
−1
at the lowest calcium concentration studied (pCa 5.9) to 9.5±1.3 s
−1
during maximal activation (pCa 4.5). The relationship between relative

k
tr

and relative tension was approximately linear over a wide range of [Ca
2+
] (
r
2
=.94). This result differs quantitatively from results in skeletal muscle, in which

k
tr

is sensitive to [Ca
2+
] primarily at higher activation levels. This observation is also inconsistent with a recent suggestion that the rate of force development in living myocardium is independent of the activation level. Our results in skinned myocardium can be explained by a model in which calcium is a graded regulator of both the extent and rate of binding of force-generating crossbridges to the thin filament.
Differentiation of Vascular Smooth Muscle Cells and the Regulation of Protein Kinase C-α
Authors: Haller Hermann , Lindschau Carsten , Quass Petra , Distler Armin , Luft Friedrich C.
Published: June 2012

Abstract
Dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) are important features of atherosclerosis. The molecular mechanisms are largely unclear; however, protein kinase C (PKC) is a key enzyme in the intracellular signaling pathways that mediate this process. We studied the activity and immunoreactivity of PKC-α in primary cultures of VSMCs from rat aortas under different conditions of growth and differentiation. PKC-α was determined under the following conditions: (1) during the growth phase and after confluence of cultured (passages 1 through 3) VSMCs, (2) before and after induction of differentiation in VSMCs by retinoic acid, and (3) in primary cultures of VSMCs from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats during early passages. PKC activity was measured by in vitro substrate phosphorylation. PKC-α immunoreactivity was assessed by Western blot using specific polyclonal antibodies and by immunostaining with confocal microscopy. Cell proliferation was measured by direct count. The cell phenotype was characterized by immunostaining and Western blot for α-actin and desmin. PKC-α expression and PKC activity during VSMC growth showed a decrease during rapid growth and an increase in confluent cells. This pattern was associated with the respective changes in cell differentiation. Retinoic acid induced an increase in PKC-α expression together with a more differentiated phenotype. Subcultured, rapidly growing VSMCs from SHR showed a decreased PKC-α expression compared with cells from WKY rats. To establish cause and effect, we next microinjected either PKC-α or inactivated material directly into dedifferentiated cells. We found that cells injected with active PKC-α expressed increased amounts of actin compared with control cells. We identified a close correlation between PKC-α and actin immunofluorescence. We conclude that PKC-α is downregulated in rapidly growing VSMCs. Our findings demonstrate an inverse association between PKC-α expression and VSMC differentiation. They suggest a role for downregulation of PKC-α in the proliferative response of these cells.
Role of Extracellular and Intracellular Acidosis for Hypercapnia-Induced Inhibition of Tension of Isolated Rat Cerebral Arteries
Authors: Tian Rong , Vogel Pia , Lassen Niels A. , Mulvany Michael J. , Andreasen Frederik , Aalkjær Christian
Published: June 2012

Abstract
The importance of smooth muscle cell pH
i
and pH
o
for the hypercapnic vasodilation of rat cerebral arteries was evaluated in vitro. Vessel segments were mounted in a myograph for isometric tension recording; pH
i
was measured by loading the smooth muscle cells with the fluorescent dye BCECF, and pH
o
was measured with a glass electrode. In all studies, Ca
2+
-dependent basal tension (in the absence of any agonist) and tension in the presence of arginine vasopressin were investigated. Control solution was physiological saline bubbled with 5% CO
2
and containing 25 mmol/L HCO
3
−
(pH 7.45 to 7.50). Induction of hypercapnic acidosis (10% CO
2
) or normocapnic acidosis (15 mmol/L HCO
3
−
) caused significant inhibition of smooth muscle tension, and both conditions reduced pH
i
as well as pH
o
.
N
-Nitro-
l
-arginine significantly inhibited the relaxation to hypercapnic acidosis but had no significant effect on relaxation to normocapnic acidosis. Predominant extracellular acidosis, induced by reducing [HCO
3
−
] from 25 to 9 mmol/L and CO
2
from 5% to 2.5%, also caused inhibition of tension in steady state. By contrast, predominant intracellular acidosis, induced by increasing [HCO
3
−
] from 25 to 65 mmol/L and CO
2
from 5% to 15%, induced a small increase of basal tension and a small decrease of tension in the presence of arginine vasopressin. The responses to predominant intracellular or extracellular acidosis were qualitatively similar in the presence and absence of endothelium and in the presence and absence of
N
-nitro-
l
-arginine. It is concluded that the extracellular acidosis and not smooth muscle intracellular acidosis is responsible for the relaxation to hypercapnic acidosis.
Expression of Multiple Connexins in Cultured Neonatal Rat Ventricular Myocytes
Authors: Darrow Bruce J. , Laing James G. , Lampe Paul D. , Saffitz Jeffrey E. , Beyer Eric C.
Published: June 2012

