bluuebunny/crossref_metadata_2025 huggingface dataset:
Acetylcholine Elicits a Rebound Stimulation of Ca
2+
Current Mediated by Pertussis Toxin–Sensitive G Protein and cAMP-Dependent Protein Kinase A in Atrial Myocytes
Authors: Wang Yong Gao , Lipsius Stephen L.
Published: June 2012

Abstract
Cholinergic inhibition of atrial contraction is typically followed by a rebound positive inotropic response. In the present study, we used a nystatin–perforated patch whole-cell recording method to determine whether acetylcholine (ACh) elicits a rebound stimulation of L-type Ca
2+
current (I
Ca,L
) in cat atrial myocytes. ACh (1 μmol/L) decreased basal I
Ca,L
(−19±2%). Within ≈30 s of returning to ACh-free solution, basal I
Ca,L
exhibited a rebound increase above the control level (+61±7%) that returned to the control level within 4 to 5 minutes. ACh elicited concomitant changes in cell shortening, ie, a decrease followed by a rebound increase. The EC
50
and maximal response of ACh-induced inhibition and rebound stimulation of I
Ca,L
were 1.9×10
−9
mol/L and −30%, respectively, and 2.9×10
−8
mol/L and +64%, respectively. All effects of ACh on I
Ca,L
were blocked by prior exposure to 1 μmol/L atropine or 100 μmol/L AFDX116 and unaffected by 0.2 μmol/L pirenzepine or 1 μmol/L propranolol. In the presence of ACh, exposure to atropine elicited stimulation of I
Ca,L
. ACh-induced inhibition and rebound stimulation of current were independent of external Ca
2+
. Rebound stimulation of I
Ca,L
was associated with a negative shift in the voltage dependence of I
Ca,L
activation. Inhibition of protein kinase A by 50 μmol/L Rp-cAMPs decreased basal I
Ca,L
by 36±1% and abolished the rebound stimulation of I
Ca,L
. Forskolin (0.01 μmol/L) or isoproterenol (0.01 μmol/L) had no effect on basal I
Ca,L
, but each accentuated the rebound increase in I
Ca,L
. When adenylate cyclase was maximally stimulated with 1 μmol/L isoproterenol plus 2 μmol/L forskolin, ACh decreased I
Ca,L
but failed to elicit rebound stimulation of I
Ca,L
. Milrinone (10 μmol/L) increased basal I
Ca,L
by 70±7% and significantly attenuated the rebound stimulation of I
Ca,L
. Exposure to 1 mmol/L 8-bromo-cGMP elicited a small decrease in basal I
Ca,L
, attenuated ACh-induced inhibition, and enhanced the rebound stimulation of I
Ca,L
. Incubation in pertussis toxin prevented all ACh-induced changes in I
Ca,L
. Inhibition of nitric oxide synthase by 100 μmol/L
N
G
-monomethyl-
l
-arginine (L-NMMA) decreased basal I
Ca,L
by −20±5%, prevented ACh-induced inhibition, and markedly attenuated the rebound stimulation of I
Ca,L
. We conclude that in cat atrial myocytes ACh acts via M
2
muscarinic receptors and pertussis toxin–sensitive G protein to inhibit basal I
Ca,L
and that on withdrawal ACh elicits a rebound stimulation of I
Ca,L
. Rebound stimulation of I
Ca,L
is mediated via cAMP-dependent protein kinase A enhanced by ACh-induced inhibition of phosphodiesterase. This mechanism contributes directly to the positive inotropic response that follows cholinergic inhibition of atrial contraction.
In Vivo Adenovirus-Mediated Gene Transfer Via the Pulmonary Artery of Rats
Authors: Schachtner Susan K. , Rome Jonathan J. , Hoyt Robert F. , Newman Kurt D. , Virmani Renu , Dichek David A.
Published: June 2012

