bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and Function of Recombinant Endothelial Nitric Oxide Synthase Gene in Canine Basilar Artery
Authors: Chen Alex F.Y. , O’Brien Timothy , Tsutsui Masato , Kinoshita Hiroyuki , Pompili Vincent J. , Crotty Thomas B. , Spector David J. , Katusic Zvonimir S.
Published: June 2012

Endothelial NO synthase (eNOS) is an enzyme responsible for the production of a potent vasodilator and a key regulator of vascular tone, NO. In peripheral arteries, expression of a recombinant eNOS gene increases production of NO in the blood vessel wall. This approach appears to be a promising strategy for gene therapy of cerebrovascular disease. The major objective of the present study was to determine whether a recombinant eNOS gene (AdCMVNOS) can be functionally expressed in cerebral arteries. Replication-defective recombinant adenovirus vectors encoding bovine eNOS and Escherichia coli beta-galactosidase (AdCMVLacZ) genes, driven by the cytomegalovirus promoter, were used for ex vivo gene transfer. Rings of canine basilar artery were incubated with increasing titers of the vectors in MEM. Twenty-four or forty-eight hours after gene transfer, expression and function of AdCMVNOS were evaluated by (1) immunohistochemical staining, (2) isometric tension recording, and (3) cGMP radioimmunoassay. Transfection with AdCMVNOS resulted in the expression of recombinant eNOS protein in the vascular adventitia and endothelium, associated with significantly reduced contractile responses to UTP and enhanced endothelium-dependent relaxation to calcium ionophore A23187. Basal production of cGMP was significantly increased in the transfected vessels. The reduced contractions to UTP with increased cGMP production were reversed by a NOS inhibitor, N
G
-monomethyl-L-arginine. Contractions to UTP or production of cGMP were not affected in arteries transfected with AdCMVLacZ reporter gene. The results of the present study represent the first successful transfer and functional expression of recombinant eNOS gene in cerebral arteries. Our findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall. (Circ Res. 1997;80:327-335.)
Transgenic Remodeling of the Regulatory Myosin Light Chains in the Mammalian Heart
Authors: Gulick James , Hewett Timothy E. , Klevitsky Raisa , Buck Scott H. , Moss Richard L. , Robbins Jeffrey
Published: June 2012

Abstract
The regulatory myosin light chain (MLC) regulates contraction in smooth muscle. However, its function in striated muscle remains obscure, and the different functional activities of the various isoforms that are expressed in the mammalian heart (ventricle- and atrium-specific MLC2) remain undefined. To begin to explore these issues, we used transgenesis to determine the feasibility of effecting a complete or partial replacement of the cardiac regulatory light chains with the isoform that is normally expressed in fast skeletal muscle fibers (fast muscle–specific MLC2). Multiple lines of transgenic mice were generated that expressed the transgene at varying levels in the heart in a copy number–dependent fashion. There is a major discordance in the manner in which the different cardiac compartments respond to high levels of overexpression of the transgene. In atria, isoform replacement with the skeletal protein was quite efficient, even at low copy number. The ventricle is much more refractory to replacement, and despite high levels of transgenic transcript, protein replacement was incomplete. Replacement could be further increased by breeding the transgenic lines with one another. Despite very high levels of transgenic transcript in these mice, the overall level of the regulatory light chain in both compartments remained essentially constant; only the protein isoform ratios were altered. The partial replacement of the ventricular with the skeletal isoform reduced both left ventricular contractility and relaxation, although the unloaded shortening velocity of isolated ventricular cardiomyocytes was not significantly different.
Skeletal Muscle–Specific Myosin Binding Protein-H Is Expressed in Purkinje Fibers of the Cardiac Conduction System
Authors: Alyonycheva Tatiana , Cohen-Gould Leona , Siewert Christiana , Fischman Donald A. , Mikawa Takashi
Published: June 2012

Abstract
Heart contraction is coordinated by conduction of electrical excitation through specialized tissues of the cardiac conduction system. By retroviral single-cell tagging and lineage analyses in the embryonic chicken heart, we have recently demonstrated that a subset of cardiac muscle cells terminally differentiates as cells of the peripheral conduction system (Purkinje fibers) and that this occurs invariably in perivascular regions of developing coronary arteries.
Cis
regulatory elements that function in transcriptional regulation of cells in the conducting system have been distinguished from those in contractile cardiac muscle cells; eg, 5′ regulatory sequences of the desmin gene act as enhancer elements in skeletal muscle and in the conduction system but not in cardiac muscle. We hypothesize that Purkinje fiber differentiation involves a switch of the gene expression program from that characteristic of cardiac muscle to one typical of skeletal muscle. To test this hypothesis, we examined the expression of myosin binding protein-H (MyBP-H) in Purkinje fibers of chicken hearts. This unique myosin binding protein is present in skeletal but not cardiac myocytes. A site-directed polyclonal antibody (AB105) was generated against MyBP-H. Immunohistological analysis of the myocardium mapped the AB105 antigen predominantly to A bands of myofibrils within Purkinje fibers. Western blot analysis of whole extracts from the ventricular wall of adult chicken hearts revealed that the AB105 epitope was restricted to a single protein of ≈86 kD, the same size as MyBP-H in skeletal muscle. Biochemical properties of the Purkinje fiber 86-kD protein and RNase protection analyses of its mRNA indicate that Purkinje fiber 86-kD protein is indistinguishable from skeletal muscle MyBP-H. The results provide evidence that skeletal muscle MyBP-H is expressed in a subset of cardiac muscle cells that differentiate into Purkinje fibers of the heart.
Hypercholesterolemia Impairs a Detoxification Mechanism Against Peroxynitrite and Renders the Vascular Tissue More Susceptible to Oxidative Injury
Authors: Ma Xin L. , Lopez Bernard L. , Liu Gao-Lin , Christopher Theodore A. , Gao Feng , Guo Yaping , Feuerstein Giora Z. , Ruffolo Robert R. , Barone Frank C. , Yue Tian-Li
Published: June 2012