Abstract
Three gap junction proteins have been identified in mammalian cardiac myocytes: connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40). These proteins form channels with different electrophysiological properties and have different distributions in cardiac tissues with disparate conduction properties. We characterized the expression, phosphorylation, turnover, and subcellular distribution of these connexins in primary cultures of neonatal rat ventricular myocytes. Cx43, Cx45, and Cx40 mRNA were specifically detected in RNA blots. Immunofluorescent staining with antibodies specific for Cx43 and Cx45 revealed punctate labeling at appositional membranes, but no immunoreactive Cx40 was detected. Double-label immunofluorescence confocal microscopy of cultured myocytes revealed colocalization of Cx43 and Cx45. Cx43 and Cx45 were both identified by immunoprecipitation from [
35
S]methionine-labeled cultures, but anti-Cx40 antibodies did not precipitate any radiolabeled protein. Phosphorylated forms of both Cx45 and Cx43 were immunoprecipitated from cultures metabolically labeled with [
32
P]orthophosphate. Phosphoamino acid analysis demonstrated that Cx45 was modified on serine residues, and Cx43 was phosphorylated on serine and threonine residues. Pulse-chase labeling experiments demonstrated that the half-lives of Cx43 and Cx45 were 1.9 and 2.9 hours, respectively. Thus, both Cx43 and Cx45 turn over relatively rapidly, suggesting that myocardial gap junctions have the potential for dynamic remodeling. The results implicate multiple mechanisms of gap junction regulation that may differ for different connexins.
Heparin Increases Exercise-Induced Collateral Blood Flow in Rats With Femoral Artery Ligation
Authors: Yang H. T. , Ogilvie Robert W. , Terjung Ronald L.
Published: June 2012

Abstract
The potential for heparin to enhance the training-induced increase in collateral-dependent blood flow to the distal hind-limb muscles was evaluated after bilateral femoral artery ligation in adult male rats (≈350 g). Rats received either saline (n=34) or heparin (n=36) injections and were kept sedentary (limited to cage activity) or exercised on a treadmill 5 days per week up a 15% incline by one of two protocols: (1) exercise at a constant moderate speed (20 m/min) for ≈6 wks or (2) exercise at a progressively increased speed for 7 to 8 weeks (started at 20 m/min, increased at 15 minutes to 25 m/min, and then increased at 30 minutes to 30 m/min). Heparin- and saline-treated rats, exercised by the moderate-speed protocol, were run for the same time each day. Collateral-dependent blood flow to the distal limb tissue was determined by using 15-μm
85
Sr-labeled microspheres in an isolated hindquarter preparation perfused in the descending aorta at 100 mm Hg. For comparison with the above groups, sedentary animals with acute femoral artery ligation and without femoral obstruction were included. Exercise tolerance increased from ≈7 minutes initially to 30 to 40 minutes per bout; tolerance was greater in the heparin-injected rats than in the saline-injected rats (
P
<.05). Muscle performance of the gastrocnemius-plantaris-soleus muscle group (GPS) during isometric contractions in situ improved with training, was further increased by heparin administration (
P
<.001), and generally scaled with recovery of blood flow. Collateral-dependent blood flow in the GPS of the heparin-injected exercised groups (40±4 and 56±3 mL/min per 100 g) was greater than that in the saline-injected exercised groups (19±3 and 30±5 mL/min per 100 g), and blood flow in both of these groups was greater than that in the sedentary groups (14±4 and 12±2 mL/min per 100 g) (
P
<.005). Heparin injections did not alter collateral-dependent blood flow in the absence of exercise training. Thus, exercise appears to impart an essential stimulus for collateral vessel development that is enhanced by heparin administration.
Angiotensin II–Induced Growth Responses in Isolated Adult Rat Hearts
Authors: Schunkert Heribert , Sadoshima Jun-ichi , Cornelius Torsten , Kagaya Yutaka , Weinberg Ellen O. , Izumo Seigo , Riegger Günter , Lorell Beverly H.
Published: June 2012