Abstract
Gene transfer into the pulmonary vasculature has the potential to be a powerful technique for both investigation of pulmonary pathophysiology and development of genetic therapies for pulmonary vascular disease. To evaluate the potential for in vivo pulmonary arterial gene transfer, we infused adenoviral vectors into the left pulmonary artery of Sprague-Dawley and cotton rats. Access to the left pulmonary artery was obtained by a percutaneous transcatheter approach or through thoracotomy and pulmonary arteriotomy. With the thoracotomy approach, both pulmonary arterial inflow and pulmonary venous outflow were occluded during vector infusion and throughout a subsequent 20-minute dwell period. The success of gene transfer was assessed by staining for evidence of recombinant gene expression in lungs excised at time points ranging from 48 to 72 hours after virus infusion. With the thoracotomy technique, pulmonary gene transfer was successful in 15% of surviving Sprague-Dawley rats and 30% of surviving cotton rats. Percutaneous catheter-based pulmonary gene transfer was not successful. In rats with pulmonary gene transfer, 1% to 8% of total left lung cells expressed the recombinant gene. Recombinant gene expression was found in endothelial cells (0.2% to 18% of total transduced cells), smooth muscle cells (0% to 3%), macrophages (1% to 7%), airway epithelial cells (2% to 50%), and alveolar epithelial cells (38% to 94%). Investigation of the low rate of successful gene transfer in individual animals suggested that insufficient physical contact between the virions and pulmonary cells was the most likely cause. In vivo gene transfer into the rat pulmonary vasculature can be accomplished with adenovirus vectors. The overall efficiency is low, however, and pulmonary arterial infusion of the vectors results in gene transfer primarily into nonvascular cells.
A Retinoic Acid–Induced Clonal Cell Line Derived From Multipotential P19 Embryonal Carcinoma Cells Expresses Smooth Muscle Characteristics
Authors: Blank Randal S. , Swartz Ellen A. , Thompson Maria M. , Olson Eric N. , Owens Gary K.
Published: June 2012

Abstract
Despite intense interest in understanding the differentiation of vascular smooth muscle, very little is known about the cellular and molecular mechanisms that control differentiation of this cell type. Progress in this field has been hampered by the lack of an inducible in vitro system for study of the early steps of smooth muscle differentiation. In this study, we describe a model system in which multipotential mouse P19 embryonal carcinoma cells (P19s) can be induced to express multiple characteristics of differentiated smooth muscle. Treatment of P19s with retinoic acid was associated with profound changes in cell morphology and with the appearance at high frequency of smooth muscle α-actin–positive cells that were absent or present at extremely low frequency in parental P19s. A clonal line derived from retinoic acid–treated P19s (9E11G) stably expressed multiple characteristics of differentiated smooth muscle, including smooth muscle–specific isoforms of α-actin and myosin heavy chain, as well as functional responses to the contractile agonists phenylephrine, angiotensin II, ATP, bradykinin, histamine, platelet-derived growth factor (PDGF)-AA, and PDGF-BB. Additionally, 9E11G cells expressed transcripts for
MHox
, a muscle homeobox gene expressed in smooth, cardiac, and skeletal muscles, but not the skeletal muscle–specific regulatory factors,
MyoD
and myogenin. Results demonstrate that retinoic acid treatment of multipotential P19 cells is associated with formation of cell lines that stably express multiple properties of differentiated smooth muscle. It remains to be determined whether retinoic acid has induced commitment to a smooth muscle cell lineage as opposed to directly (or indirectly) activating genes characteristic of differentiated smooth muscle cells. However, results suggest that this cell system may be of use in attempting to identify genes involved in controlling smooth muscle differentiation and/or lineage determination.
Na
+
-H
+
Exchanger Isoform 1 Phosphorylation in Normal Wistar-Kyoto andSpontaneously Hypertensive Rats
Authors: Siczkowski Martin , Davies Joan E. , Ng Leong L.
Published: June 2012