Abstract
Previous studies have shown that glutathione (GSH) plays a central role in the protection against peroxynitrite (ONOO
−
) toxicity. The present study evaluated the changes of the GSH cytoprotective system against ONOO
−
in hypercholesterolemia and determined the effects of carvedilol, a β-blocker with free radical–scavenging activity, on these hypercholesterol-induced changes. New Zealand White rabbits were fed either a normal diet, a high-cholesterol diet, or a high-cholesterol diet supplemented with either carvedilol or propranolol. Eight weeks later, the rabbits were killed, and the thoracic aortas were isolated. Total GSH content of aortic tissue, vasorelaxation response of aortic rings to exogenous ONOO
−
,
·
NO regeneration from ONOO
−
by aortic homogenate, and ONOO
−
-induced aortic tissue injury were examined. Hypercholesterolemia decreased tissue GSH content (0.52±0.08 versus 0.86±0.04 μmol/g in control,
P
<.01), attenuated the vasorelaxation response to ONOO
−
(40±4.1% versus 76±3.2%,
P
<.01), reduced
·
NO regeneration from ONOO
−
(387±40 versus 662±51 pmol,
P
<.01), and potentiated ONOO
−
-induced vascular tissue injury (37±4.4% versus 14±2.6% of increase in lactate dehydrogenase release after 3-morpholinosydnonimine exposure,
P
<.01). Treatment of the hypercholesterolemic rabbits with carvedilol, but not propranolol, significantly preserved tissue GSH content (0.79±0.05 μmol/g,
P
<.01 versus nontreated hypercholesterolemic rabbits), restored the vasorelaxation to ONOO
−
(61±2%,
P
<.01), increased
·
NO regeneration from ONOO
−
(583±39 pmol,
P
<.01), and attenuated ONOO
−
-induced tissue injury (19±1.8%,
P
<.01). These results suggest that hypercholesterolemia impairs the GSH-mediated detoxification mechanism against ONOO
−
and renders the vascular tissue more susceptible to oxidative injury. Carvedilol, a novel vasodilating β-blocker with antioxidant activity, significantly preserved this self-defense system and protected tissue from oxidant injury.
Regional Localization of
ERG
, the Channel Protein Responsible for the Rapid Component of the Delayed Rectifier, K
+
Current in the Ferret Heart
Authors: Brahmajothi Mulugu V. , Morales Michael J. , Reimer Keith A. , Strauss Harold C.
Published: June 2012

Abstract
Repolarization of the cardiac action potential varies widely throughout the heart. This could be due to the differential distribution of ion channels responsible for repolarization, especially the K
+
channels. We have therefore studied the cardiac localization of ERG, a channel protein known to play an important role in generation of the rapid component of the delayed rectifier K
+
current (I
Kr
), an important determinant of the repolarization waveform. Cryosections of the ferret atrium and ventricle were prepared to determine the localization of
ERG
by fluorescence in situ hybridization (FISH) and immunofluorescence. We found that in the ferret,
ERG
transcript and protein expression was most abundant in the epicardial cell layers throughout most of the ventricle, except at the base. In the atrium, we found that
ERG
is most abundant in the medial right atrium, especially in the trabeculae and the crista terminalis of the right atrial appendage. It also is present in areas within the sinoatrial node. In all regions studied, FISH and immunofluorescence showed concordant localization patterns. These data suggest that repolarization mediated by I
Kr
is not uniform throughout the ferret heart and provide a molecular explanation for heterogeneity in action potential repolarization throughout the mammalian heart.
Type B Atrial Natriuretic Peptide Receptor in Cardiac Myocyte Caveolae
Authors: Doyle Donald D. , Ambler S. Kelly , Upshaw-Earley Judy , Bastawrous Amir , Goings Gwendolyn E. , Page Ernest
Published: June 2012

Abstract
We have previously shown that atrial natriuretic peptide (ANP) is present in caveolae of in situ rat atrial myocytes. To investigate whether intracaveolar ANP of rat atrial myocytes exists within caveolae bound to type B ANP receptors (ANP-RB, a guanylyl cyclase), we have used confocal immunofluorescence microscopy applied to primary cultures of atrial myocytes from adult rats and to freshly dissociated rat atrial myocytes (not cultured). These experimental designs tested whether atrial myocyte ANP-RB colocalizes at the plasmalemma and elsewhere in the cell with the muscle-specific isoform of the caveolar coating protein, caveolin-3, and with a fraction of cellular ANP. The experiments showed that cellular caveolin-3, a fraction of cellular ANP-RB, and a fraction of cellular ANP colocalize at the plasmalemma of cultured atrial myocytes and of freshly dissociated atrial myocytes. The observations support the hypothesis that in rat atrial myocytes, intracaveolar ANP is bound to ANP-RB, a protein whose cytosolic amino acid sequences are known to encode guanylyl cyclase activity. We suggest that among the (probably multiple) effects of the cGMP thus generated in the cytoplasmic microdomain underlying atrial myocyte caveolae may be the activation of cGMP-dependent protein kinase, which would thereby inhibit plasma membrane Ca
2+
channel activity and contribute to a negative inotropic effect of ANP.
Mechanical Transduction of Nitric Oxide Synthesis in the Beating Heart
Authors: Pinsky David J. , Patton Stephen , Mesaros Stefan , Brovkovych Viktor , Kubaszewski Eugeniusz , Grunfeld Saul , Malinski Tadeusz
Published: June 2012