Abstract
Cardiac myocyte hypertrophy often occurs in response to both hemodynamic and neurohumoral factors. To study whether activation of the renin-angiotensin system by itself may induce a cardiac growth response, the acute effects of angiotensin II on cardiac protein synthesis were studied in isolated rat hearts. New protein synthesis in isolated buffer-perfused adult rat hearts was measured by incorporation of [
3
H]phenylalanine into cardiac proteins during a 3-hour perfusion protocol. Angiotensin II (1×10
−8
mol/L), administered alone or in combination with the α
1
-blocker prazosin (1×10
−7
mol/L), stimulated protein synthesis in both ventricles. The rate of [
3
H]phenylalanine incorporation into cardiac proteins was 3.9-fold (
P
<.005) and 2.6-fold (
P
<.01) higher in angiotensin II–perfused (n=6) than in vehicle-perfused (n=6) left and right ventricles, respectively. The induction of new protein synthesis by angiotensin II was blocked by the angiotensin II type 1 (AT
1
) receptor antagonist losartan (1×10
−7
mol/L, n=5). To study the pathways of angiotensin signal transduction, protein kinase C (PKC)-ε as well as cardiac c-
fos
and c-
jun
mRNA levels were analyzed. Angiotensin II (1×10
−8
mol/L, n=20) resulted in a transient translocation of PKC-ε from the cytosol to the cellular membrane. However, compared with phorbol ester stimulation (phorbol 12-myristate 13-acetate [PMA], 1×10
−7
mol/L; n=20), angiotensin II effects on PKC translocation were significantly less pronounced and required a more prolonged stimulation. There was no effect of angiotensin II in concentrations from 10
−9
to 10
−6
mol/L (n=22) on c-
fos
and c-
jun
mRNA levels in intact adult rat hearts studied over a time course from 15 to 120 minutes of perfusion. In contrast, norepinephrine (10
−6
mol/L, n=6), phorbol ester (PMA, 10
−7
mol/L; n=5), and calcium ionophore (A23187, 2.5×10
−6
mol/L; n=5) infusion as well as elevated left ventricular systolic wall stress (n=6) were all followed by a threefold to fourfold induction of cardiac c-
fos
and c-
jun
mRNA levels (
P
<.005) compared with respective angiotensin II–infused or vehicle-infused rat hearts (n=12). In contrast, administration of angiotensin II in concentrations >10
−9
mol/L caused a significant induction of c-
fos
in adult and neonatal cardiac myocytes. In conclusion, angiotensin II acutely stimulates protein synthesis in cultured adult isolated perfused rat hearts. Angiotensin II–activated signal transduction appears to involve AT
1
receptors and activation of PKC. However, further downstream signaling mechanisms remain elusive, since angiotensin II may stimulate protein synthesis in the adult intact heart without preceding c-
fos
and c-
jun
proto-oncogene induction.
Calcium Signaling in Endothelial Cells Involves Activation of Tyrosine Kinases and Leads to Activation of Mitogen-Activated Protein Kinases
Authors: Fleming Ingrid , Fisslthaler Beate , Busse Rudi
Published: June 2012