Abstract
Increased activity of the cellular Na
+
-H
+
exchanger (NHE) has been documented in various cell types in essential hypertension and in vascular myocytes of the spontaneously hypertensive rat (SHR). The mechanism underlying this abnormality is unclear. Because the NHE can be activated by phosphorylation, we examined phosphorylation of the Na
+
-H
+
exchanger isoform 1 (NHE-1) as one possible mechanism for its increased turnover number in cultured vascular myocytes of the SHR. A polyclonal rabbit antibody against a fusion protein consisting of β-galactosidase and the C-terminus of NHE-1 was used to immunoprecipitate
32
P-labeled NHE-1 from cell extracts of SHR and Wistar-Kyoto (WKY) rat vascular myocytes in the absence and presence of 10% fetal calf serum. Immunoprecipitates were separated by SDS-PAGE, and
32
P-labeled NHE-1 was quantified from autoradiographs. Similar amounts of NHE-1 protein were detected on Western blots of the cultured vascular myocytes from SHR and WKY rats. In quiescent cells, NHE-1 was significantly more phosphorylated in SHR myocytes than in WKY myocytes (2.17±0.06-fold enhancement [mean±SEM];
P
<.001, n=8). The addition of fetal calf serum to quiescent cells had no significant effect on the phosphorylation of NHE-1 in SHR myocytes. However, NHE-1 phosphorylation fell transiently in serum-treated WKY myocytes, with recovery to control levels after 20 minutes. Measurement of NHE activity using fluorometry confirmed elevated activity in the quiescent SHR myocytes compared with WKY myocytes. Fetal calf serum led to further enhancement of NHE activity in both cell types. These findings suggest that the increased NHE activity in quiescent SHR myocytes may be correlated with enhanced NHE-1 phosphorylation and that serum stimulates NHE activity in both cell types without a further increase in total NHE-1 phosphorylation, indicating a role for non–phosphorylation-dependent regulatory mechanisms.
Quantification of Myotrophin From Spontaneously Hypertensive and Normal Rat Hearts
Authors: Sil Parames , Mukherjee Debabrata , Sen Subha
Published: June 2012

Abstract
A novel peptide, myotrophin, has been isolated, purified, cloned, and sequenced from the hearts of spontaneously hypertensive rats (SHR) and from dilated cardiomyopathic human heart tissue. Myotrophin accelerates myocyte growth by stimulating protein synthesis (not by altering myocardial cell division). Our successive studies were conducted to evaluate the pathophysiological significance of myotrophin; a solid-phase radioimmunoassay technique was developed for quantifying the protein in hypertrophied and normal hearts. Specific antipeptide antibody was raised in rabbits against a peptide that represents a selected amino acid sequence of a 17–amino acid myotrophin segment by using the multiple antigenic peptide technique. The specificity of the antibody was evaluated by determining the affinity constant after constructing the Scatchard plot obtained from the ratio of bound to free myotrophin against bound myotrophin. The value obtained was 2.61×10
7
L/mol. The specificity was further demonstrated by Western blot analysis, in which a single protein band was obtained in the region of 12 kD. Pretreatment of the antibody with myotrophin completely blocked the binding sites, because no protein band was detected on the immunoblot. The antibody prevented the myocardial protein synthesis induced by myotrophin as revealed by the blockage of the stimulation of [
3
H]leucine incorporation into myocyte protein. Quantification of myotrophin from different heart tissues was achieved by Western blot and dot blot analyses. Amounts of myotrophin present in different dots were determined by using a video image analyzer. The level of myotrophin in the embryonic tissue was found to be similar in male normal and SHR hearts. The level of myotrophin concentration then starts increasing by as early as 3 days of age in SHR. In 9-day-old SHR, the change is statistically significant. In 4-, 8-, 11-, and 17-week-old SHR, myotrophin concentration increased linearly and significantly compared with age- and sex-matched normal control rats. No detectable amount of myotrophin was found in kidney or lung. Our data suggest that myotrophin may play an important role in the pathogenesis of cardiac hypertrophy.
Relationship Between Intracellular Calcium and Contractile Force in Stunned Myocardium
Authors: Gao Wei Dong , Atar Dan , Backx Peter H. , Marban Eduardo
Published: June 2012

Abstract
To elucidate the abnormalities of excitation-contraction coupling in stunned myocardium, we measured [Ca
2+
]
i
and force in thin fura 2–loaded ventricular trabeculae from control or stunned (20 minutes ischemia followed by 20 minutes reflow at 37°C) rat hearts. At any given [Ca
2+
]
o
, force development was significantly lower in the stunned trabeculae than in control trabeculae. In contrast, there was no difference in the amplitude of Ca
2+
transients between the two groups. The steady state force-[Ca
2+
]
i
relationship, assessed by tetanization in the presence of ryanodine, revealed both a decrease in maximal Ca
2+
-activated force and an increase in the [Ca
2+
]
i
required for 50% activation in stunned trabeculae. Postischemic myocardium also exhibited an accelerated rate of diastolic relaxation that was not due to changes in the rate of Ca
2+
transient decay. Destabilization of attached cross-bridges in a quantitative model of cardiac myofibrils accurately reproduced the salient systolic and diastolic features of the stunned phenotype, suggesting an abnormality of the thin filaments. In response to supraphysiological increases in [Ca
2+
]
o
, diastolic [Ca
2+
]
i
and diastolic tone increased much more in stunned trabeculae than in controls, with the frequent occurrence of aftercontractions. This novel experimental model lends further support to the hypothesis that the primary lesion of excitation-contraction coupling resides at the level of the contractile proteins. The finding of enhanced susceptibility to calcium overload helps to rationalize the functional deterioration of stunned myocardium during intense inotropic stimulation and additionally suggests that stunned myocardium may represent a favorable substrate for triggered arrhythmias.
Intraischemic Preconditioning
Authors: Schulz Rainer , Post Heiner , Sakka Samir , Wallbridge David R. , Heusch Gerd
Published: June 2012