Abstract
NO alters contractile and relaxant properties of the heart. However, it is not known whether changes in ventricular loading conditions affect cardiac NO synthesis. To understand this potential contractile-relaxant autoregulatory mechanism, production of cardiac NO in response to mechanical stimuli was measured in vivo using a porphyrinic sensor placed in the left ventricular myocardium. The beating rabbit heart exhibited cyclic changes in [NO], peaking at 2.7±0.1 μmol/L near the endocardium and 0.93±0.20 μmol/L in the midventricular myocardium (concentrations were 15±4% lower in the rat heart). In the present study, we demonstrate for the first time that increasing or decreasing ventricular preload in vivo is followed by parallel changes in [NO], which may represent a novel autoregulatory mechanism to adjust cardiac performance or perfusion on a beat-to-beat basis. To quantify the relationship between applied force and NO synthesis, intermittent compressive or distending forces applied to ex vivo nonbeating hearts were shown to cause bursts of NO synthesis, with peak [NO] linearly related to ventricular transmural pressure. Experiments in which denuding cardiac endothelial and endocardial cells abrogated the NO signal indicate that these cells transduce mechanical stimulation into NO production in the heart. Taken together, these studies may help explain load-dependent relaxation, cardiac memory for mechanical events of preceding beats, diseases associated with myocardial distension, autoregulation of myocardial perfusion, and protection from thrombosis in the turbulent flow environment within the beating heart.
Skeletal Muscle Sarcoplasmic Reticulum Ca
2+
-ATPase Gene Expression in Congestive Heart Failure
Authors: Peters David G. , Mitchell Heather L. , McCune Sylvia A. , Park Sonhee , Williams Jay H. , Kandarian Susan C.
Published: June 2012

Abstract
Congestive heart failure leads to skeletal muscle abnormalities, one of which is a prolongation of sarcoplasmic reticulum Ca
2+
flux. The purpose of this study was to determine whether skeletal muscle of spontaneous hypertensive and heart failure rats have alterations in the expression of the sarcoplasmic (or endoplasmic) reticulum Ca
2+
-ATPase (SERCA) gene. Northern analysis revealed that SERCA1, the predominant skeletal muscle isoform, was decreased by 45%, 43%, and 58% in the tibialis anterior, plantaris, and diaphragm muscles, respectively. Ribonuclease protection assay showed that the decrease was due to the adult isoform, SERCA1a, with minor changes in the alternatively spliced neonatal isoform, SERCA1b. There was no change in SERCA1 mRNA levels in gastrocnemius muscles. No change was found in SERCA2a (cardiac/slow skeletal isoform) mRNA or protein levels or in SERCA2b (smooth muscle isoform), dihydropyridine receptor, or α-actin mRNA levels in diaphragm muscle. Northern blot and ribonuclease protection assays showed that SERCA2a decreased 61% in the heart while the alternatively spliced isoform, SERCA2b, decreased 27%. Western analysis of the tibialis anterior, diaphragm, and gastrocnemius muscles showed a decrease in SERCA1 protein levels by 46%, 64%, and 42%, respectively, whereas sarcoplasmic reticulum Ca
2+
-ATPase activity, a functional correlate of SERCA expression, was decreased by 38%, 38%, and 40% in the same muscles. SERCA2 protein expression decreased by 36% in the failing heart. Decreases in both mRNA and protein suggest pretranslational control of SERCA1 expression, whereas the lack of decreased SERCA1 mRNA in gastrocnemius muscle suggests translational regulation. The decreased SERCA1 protein expression in all muscles studied probably contributes to contractile abnormalities related to excitation-contraction coupling function in heart failure.
Unexpected and Differential Effects of Cl
−
Channel Blockers on the Kv4.3 and Kv4.2 K
+
Channels
Authors: Wang Hong-Sheng , Dixon Jane E. , McKinnon David
Published: June 2012

Abstract
The Kv4.3 K
+
channel is thought to underlie the Ca
2+
-insensitive transient outward current (
I
to1
) in ventricular myocytes of canine and human heart and to contribute to the
I
to1
in rat myocytes. It has been suggested that there is a second component of the transient outward current in some species that is contributed by a Ca
2+
-activated Cl
−
current (known as
I
to2
). The evidence for the existence of the
I
to2
current is based, in part, on the pharmacological effects of various Cl
−
channel blockers. To test for possible interactions between these compounds and
I
to1
, the effect of several different Cl
−
channel blockers on the Kv4.3 channel was examined. The fenamates (niflumic and flufenamic acid) were found to have large effects on the position of the steady state inactivation curve of the Kv4.3 channel. The disulfonic stilbenes (DIDS and SITS) had markedly different effects and were found to greatly reduce the rate of recovery from inactivation of the Kv4.3 channel without large changes in the position of the activation and steady state inactivation curves. Both classes of drugs produced an apparent blockade of the Kv4.3 channel under some recording conditions. Surprisingly, the closely related Kv4.2 channel was found to be markedly less sensitive to these drugs. Caffeine was found to block both the Kv4.3 and Kv4.2 channels to a similar extent. These nonspecific drug effects have implications for the study of the two components of the transient outward current and suggest that purely pharmacological criteria cannot be used to define the physiological role of
I
to2
.
Phenylephrine-Induced Ca
2+
Oscillations in Canine Pulmonary Artery Smooth Muscle Cells
Authors: Hamada Hiroshi , Damron Derek S. , Hong Sung Jin , Van Wagoner David R. , Murray Paul A.
Published: June 2012