Abstract
The activation of endothelial cells following exposure to a variety of receptor-dependent and -independent stimuli is associated with the release of Ca
2+
from intracellular stores as well as the influx of Ca
2+
from the extracellular space. In the present study, we investigated the interaction between Ca
2+
signaling in cultured human umbilical vein endothelial cells and tyrosine phosphorylation. Stimulation of endothelial cells with either bradykinin (100 nmol/L), histamine (1 μmol/L), or the Ca
2+
-ATPase inhibitor thapsigargin (30 nmol/L) resulted in a slightly delayed but prolonged tyrosine phosphorylation of two low molecular weight proteins (≈42 and ≈44 kD). These proteins were identified by immunoprecipitation as the 42- and 44-kD isoforms of mitogen-activated protein kinase (MAP kinase). The agonist-induced tyrosine phosphorylation of the 42-/44-kD doublet was sensitive to the tyrosine kinase inhibitors genistein (100 μmol/L) and piceatannol (10 μmol/L) and was inhibited by the removal of Ca
2+
from the extracellular medium. In fura 2–loaded endothelial cells, inhibition of tyrosine kinases attenuated Ca
2+
signaling after stimulation with either bradykinin (30 nmol/L) or thapsigargin (30 nmol/L). Since inhibition of tyrosine kinases specifically attenuates the plateau phase of the Ca
2+
response after stimulation, the effect of tyrosine kinase inhibition appeared to be mostly associated with the influx of Ca
2+
from the extracellular space. These data demonstrate that the signal transduction cascade initiated by receptor-dependent and -independent stimulation of endothelial cells includes the following: a tyrosine kinase inhibitor–sensitive transmembranous influx of Ca
2+
and the tyrosine phosphorylation of two cytosolic protein substrates identified as MAP kinases. Furthermore, on one hand, an increase in [Ca
2+
]
i
was essential for tyrosine phosphorylation; on the other, the Ca
2+
influx was modulated by tyrosine phosphorylation. This finding documents the mutual dependence of these two crucial signaling pathways in endothelial cells.
Patterns of Expression of Sarcoplasmic Reticulum Ca
2+
-ATPase and Phospholamban mRNAs During Rat Heart Development
Authors: Moorman Antoon F. M. , Vermeulen Jacqueline L. M. , Koban Maren U. , Schwartz Ketty , Lamers Wouter H. , Boheler Kenneth R.
Published: June 2012

Abstract
This study reports the clonal analysis and sequence of rat phospholamban (PLB) cDNA clones and the temporal appearance and patterns of distribution of the mRNAs encoding sarcoplasmic/endoplasmic reticulum Ca
2+
-ATPase (SERCA2) and PLB in the developing rat heart determined by in situ hybridization. Both proteins play a critical role in the contraction-relaxation cycle of the heart. SERCA2 mRNA is already abundantly present in the first stage studied, in the cardiogenic plate of the 9-day-old presomite embryo, before the occurrence of the first contractions. This very early expression makes it an excellent marker for the study of early heart development. Subsequently, SERCA2 mRNA becomes expressed in a craniocaudal gradient, being highest at the venous pole and decreasing in concentration toward the arterial pole of the heart. PLB mRNA can be detected in hearts from 12 days of development onward in a virtually opposite gradient. In essence, these patterns do not change during further development. PLB mRNA levels remain highest in the ventricle and outflow tract, whereas SERCA2 mRNA prevails in the inflow tract and atrium, although the difference between atrium and ventricle becomes less pronounced. These observations are compatible with a model in which the upstream part of the heart (inflow tract and atrium) would have a greater capacity to clear calcium and hence would have a longer duration of the diastole than the downstream compartments (atrioventricular canal, ventricle, and outflow tract), similar to the observed pattern of contraction of the embryonic heart. The sinoatrial and atrioventricular nodes do not reveal an expression pattern of SERCA2 and PLB mRNA that allows one to distinguish them from the surrounding atrial working myocardium. However, the ventricular part of the conduction system, comprising atrioventricular bundle and bundle branches, are almost devoid of SERCA2 mRNA.
Inhibition of the Spontaneous Rate of Contraction of Neonatal Cardiac Myocytes by Protein Kinase C Isozymes
Authors: Johnson John A. , Mochly-Rosen Daria
Published: June 2012