Abstract
Ischemic preconditioning (IP) and myocardial hibernation (MH) are both adaptive phenomena during acute myocardial ischemia, characterized by preserved myocardial viability and attenuated alterations of energy metabolism. Recent data from isolated buffer-perfused rabbit hearts pointed to a further link between IP and MH, in that an initial stimulus of no-flow ischemia was required to permit the development of MH during subsequent sustained low-flow ischemia. In the present study, we therefore investigated in the in situ pig heart whether a brief episode of no-flow ischemia enhances the myocardial tolerance to subsequent sustained low-flow ischemia. By blocking ATP-dependent potassium channels, we attempted to further determine whether such increased tolerance to ischemia is related to IP or MH, since blockade of ATP-dependent potassium channels abolishes the cardioprotection achieved by IP but not by MH. In 8 enflurane-anesthetized pigs serving as controls (group 1), the inflow into the cannulated left anterior descending coronary artery was reduced to achieve a 90% reduction in the anterior myocardial work index (sonomicrometry) for 90 minutes. In 15 pigs (group 2), a 10-minute no-flow ischemic episode preceded 80 minutes of sustained ischemia at a blood flow reduction identical to that in pigs of group 1. In 8 additional pigs (group 3), glibenclamide was administered before the 10-minute no-flow ischemic episode. In all pigs after 120 minutes of reperfusion, infarct size (IS, percentage of area at risk) was determined by triphenyltetrazolium chloride staining. In group 2, IS was reduced (6.8±6.0% [mean±SD],
P
<.05) when compared with groups 1 (13.2±9.8%) and 3 (16.7±8.3%). In group 2, subendocardial blood flow of tissue that remained viable averaged 0.06±0.02 mL · min
−1
· g
−1
. This blood flow was lower than that in groups 1 (0.11±0.04 mL · min
−1
· g
−1
,
P
<.05) and 3 (0.10±0.06 mL · min
−1
· g
−1
,
P
=NS), indicating an increased ischemic tolerance of the myocardium in pigs of group 2. Conclusions are as follows: (1) A brief episode of no-flow myocardial ischemia without intermittent reperfusion increases the tolerance to sustained low-flow ischemia. (2) The cardioprotective effect is mediated by activation of ATP-dependent potassium channels and therefore relates to IP rather than to MH.
Low Concentration of Oxidized Low-Density Lipoprotein and Lysophosphatidylcholine Upregulate Constitutive Nitric Oxide Synthase mRNA Expression in Bovine Aortic Endothelial Cells
Authors: Hirata Ken-ichi , Miki Nobuhiko , Kuroda Yuichi , Sakoda Tsuyoshi , Kawashima Seinosuke , Yokoyama Mitsuhiro
Published: June 2012

Abstract
Endothelium-dependent relaxation is markedly reduced in atherosclerotic arteries. Recently, the endothelium-dependent relaxing factor has been identified as nitric oxide (NO). We used RNase protection assay and immunoblotting to elucidate the effect of atherogenic lipoprotein on the expression of constitutive NO synthase (cNOS) mRNA and protein levels in bovine aortic endothelial cells. Twenty-four-hour exposure to a low concentration of oxidized low-density lipoprotein (10 μg protein/mL) upregulated cNOS mRNA levels (2.4±0.4-fold,
P
<.01). However, native low-density lipoprotein and high-density lipoprotein did not have any effect on cNOS mRNA levels. Furthermore, 5 μg/mL of lysophosphatidylcholine (LPC) also upregulated cNOS mRNA levels (2.6±0.5-fold,
P
<.01) at 8 hours. This action of LPC was abolished with cycloheximide but not with staurosporine. We concluded that atherogenic lipoproteins upregulate cNOS mRNA and protein levels in bovine aortic endothelial cells. This observation supports the hypothesis that an impairment of endothelium-dependent vasodilatation in atherosclerotic vessels may not be due to a decrease in cNOS expression. Moreover, the LPC action on cNOS mRNA levels requires new protein synthesis.
Nitric Oxide Modulates the Expression of Monocyte Chemoattractant Protein 1 in Cultured Human Endothelial Cells
Authors: Zeiher Andreas M. , Fisslthaler Beate , Schray-Utz Beate , Busse Rudi
Published: June 2012