Abstract
Modulation of [Ca
2+
]
i
in response to receptor activation is a critical determinant of vascular smooth muscle tone. In this study, we examined the effect of continuous stimulation of α
1
-adrenoceptors with phenylephrine (PE) on [Ca
2+
]
i
in single pulmonary artery smooth muscle cells (PASMCs) cultured from explants of canine intrapulmonary artery. Fura 2–loaded PASMCs pretreated with propranolol (5 μmol/L) were continuously superfused with PE at 37°C on the stage of an inverted fluorescence microscope, and [Ca
2+
]
i
was measured using a dual-wavelength spectrofluorometer. Resting values of [Ca
2+
]
i
were 96±4 nmol/L. PE (10 μmol/L) stimulated oscillations in [Ca
2+
]
i
at a frequency of 1.35±0.07/min, which reached a peak [Ca
2+
]
i
of 650±26 nmol/L (n=69 cells). The oscillations lasted for >30 minutes and were constant in amplitude and frequency. Both the amplitude and frequency of PE-induced [Ca
2+
]
i
oscillations increased in a dose-dependent (3×10
−8
to 10
−4
mol/L) manner. Pretreatment with the α
1
-adrenoceptor antagonist prazosin (50 nmol/L) or removal of extracellular Ca
2+
abolished the repetitive [Ca
2+
]
i
oscillations induced by PE. The voltage-operated Ca
2+
channel blockers nifedipine (1 μmol/L) and verapamil (1 μmol/L) had no effect on the [Ca
2+
]
i
oscillations. In contrast, inhibition of phospholipase C with U73122 (10
−7
to 10
−5
mol/L) attenuated the oscillations in a dose-dependent fashion. The nonselective protein kinase inhibitor staurosporine (10
−9
to 10
−7
mol/L) had a minimal inhibitory effect on the oscillations. Caffeine (30 mmol/L) and thapsigargin (1 μmol/L) abolished the oscillations, whereas pretreatment with ryanodine (1 to 100 μmol/L) had no effect. In freshly dispersed PASMCs, PE (10 μmol/L) induced oscillations in [Ca
2+
]
i
similar to those observed in cultured cells, and patch-clamp experiments revealed oscillations in membrane potential. These results indicate that PE induces [Ca
2+
]
i
oscillations in PASMCs via stimulation of α
1
-adrenoceptors coupled to phospholipase C activation. Voltage-operated Ca
2+
channels and protein kinases are not required for the oscillations. The requirement for extracellular Ca
2+
and intracellular Ca
2+
stores indicates that both Ca
2+
influx and intracellular Ca
2+
release play a role in the maintenance of the oscillations.
Actions of cADP-Ribose and Its Antagonists on Contraction in Guinea Pig Isolated Ventricular Myocytes
Authors: Iino Shigeo , Cui Yi , Galione Antony , Terrar Derek A.
Published: June 2012

Abstract
Although it is becoming widely accepted that cADP-ribose (cADPR) can regulate calcium release from the endoplasmic reticulum in sea urchin eggs and in a variety of mammalian cell types, it remains controversial whether this substance might influence calcium release during excitation-contraction coupling in cardiac muscle. We have investigated possible actions of cADPR in intact cells isolated from guinea pig ventricle, paying particular attention to the possible influence of temperature. At 36°C, myocyte contraction was influenced by cytosolic application of cADPR in a concentration-dependent manner (showing an ≈30% increase in contraction with 5 μmol/L cADPR applied via a patch pipette in myocytes stimulated to fire action potentials at 1 Hz). Calcium transients measured with fura 2 were also increased by 5 μmol/L cADPR. Antagonists of cADPR reduced contraction at 36°C (by ≈35% with either 50 μmol/L 8-Br-cADPR or 5 μmol/L 8-amino-cADPR applied via the patch pipette). At room temperature (≈20°C to 24°C), no significant effects on contraction were detected with either cADPR or its antagonists. At 36°C, treatment of the cells with a mixture of 2 μmol/L ryanodine and 1 μmol/L thapsigargin to suppress function of the sarcoplasmic reticulum stores of calcium prevented the action of 5 μmol/L cADPR applied via a patch pipette. These observations are consistent with an action of cytosolic cADPR to enhance calcium-induced calcium release from the sarcoplasmic reticulum in guinea pig ventricular myocytes at 36°C. The observed influence of temperature under the conditions of our experiments is one factor that might help to account for failure to detect actions of cADPR and its analogues in some previous studies.
Low Efficiency of Ca
2+
Entry Through the Na
+
-Ca
2+
Exchanger as Trigger for Ca
2+
Release From the Sarcoplasmic Reticulum
Authors: Sipido Karin R. , Maes Micheline , Van de Werf Frans
Published: June 2012

Abstract
It has been proposed that Ca
2+
entry through the Na
+
-Ca
2+
exchanger can contribute significantly to the trigger for Ca
2+
release from the sarcoplasmic reticulum (SR). We have compared the characteristics of Ca
2+
release triggered by reverse-mode Na
+
-Ca
2+
exchange and by L-type Ca
2+
current (
I
CaL
) during depolarizing steps in single guinea pig ventricular myocytes (whole-cell voltage clamp, fluo 3 and fura-red as [Ca
2+
]
i
indicators, 36±1°C, K
+
-based pipette solution with 20 mmol/L [Na
+
]). Conditioning pulses to +60 mV ensured comparable Ca
2+
loading of the SR. In the presence of
I
CaL
, [Ca
2+
]
i
transients typically have an early and rapid rising phase reflecting Ca
2+
release, which has a bell-shaped voltage dependence with a peak at +10 mV. With Ca
2+
entry through Na
+
-Ca
2+
exchange only (20 μmol/L nisoldipine), Ca
2+
release flux from the SR is decreased and directly related to the amplitude of the depolarizing step. Ca
2+
release is preceded by a significant delay (81±21 ms at +20 mV, 24±4 ms at +70 mV) related to Ca
2+
entry through the exchanger. Triggered release interrupts Ca
2+
entry, as evidenced by reversal of the exchanger current. At potentials positive to +40 mV, Ca
2+
influx through Na
+
-Ca
2+
exchange, calculated from the outward exchange current, reaches magnitudes comparable to
I
CaL
, but Ca
2+
release due to reverse-mode Na
+
-Ca
2+
exchange still has a significant delay. We calculated trigger efficiency as the ratio between the maximal rate of Ca
2+
release and the Ca
2+
influx preceding this release; efficiency of reverse-mode Na
+
-Ca
2+
exchange is approximately four times less than that of
I
CaL
. With both
I
CaL
and reverse-mode Na
+
-Ca
2+
exchange present, Ca
2+
release is triggered by
I
CaL
, and a contribution of reverse-mode Na
+
-Ca
2+
exchange to the trigger could not be detected at potentials below +60 mV. These characteristics of reverse-mode Na
+
-Ca
2+
exchange predict that its role as a trigger for Ca
2+
release during the action potential is likely to be negligible.
Transcriptional Regulation of Inducible Nitric Oxide Synthase in Cultured Neonatal Rat Cardiac Myocytes
Authors: Kinugawa Koh-ichiro , Shimizu Tatsuya , Yao Atsushi , Kohmoto Osami , Serizawa Takashi , Takahashi Toshiyuki
Published: June 2012