Abstract
Protein kinase C (PKC) enzymes regulate numerous cardiac functions. In the present study, we determined the effects of the PKC-activating drug 4-β phorbol 12-myristate 13-acetate (4-β PMA) on the rate of contraction and correlated these changes with the distribution and levels of α-, β-, δ-, ε-, and ζ-PKC isozymes by using neonatal rat cardiac myocytes in culture. Treatment with 0.3 to 100 nmol/L 4-β PMA caused negative chronotropic effects on contraction. This effect was maximal at a concentration of 3 nmol/L 4-β PMA and correlated with redistribution of the α- and ε-PKC isozymes from the cytosolic to the particulate cell fraction. After a 1-hour treatment with 100 nmol/L PMA, the α- and β-PKC isozymes and an 80-kD ζ-like PKC isozyme were greatly diminished (downregulated), yet the negative chronotropic effect was sustained. Therefore, our results are most consistent with a role for the ε-PKC isozyme in suppressing the contraction rate of neonatal rat cardiac myocytes. Understanding the role(s) of individual PKC isozymes in the modulation of cardiac functions may ultimately yield more selective targets for therapies of cardiac disorders.
Contribution of Na
+
-Ca
2+
Exchange to Stimulation of Transient Inward Current by Isoproterenol in Rabbit Cardiac Purkinje Fibers
Authors: Han Xinqiang , Ferrier Gregory R.
Published: June 2012

Abstract
Cellular mechanisms underlying β-adrenergic stimulation of the arrhythmogenic transient inward current (TI) were investigated by using a two-microelectrode voltage-clamp technique in rabbit cardiac Purkinje fibers. TI induced by elevating [Ca
2+
]
o
to 30 mmol/L and substituting [Na
+
]
o
with
N
-methyl-
d
-glucamine (NMG) chloride had a distinct reversal potential (E
REV
) of −25 mV, suggesting that Na
+
-Ca
2+
exchange was not the charge carrier for TI. In the absence of [Na
+
]
o
, isoproterenol (ISO, 0.01 to 5.0 μmol/L) had no effect on either inward or outward TI or on the current-voltage relation of TI. However, ISO (0.1 μmol/L) significantly increased both inward and outward TIs without affecting the E
REV
of TI, if [Na
+
]
o
was present. Pretreatment with propranolol (0.2 μmol/L) or atenolol (0.2 μmol/L) abolished the stimulatory effects of ISO. Addition of propranolol (0.2 to 0.5 μmol/L) after the effects of ISO had developed caused only partial reversal of TI stimulation. This indicates persistence of stimulatory effects downstream from the initial agonist-receptor interaction. Forskolin (1 μmol/L), a direct adenylate cyclase activator, also strongly increased both inward and outward TI in the presence of [Na
+
]
o
. These effects also were abolished when [Na
+
]
o
was substituted by NMG. Inward and outward TIs enhanced by either ISO or forskolin were reversed by two putative Na
+
-Ca
2+
exchange blockers, dodecylamine (20 μmol/L) and quinacrine (20 μmol/L). These results suggest that β-adrenergic stimulation of TI is mediated by the Na
+
-Ca
2+
exchange; stimulation likely involves phosphorylation of the exchanger or some factor that modulates exchanger activity.
Molecular Basis of Human Cardiac Troponin T Isoforms Expressed in the Developing, Adult, and Failing Heart
Authors: Anderson Page A. W. , Greig Ann , Mark Trisha M. , Malouf Nadia N. , Oakeley Annette E. , Ungerleider Ross M. , Allen Paul D. , Kay Brian K.
Published: June 2012

Abstract
Cardiac troponin T (cTnT), a protein essential for calcium-regulated myofibrillar ATPase activity, is expressed in the human heart as four isoforms (cTnT
1
through cTnT
4
, numbered in the order of decreasing molecular size). The expression of these isoforms at the protein level has previously been found by us to differ in the normal and failing adult and fetal human heart. In the present study, we have cloned and sequenced four full-length cDNAs corresponding to the four native cTnT protein isoforms and have expressed these cDNAs in an in vitro transcription and translation system. The cDNAs differ by the variable inclusion of a 15- and a 30-nt exon in the 5′ half of the coding region. These cDNAs yielded proteins that comigrate with the native isoforms, cTnT
1
through cTnT
4
. Polyclonal antisera, raised against a synthetic peptide corresponding to the 10-residue peptide encoded by the 30-nt exon, reacted with the two human isoforms largest in molecular size (cTnT
1
and cTnT
2
) and the two largest cTnT isoforms of the rabbit and rat. The isoforms cTnT
1
and cTnT
2
, containing either both peptides encoded by the 30- and 15-nt exons or the peptide encoded by the 30-nt exon alone, are expressed in the fetal heart, with cTnT
2
being expressed at a very low level. cTnT
4
, lacking both of these sequences, is expressed in the fetal heart and is reexpressed in the failing adult heart, whereas cTnT
3
, containing the 5-residue peptide, is the dominant isoform in the adult heart. The identification and acquisition of these full-length clones that encode the four human cTnT isoforms should prove valuable in analyzing genomic DNA, identifying cTnT mutations in these alternatively spliced coding sequences and their splice sites, and studying the functional role of these isoform sequence differences in the normal and the diseased human heart.
Induction of Monocyte Chemoattractant Protein-1 Synthesis in Human Monocytes During Transendothelial Migration In Vitro
Authors: Takahashi Masafumi , Masuyama Jun-Ichi , Ikeda Uichi , Kasahara Tadashi , Kitagawa Sei-Ichi , Takahashi Yu-Ichi , Shimada Kazuyuki , Kano Shogo
Published: June 2012

Abstract
Monocyte chemoattractant protein-1 (MCP-1, or monocyte chemotactic and activating factor) plays important roles in the recruitment of monocytes and thus in the development of atherosclerosis. In this study, we determined whether MCP-1 synthesis was induced by the cellular interaction between monocytes and endothelial cells during the process of transendothelial migration. We found that when human peripheral blood monocytes (2.5×10
6
cells) and umbilical vein endothelial cells (HUVECs; 5.0×10
5
cells) were cocultured for 5 hours, 7.9 ng/mL MCP-1 was secreted into the medium, whereas when the two were cultured separately, MCP-1 levels were 1.0 and 0.9 ng/mL, respectively. Furthermore, the use of interleukin-1β (IL-1β)–pretreated HUVECs in cocultures induced twice the levels of MCP-1 as in unstimulated HUVEC culture. Conditioned medium had transendothelial chemotactic activity for monocytes, and this activity was completely abolished by addition of anti–MCP-1 antibody. Although MCP-1 mRNA levels were very low or undetectable in HUVECs or monocytes alone, message could be detected after 2 hours of coculture in total mRNA preparations from both monocytes and HUVECs. mRNA levels increased by 4 hours and had declined slightly by 24 hours. The rapid induction of message suggests that cell contact between monocytes and HUVECs induces the de novo synthesis of MCP-1 protein. Anti–interleukin (IL)-1α/β and anti–tumor necrosis factor-α antibodies, or anti–lymphocyte function–associated antigen-1 and very late antigen-4 antibodies, had little or no inhibitory effects on MCP-1 secretion by cocultures. Immunohistochemistry revealed that monocytes adherent to or having migrated across unstimulated HUVEC monolayers as well as the HUVECs themselves expressed MCP-1 protein. However, nonadherent monocytes failed to express it. This finding suggests that the monocyte–endothelial cell adhesive interaction results in an MCP-1–inductive signal to each cell type. MCP-1 expression by migrated monocytes may indicate that monocytes are primed to produce MCP-1 during transmigration and can secrete it in normal tissue in which inflammatory cytokines that induce MCP-1 are otherwise absent.
Regulation of Vascular Endothelial Growth Factor in Cardiac Myocytes
Authors: Levy Andrew P. , Levy Nina S. , Loscalzo Joseph , Calderone Angelino , Takahashi Nobuyuki , Yeo Kiang-Teck , Koren Gideon , Colucci Wilson S. , Goldberg Mark A.
Published: June 2012