Abstract
Previous work has demonstrated that inducible NO synthase (iNOS) can be expressed in cardiac myocytes. In this study, we investigated transcriptional regulation of the iNOS gene in these cells. Lipopolysaccharide (LPS) induced iNOS mRNA and protein in cultured neonatal rat cardiac myocytes. H-89, dexamethasone, herbimycin, genistein, staurosporine, or pyrrolidine dithiocarbamate (PDTC) attenuated the iNOS induction by LPS. Forskolin, interleukin (IL)-6, tumor necrosis factor (TNF)-α, or interferon (IFN)-γ enhanced the LPS-induced iNOS expression. Combined stimulation of IL-6 and TNF-α also induced iNOS. The 5′-upstream sequence of the rat iNOS gene contains the nuclear factor-κB (NF-κB) site, CAAT box, IFN-γ activation site (GAS), and IFN regulatory factor (IRF) site. DNase I footprinting assay revealed that the nuclear factors binding to these elements were increased by LPS exposure. Transient transfection assay suggested that these elements were indispensable for transcriptional regulation of the iNOS induction. Electrophoretic mobility shift assay revealed that LPS or TNF-α increased binding activity for the NF-κB site. A slower-migrating complex binding to the CAAT box gave rise after exposure to LPS or forskolin. Competition assay suggested that this slower-migrating complex consisted of a heterodimer between a member of CAAT box/enhancer binding (C/EBP) protein family and cAMP responsive element binding protein (CREB). LPS or IL-6 increased binding complexes for the IRF site, which was compatible with induction of IRF-1. LPS, IL-6, or IFN-γ induced a novel binding complex for GAS, which also existed in the 5′-flanking region of the IRF-1 gene. These data suggest that (1) iNOS induction simultaneously requires both NF-κB activation and IRF-1 induction, and (2) the heterodimer between C/EBP and CREB has synergistic effects on the iNOS induction via the CAAT box.
Smooth Muscle Cells Isolated From Discrete Compartments of the Mature Vascular Media Exhibit Unique Phenotypes and Distinct Growth Capabilities
Authors: Frid M. G. , Aldashev A. A. , Dempsey E. C. , Stenmark K. R.
Published: June 2012

Abstract
Heterogeneity of smooth muscle cell (SMC) phenotype and function is rapidly emerging as an important concept. We have recently described that phenotypically distinct SMC subpopulations in bovine pulmonary arteries exhibit unique proliferative and matrix-producing responses to hypoxic pulmonary hypertension. To provide better understanding of the molecular mechanisms contributing to this phenomenon, experimental studies will require a reliable in vitro model. The purpose of the present study was first to determine if distinct SMC subpopulations, similar to those observed in vivo, could be selectively isolated from the mature arterial media, and then to evaluate whether select SMC subpopulations would exhibit heightened responses to growth-promoting stimuli and hypoxia. We were able to reproducibly isolate at least four phenotypically unique cell subpopulations from the inner, middle, and outer compartments of the arterial media. Differences in cell phenotype were demonstrated by morphological appearance and differential expression of muscle-specific proteins. The isolated cell subpopulations exhibited markedly different growth capabilities. Two SMC subpopulations grew slowly in 10% serum and were quiescent in plasma-based medium. The other two cell subpopulations, exhibiting nonmuscle characteristics, grew rapidly in 10% serum and proliferated in plasma-based medium and in response to hypoxia. Certain colonies of the nonmuscle-like cell subpopulations were found to grow autonomously under serum-deprived conditions and to secrete mitogenic factors. Our data, demonstrating that phenotypically distinct cells with enhanced growth potential exist within the normal arterial media, support the idea that these unique cells could contribute selectively to the pathogenesis of vascular disease.
A Mutant Tropomyosin That Causes Hypertrophic Cardiomyopathy Is Expressed In Vivo and Associated With an Increased Calcium Sensitivity
Authors: Bottinelli R. , Coviello D. A. , Redwood C. S. , Pellegrino M. A. , Maron B. J. , Spirito P. , Watkins H. , Reggiani C.
Published: June 2012