Abstract
Collateral blood vessels supplement normal coronary blood flow and coronary blood flow compromised by coronary artery disease, thereby protecting the myocardium from ischemia. Collateral vessel formation is the result of angiogenesis. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a secreted mitogen specific for endothelial cells and an extremely potent angiogenic factor. In the present study, VPF/VEGF mRNA and protein were demonstrated to be markedly stimulated in primary rat cardiac myocytes in vitro in response to reduction of the oxygen tension to 1% or inhibition of the electron transport chain. Four isoforms of VPF/VEGF were coordinately regulated by hypoxia, including a novel isoform not previously described. Phorbol ester and the depolarizing agent veratridine, stimulators of protein kinase C and calcium influx, respectively, were found to markedly increase VPF/VEGF mRNA expression in cardiac myocytes. Forskolin, a potent stimulator of adenylate cyclase, produced a small but significant increase in VPF/VEGF mRNA expression in the cardiac myocytes. However, only H7, an inhibitor of protein kinase C, inhibited the hypoxic induction of VPF/VEGF mRNA; inhibitors of calcium influx and the calcium-calmodulin–dependent protein kinase II as well as inhibition of protein kinase A did not block the hypoxic induction of VPF/VEGF mRNA. This suggests that more than one signal transduction pathway is involved in regulating VPF/VEGF expression. The sensor that regulates the expression of hypoxia-responsive genes has been proposed to be a heme protein. Consistent with this model, transition metals initiate a genetic program similar to hypoxia. In the present study, the transition metals cobalt and manganese increased VPF/VEGF mRNA in cardiac myocytes in vitro and myocardial tissue in vivo, providing evidence that a similar sensor may regulate VPF/VEGF in the cardiac myocyte. These data suggest a novel mechanism by which VPF/VEGF induction contributes to collateral vessel formation in ischemic myocardium and also suggest strategies to increase VPF/VEGF production in vivo.
Enhanced Preservation of Orthotopically Transplanted Rat Lungs by Nitroglycerin but Not Hydralazine
Authors: Naka Yoshifumi , Chowdhury Nepal C. , Liao Hui , Roy Dilip K. , Oz Mehmet C. , Michler Robert E. , Pinsky David J.
Published: June 2012

Abstract
Nitric oxide (NO) produced within the lungs maintains pulmonary vascular homeostatic properties, modulating leukocyte traffic, platelet aggregation, and vasomotor tone. Because reactive oxygen intermediates generated during reperfusion react rapidly with available NO, we hypothesized that the NO donor nitroglycerin (NTG) would enhance lung preservation for transplantation by improving graft blood flow and reducing graft neutrophil and platelet sequestration. By use of an orthotopic rat left lung transplant model, with ligation of the native right pulmonary artery to ensure that recipient survival and physiological measurements depend entirely on the transplanted lung, transplants were performed in 70 male Lewis rats after 6-hour 4°C preservation in Euro-Collins solution (EC) alone or EC with supplemental NTG. Compared with EC alone, supplemental NTG significantly increased pulmonary arterial flow (2.2±1.4 to 21.4±2.9 mL/min,
P
<.01), decreased pulmonary vascular resistance (7.4±2.0 to 1.4±0.1×10
3
Woods units,
P
<.05), improved arterial oxygenation (163±57 to 501±31 mm Hg,
P
<.01), and enhanced recipient survival (17% to 100%,
P
<.001). These beneficial effects of NTG were dose dependent over a range of 0.001 to 0.1 mg/mL. Although NTG caused significant pulmonary vasodilation during the harvest/flushing period, the direct-acting vasodilator hydralazine caused greater vasodilation than did NTG but was associated with poor graft function, elevated pulmonary vascular resistance, and poor recipient survival. To explore nonvasodilator protective mechanisms of NTG, graft neutrophil and platelet sequestration were studied; supplemental NTG significantly reduced both neutrophil and platelet accumulation compared with either hydralazine or EC alone. These findings suggest that vasodilation alone at the time of harvest is insufficient to protect the lungs. NTG, which produces antineutrophil and antiplatelet effects as well as harvest vasodilation, appears to be a simple and effective additive that will improve lung preservation for transplantation.
Simultaneous Measurements of Ca
2+
and Nitric Oxide in Bradykinin-Stimulated Vascular Endothelial Cells
Authors: Blatter Lothar A. , Taha Ziad , Mesaros Stefan , Shacklock Philip S. , Wier Withrow G. , Malinski Tadeusz
Published: June 2012