Abstract
—Mutant contractile protein genes that cause familial hypertrophic cardiomyopathy (FHC) are presumed to encode mutant proteins that interfere with contractile function. However, it has generally not been possible to show mutant protein expression and incorporation into the sarcomere in vivo. This study aimed to assess whether a mutant α-fast tropomyosin (TM) responsible for FHC is actually expressed and determines abnormal contractile function. Since α-fast TM is expressed in heart and skeletal muscle, samples from vastus lateralis muscles were studied from two FHC patients carrying an Asp
175
Asn α-fast TM mutation and two healthy control subjects. TM isoforms from whole biopsy samples and single fibers were identified by gel electrophoresis and Western blot analysis. An additional faster-migrating TM band was observed in both FHC patients. The aberrant TM was identified as the Asp
175
Asn α-fast TM by comigration with purified recombinant human Asp
175
Asn α-fast TM. Densitometric quantification of mutant and wild-type α-fast TMs suggested equal expression of the two proteins. Contractile parameters of single skinned muscle fibers from FHC patients and healthy control subjects were compared. Calcium sensitivity was significantly increased in muscle fibers containing Asp
175
Asn α-fast Tm compared with fibers lacking the mutant TM. No discernible difference was found regarding cooperativity, maximum force, and maximum shortening velocity. This is the first demonstration that the mutant TM that causes FHC is indeed expressed and almost certainly incorporated into muscle in vivo and does result in altered contractile function; this confirms a dominant-negative, rather than null allele, action. Since the mutant TM was associated with increased calcium sensitivity, this mutation might be associated with an enhancement and not a depression of cardiac contractile performance. If so, this contrasts with the hypothesis that FHC mutations induce contractile impairment followed by compensatory hypertrophy.
Adenosine Inhibits Lipopolysaccharide-Induced Cardiac Expression of Tumor Necrosis Factor-α
Authors: Wagner Daniel R. , Combes Alain , McTiernan Charles , Sanders Virginia J. , Lemster Bonnie , Feldman Arthur M.
Published: June 2012

Abstract
—Tumor necrosis factor-α (TNF-α) is elevated in the failing heart. Very little is known about regulation of TNF-α in cardiomyocytes. TNF-α expression by macrophages is diminished by adenosine. Therefore, we hypothesized that a similar mechanism might occur in the heart. Neonatal rat myocytes were stimulated with lipopolysaccharide (LPS), and TNF-α was measured by ELISA. In the absence of LPS, myocytes did not release TNF-α in the medium. After exposure to LPS, TNF-α increased to 70.1±3.5 pg/mL at 6 hours. Immunofluorescent staining confirmed that TNF-α was expressed in myocytes. Adenosine decreased TNF-α in a dose-dependent manner (1 to 100 μmol/L, 37% to 65% decrease,
P
<.01). Adenosine also decreased TNF-α in cell homogenates by 78% (
P
<.0001). The effect of adenosine could be replicated by the A
2
agonist PD-125944 (DPMA), by cAMP agonists 8-bromo-cAMP, forskolin, and Ro 20–1724, but not by A
1
and A
3
agonists. Conversely, the effect of adenosine could be suppressed by the adenylate cyclase inhibitor MDL-12,330. Adenosine also inhibited TNF-α in adult rat ventricular myocytes (−60%,
P
<.005) and rat papillary muscles (−55%,
P
<.05). In neonatal myocytes, adenosine normalized LPS-induced calcium changes and improved LPS-induced negative inotropic (
P
<.01) and negative lusitropic (
P
<.01) effects. Our results demonstrate that adenosine can significantly diminish TNF-α levels in the heart. The effect appears to be mediated by the A
2
receptor and transduced through a G protein–adenylyl cyclase pathway. These results may explain some cardioprotective effects of adenosine and provide a novel pharmacological intervention in congestive heart failure.
Role of Nitric Oxide in the Control of Renal Oxygen Consumption and the Regulation of Chemical Work in the Kidney
Authors: Laycock Sarra K. , Vogel Traci , Forfia Paul R. , Tuzman Joshua , Xu Xiaobin , Ochoa Manuel , Thompson Carl I. , Nasjletti Alberto , Hintze Thomas H.
Published: June 2012

Abstract
—Inhibition of NO synthesis has recently been shown to increase oxygen extraction in vivo, and NO has been proposed to play a significant role in the regulation of oxygen consumption by both skeletal and cardiac muscle in vivo and in vitro. It was our aim to determine whether NO also has such a role in the kidney, a tissue with a relatively low basal oxygen extraction. In chronically instrumented conscious dogs, administration of an inhibitor of NO synthase, nitro-
l
-arginine (NLA, 30 mg/kg IV), caused a maintained increase in mean arterial pressure and renal vascular resistance and a decrease in heart rate (all
P
<0.05). At 60 minutes, urine flow rate and glomerular flow rate decreased by 44±12% and 45±7%, respectively; moreover, the amount of sodium reabsorbed fell from 16±1.7 to 8.5±1.1 mmol/min (all
P
<0.05). At this time, oxygen uptake and extraction increased markedly by 115±37% and 102±34%, respectively (
P
<0.05). Oxygen consumption also significantly increased from 4.5±0.6 to 7.1±0.9 mL O
2
/min. Most important, the ratio of oxygen consumption to sodium reabsorbed increased dramatically from 0.33±0.07 to 0.75±0.11 mL O
2
/mmol Na
+
(
P
<0.05), suggesting a reduction in renal efficiency for transporting sodium. In vitro, both a NO-donating agent and the NO synthase–stimulating agonist bradykinin significantly decreased both cortical and medullary renal oxygen consumption. In conclusion, NO plays a role in maintaining a balance between oxygen consumption and sodium reabsorption, the major ATP-consuming process in the kidney, in conscious dogs, and NO can inhibit mitochondrial oxygen consumption in canine renal slices in vitro.
Angiotensin II Stimulates p21-Activated Kinase in Vascular Smooth Muscle Cells
Authors: Schmitz Udo , Ishida Takafumi , Ishida Mari , Surapisitchat James , Hasham Mohammed I. , Pelech Steven , Berk Bradford C.
Published: June 2012