Abstract
The production of endothelium-derived relaxing factor (EDRF), known to be nitric oxide (NO), is triggered by a rise in the cytoplasmic calcium concentration ([Ca
2+
]
i
) subsequent to receptor binding of vasoactive agonists. In vascular endothelial cells, NO is synthesized from
l
-arginine by the Ca
2+
/calmodulin–dependent NO synthase. In this study, we report the first simultaneous measurements of [Ca
2+
]
i
and [NO] at the level of single endothelial cells. In cultured bovine aortic endothelial cells, extracellular application of bradykinin (BK, 10 to 20 μmol/L) caused transient (sometimes oscillatory) increase in [Ca
2+
]
i
, which was measured with the fluorescent Ca
2+
indicator fura 2 and fluorescence imaging microscopy. BK caused an increase in [Ca
2+
]
i
, primarily through release from intracellular stores. Under identical experimental conditions, BK caused a transient increase in [NO], which was measured by application of a porphyrinic NO microsensor. [NO] peaked at ≈0.5 μmol/L. Simultaneous measurements of [Ca
2+
]
i
and [NO] in BK-stimulated endothelial cells revealed that a transient increase in [Ca
2+
]
i
was rapidly followed by an increase in [NO] that outlasted the [Ca
2+
]
i
transient.
Contraction-Induced Cell Wounding and Release of Fibroblast Growth Factor in Heart
Authors: Clarke Mark S. F. , Caldwell Robert W. , Chiao Hsi , Miyake Katsuya , McNeil Paul L.
Published: June 2012

Abstract
The heart hypertrophies in response to certain forms of increased mechanical load, but it is not understood how, at the molecular level, the mechanical stimulus of increased load is transduced into a cell growth response. One possibility is that mechanical stress provokes the release of myocyte-derived autocrine growth factors. Two such candidate growth factors, acidic and basic fibroblast growth factor (aFGF and bFGF, respectively), are released via mechanically induced disruptions of the cell plasma membrane. In the present study, we demonstrate that transient, survivable disruption (wounding) of the cardiac myocyte plasma membrane is a constitutive event in vivo. Frozen sections of normal rat heart were immunostained to reveal the distribution of the wound event marker, serum albumin. Quantitative image analysis of these sections indicated that an average of 25% of the myocytes contained cytosolic serum albumin; ie, this proportion had suffered a plasma membrane wound. Wounding frequency increased approximately threefold after β-adrenergic stimulation of heart rate and force of contraction. Heparin-Sepharose chromatography, enzyme-linked immunosorbent assay, growth assay coupled with antibody neutralization, and two-dimensional SDS-PAGE followed by immunoblotting were used to demonstrate that both aFGF and bFGF were released from an ex vivo beating rat heart. Importantly, β-adrenergic stimulation of heart rate and force of contraction increased FGF release. Cell wounding is a fundamental but previously unrecognized aspect of the biology of the cardiac myocyte. We propose that contraction-induced cardiac myocyte wounding releases aFGF and bFGF, which then may act as autocrine growth-promoting stimuli.
Restenosis After Experimental Angioplasty
Authors: Lafont Antoine , Guzman Luis A. , Whitlow Patrick L. , Goormastic Marlene , Cornhill J. Fredrick , Chisolm Guy M.
Published: June 2012

Abstract
Predicting and preventing arterial restenosis after angioplasty has failed despite considerable research into mechanisms and techniques. We examined the roles of chronic constriction, neointimal-medial growth, and adventitial changes in restenosis in atherosclerotic rabbits. Angioplasty was performed on femoral artery lesions 4 weeks after lesion induction by air drying and cholesterol-supplemented diet. Angiographic and histological evaluation was conducted 3 to 4 weeks after angioplasty. The angiographic minimum luminal diameter (MLD) increased from 1.31±0.21 to 1.73±0.41 mm after angioplasty. Loss in MLD by 3 to 4 weeks was 0.95±0.64 mm. Initial gain and late loss correlated (
P
=.008). Late residual stenosis, defined histologically as the difference between the luminal areas of a proximal reference site and lesion site normalized by the luminal area of the reference site, was 52±32%. Histological indices of chronic constriction, neointimal-medial growth, and adventitial growth were defined on the basis of the areas of these arterial wall layers at the lesion site relative to the reference site. Another parameter defined as the ratio of adventitial area to the area of intima+media at the lesion site allowed evaluation of the relative importance of these layers. Surprisingly, late residual stenosis correlated with chronic constriction (
P
=.0003) but not with neointimal-medial growth or adventitial growth. The ratio of adventitial area to the area of intima+media at the lesion site also correlated with chronic constriction (
P
=.01). These findings suggest that factors related to arterial remodeling rather than neointimal-medial growth may dominate the response to angioplasty.