Abstract
—Angiotensin II (Ang II) has been previously shown to stimulate the extracellular signal–regulated kinase (ERK)1/2 and c-Jun
N
-terminal kinase (JNK) mitogen-activated protein (MAP) kinase family members. Little is known regarding the upstream signaling molecules involved in Ang II–mediated JNK activation. Ang II has been shown to activate the Janus kinase/signal transducer(s) and activator(s) of transcription (JAK/STAT) pathway, suggesting similarities to cytokine signaling. In response to cytokines such as interleukin-1 and tumor necrosis factor-α, the p21-activated kinase (PAK) has been identified as an upstream component in JNK activation. Therefore, we hypothesized that PAK may be involved in JNK activation by Ang II in vascular smooth muscle cells (VSMCs). αPAK activity was measured by myelin basic protein phosphorylation in rat aortic VSMCs. In response to Ang II, αPAK was rapidly stimulated within 1 minute, with a peak (5-fold increase) at 30 minutes. αPAK stimulation preceded activation of JNK in VSMCs. Ang II–mediated activation of both αPAK and JNK was Ca
2+
dependent and inhibited by downregulation of phorbol ester–sensitive protein kinase C isoforms (by pretreatment with phorbol 12,13-dibutyrate) but not by pretreatment with GF109203X. Activation of both PAK and JNK was partially inhibited by tyrosine kinase inhibitors but not by specific Src inhibitors, suggesting regulation by a tyrosine kinase other than c-Src. Finally, introduction of dominant negative PAK markedly reduced the JNK activation by Ang II in both Chinese hamster ovary and COS cells stably expressing the Ang II type 1 receptor (AT1R). Our data provide evidence for αPAK as an upstream mediator of JNK in Ang II signaling and extend the role of Ang II as a proinflammatory mediator for VSMCs.
Angiotensin II Type 1 Receptor–Induced Extracellular Signal–Regulated Protein Kinase Activation Is Mediated by Ca
2+
/Calmodulin-Dependent Transactivation of Epidermal Growth Factor Receptor
Authors: Murasawa Satoshi , Mori Yasukiyo , Nozawa Yoshihisa , Gotoh Noriko , Shibuya Masabumi , Masaki Hiroya , Maruyama Katsuya , Tsutsumi Yoshiaki , Moriguchi Yasutaka , Shibazaki Yasunobu , Tanaka Yohko , Iwasaka Toshiji , Inada Mitsuo , Matsubara Hiroaki
Published: June 2012

Abstract
—The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of growth factor stimulation, and recent evidence suggests that G protein–coupled receptors transactivate growth factor receptors to transmit mitogenic effects. In the present study, we report the involvement of epidermal growth factor receptor (EGF-R) in Ang II–induced extracellular signal–regulated kinase (ERK) activation, c-
fos
gene expression, and DNA synthesis in cardiac fibroblasts. Ang II induced a rapid tyrosine phosphorylation of EGF-R in association with phosphorylation of Shc protein and ERK activation. Specific inhibition of EGF-R function by either a dominant-negative EGF-R mutant or selective tyrphostin AG1478 completely abolished Ang II–induced ERK activation. Induction of c-
fos
gene expression and DNA synthesis were also abolished by the inhibition of EGF-R function. Calmodulin or tyrosine kinase inhibitors, but not protein kinase C (PKC) inhibitors or downregulation of PKC, completely abolished transactivation of EGF-R by Ang II or the Ca
2+
ionophore A23187. Epidermal growth factor (EGF) activity in concentrated supernatant from Ang II–treated cells was not detected, and saturation of culture media with anti-EGF antibody did not affect the Ang II–induced transactivation of EGF-R. Conditioned media in which cells were incubated with Ang II could not induce phosphorylation of EGF-R on recipient cells. Platelet-derived growth factor-β receptor was not phosphorylated on Ang II stimulation, and Ang II–induced c-
jun
gene expression was not affected by tyrphostin AG1478. Our results demonstrated that in cardiac fibroblasts Ang II–induced ERK activation and its mitogenic signals are dominantly mediated by EGF-R transactivated in a Ca
2+
/calmodulin-dependent manner and suggested that the effects of Ang II on cardiac fibroblasts should be interpreted in association with the signaling pathways regulating cellular proliferation and/or differentiation by growth factors.
Localization of Ryanodine Receptors in Smooth Muscle
Authors: Lesh Ryan E. , Nixon Graeme F. , Fleischer Sidney , Airey Judith A. , Somlyo Andrew P. , Somlyo Avril V.
Published: June 2012

Abstract
—The ryanodine receptor (RyR) in aortic and vas deferens smooth muscle was localized using immunofluorescence confocal microscopy and immunoelectron microscopy. Indirect immunofluorescent labeling of aortic smooth muscle with anti-RyR antibodies showed a patchy network-like staining pattern throughout the cell cytoplasm, excluding nuclei, in aortic smooth muscle and localized predominantly to the cell periphery in the vas deferens. This distribution is consistent with that of the sarcoplasmic reticulum (SR) network, as demonstrated by electron micrographs of osmium ferrocyanide–stained SR in the two smooth muscles. Immunoelectron microscopy of vas deferens smooth muscle showed anti-RyR antibodies localized to both the sparse central and predominant peripheral SR elements. We conclude that RyR–Ca
2+
-release channels are present in both the peripheral and central SR in aortic and vas deferens smooth muscle. This distribution is consistent with the possibility that both regions are release sites, as indicated by results of electron probe analysis, which show a decrease in the Ca
2+
content of both peripheral and internal SR in stimulated smooth muscles. The complex distribution of inositol 1,4,5-trisphosphate and ryanodine receptors (present study) is compatible with their proposed roles as agonist-induced Ca
2+
-release channels and origins of Ca
2+
sparks, Ca
2+
oscillations, and Ca
2+
waves.
Expression of Cyclin-Dependent Kinase Inhibitors in Vascular Disease
Authors: Tanner Felix C. , Yang Zhi-Yong , Duckers Eric , Gordon David , Nabel Gary J. , Nabel Elizabeth G.
Published: June 2012

Abstract
—Arterial lesions in cardiovascular diseases are characterized by proliferation and migration of smooth muscle cells as well as deposition of connective tissue matrix. Factors that stimulate vascular smooth muscle cell (VSMC) proliferation are well described; however, the role of proteins that limit intimal hyperplasia is not well understood. To examine the function of Kip/Cip and INK cyclin-dependent kinase inhibitors (CKIs) in vascular diseases, the expression of p27
Kip1
and p16
INK
was examined in VSMCs in vitro and in porcine arteries and human atherosclerosis in vivo. Western blot and fluorescence activated cell-sorting analysis demonstrated that levels of p27
Kip1
, but not p16
INK
, increased during serum deprivation of primary VSMC cultures and caused G
1
arrest. p27
Kip1
inhibited Cdk2 activity, suggesting that Kip CKIs promote G
1
arrest in VSMCs by binding cyclin E/Cdk2. In porcine arteries, p27
Kip1
, but not p16
INK
, was constitutively expressed at low levels. Immediately after balloon injury, cell proliferation increased as p27
Kip1
levels declined. Three weeks after injury, p27
Kip1
was strongly expressed in intimal VSMCs when VSMC proliferation was <2%, suggesting that p27
Kip1
functions as an inhibitor of cell proliferation in injured arteries. In contrast, p16
INK
expression was detected only transiently early after injury. CKI expression was examined in 35 human coronary arteries, ranging from normal to advanced atherosclerosis. p27
Kip1
expression was abundant in nonproliferating VSMCs and macrophages within normal (7 of 8) and atherosclerotic (25 of 27) arteries. p21
Cip1
levels were undetectable in normal arteries but were elevated in atherosclerotic (19 of 27) arteries. p16
INK
could not be detected in normal or atherosclerotic arteries (0 of 35). Thus, the Kip/Cip and INK CKIs have different temporal patterns of expression in VSMCs in vitro and in injured arteries and atherosclerotic lesions in vivo. In contrast to p16
INK
, p27
Kip1
likely contributes to the remodeling process in vascular diseases by the arrest of VSMCs in the G
1
phase of the cell cycle.
Cardiac Physiology in Transgenic Mice
Authors: James Jeanne F. , Hewett Timothy E. , Robbins Jeffrey
Published: June 2012

Abstract
—By use of gene targeting and/or transgenesis, it is now possible to make defined changes in genes whose functions underlie mammalian cardiovascular function. Because of technical and economic considerations, these experiments are largely confined to the mouse. Genetic modification of the loci responsible for aspects of cardiac development, differentiation, and function via gene targeting, as well as modulation of the cardiac protein complement using transgenesis, has begun to provide mouse models of cardiac hypertrophy, dilated cardiomyopathy, and hypertrophic cardiomyopathies. In order to use these animal models fully and explore their phenotypes at the whole organ and whole animal levels, the extension of cardiovascular physiological methodologies to the mouse is imperative. Techniques for exploring aspects of cardiovascular function are well developed for larger animal models, but their modification for the small size of the mouse heart and for the animal’s rapid cardiac cycle has proven to be a formidable challenge, requiring the combined efforts of the molecular biology, physiology, and cardiology communities. We review here the ability of present-day technology to obtain reproducible data on murine cardiac function at the whole organ and animal levels.
Increased Expression of Ca
2+
-Sensitive K
+
Channels in the Cerebral Microcirculation of Genetically Hypertensive Rats
Authors: Liu Yanping , Hudetz Antal G. , Knaus Hans-Guenther , Rusch Nancy J.
Published: June 2012

Abstract
—The Ca
2+
-sensitive K
+
channel (K
Ca
channel) plays a key role in buffering pressure-induced constriction of small cerebral arteries. An amplified current through this channel has been reported in vascular smooth muscle cells obtained from hypertensive animals, implying that the expression or properties of K
Ca
channels may be regulated by in vivo blood pressure levels. In this study, we investigated this hypothesis and its functional relevance by comparing the properties, expression levels, and physiological role of K
Ca
channels in cerebral resistance arteries from normotensive and genetically hypertensive rats. Whole-cell patch-clamp experiments revealed a 4.7-fold higher density of iberiotoxin-sensitive K
Ca
channel current at physiological membrane potentials in spontaneously hypertensive rat (SHR) compared with Wistar-Kyoto (WKY) rat cerebrovascular smooth muscle cells (n=18 and 21, respectively). However, additional single-channel analysis in detached patches showed similar levels of unitary conductance, voltage, and Ca
2+
sensitivity in K
Ca
channels from WKY and from SHR membranes. In contrast, Western analysis using an antibody directed against the K
Ca
channel α-subunit revealed a 4.1-fold increase in the corresponding 125-kD immunoreactive signal in cerebrovascular membranes from SHR compared with WKY rats. The functional impact of this enhanced K
Ca
channel expression was assessed in SHR and WKY rat pial arterioles, which were monitored by intravital microscopy through in situ cranial windows. Progressive pharmacological block of K
Ca
channels by iberiotoxin (0.1 to 100 nmol/L) dose-dependently constricted pial arterioles from SHR and WKY rats (n=6 to 8). The arterioles in SHR constricted 2- to 4-fold more intensely, and vasospasm occurred in some vessels. These data provide the first direct evidence that elevated levels of in situ blood pressure induce K
Ca
channel expression in cerebrovascular smooth muscle membranes. This homeostatic mechanism may critically regulate the resting tone of cerebral arterioles during chronic hypertension. Furthermore, the overexpression of distinct K
+
channel types during specific cardiovascular pathologies may provide for the upregulation of novel disease-specific membrane targets for vasodilator therapies.