Abstract
The recruitment of monocytes into the arterial wall is one of the earliest events in the pathogenesis of atherosclerosis. Since monocyte chemoattractant protein 1 (MCP-1) plays a key role in the subendothelial recruitment of monocytes, we tested whether nitric oxide (NO) modulates the expression of MCP-1 in cultured human endothelial cells. Inhibition of basal NO production by
N
G
-nitro-
l
-arginine (L-NAG) upregulates endothelial MCP-1 mRNA expression (250±20%) and protein secretion. Exogenous addition of NO dose-dependently decreased MCP-1 mRNA expression and secretion. Changes in MCP-1 mRNA expression and protein secretion were paralleled by corresponding changes in chemotactic activity of cell-conditioned media for monocytes. An MCP-1 antibody reduced monocyte chemotactic activity by 85% and completely abolished the increased monocyte chemotactic activity induced by the inhibition of NO production. Elevation of endothelial cGMP levels had no significant effect on MCP-1 mRNA expression. Inhibition of basal endothelial NO production by L-NAG increased binding activity of a nuclear factor κB (NF-κB)–like transcriptional regulatory factor, whereas exogenous addition of NO decreased NF-κB–like binding activity during stimulation with tumor necrosis factor-α. Thus, NO modulates MCP-1 expression and monocyte chemotactic activity secreted by human umbilical vein endothelial cells (HUVECs) in culture. The activation of NF-κB–like transcriptional regulatory proteins by inhibition of NO suggests a molecular link between an oxidant-sensitive transcriptional regulatory mechanism and NO synthesis in HUVECs.
Dithionite Increases Radical Formation and Decreases Vasoconstriction in the Lung
Authors: Archer Stephen L. , Hampl Václav , Nelson Daniel P. , Sidney Erika , Peterson Douglas A. , Weir E. Kenneth
Published: June 2012

Abstract
Dithionite is a powerful reducing agent used to deoxygenate hemoglobin and create anaerobic conditions in vitro. Recently, dithionite has been used as a convenient means of creating “hypoxia” in experiments studying the O
2
sensor in the pulmonary circulation and carotid body. We evaluated the hypothesis that hypoxia created by hypoxic ventilation and that created by dithionite have different effects on the pulmonary circulation. In vitro, dithionite (10
−5
to 10
−3
mol/L), added to oxygenated Krebs’ solution, rapidly created superoxide anion in a dose-dependent manner. Dithionite consumed O
2
in parallel with the generation of superoxide radical, with both processes peaking within seconds. Anoxia was sustained only if resupply of O
2
was prevented. In isolated rat lungs (whether perfused with autologous blood or Krebs’ solution), hypoxic ventilation alone lowered perfusate P
o
2
from ≈140 to 40 mm Hg and decreased lung levels of activated oxygen species (AOS), measured by luminol-enhanced chemiluminescence, before the onset of hypoxic pulmonary vasoconstriction. Constrictor responses to angiotensin II and KCl were not impaired by intermittent hypoxic challenges, and lung weight did not increase. In contrast, dithionite impaired constrictor responses of the Krebs’ solution–perfused lungs to all vasoconstrictors tested and increased lung weight. When given as a bolus (5×10
−3
mol/L) into the pulmonary artery during normoxic ventilation, dithionite caused no vasoconstriction and only briefly lowered P
o
2
(because of constant resupply of O
2
from the alveoli). When superimposed on hypoxic ventilation, dithionite further lowered P
o
2
from ≈40 to ≈0 mm Hg and caused additional constriction. Unlike hypoxic ventilation, dithionite increased AOS production. Antioxidant enzymes diminished dithionite-induced radical production and diminished the loss of vascular reactivity and lung edema. In conclusion, unlike hypoxic ventilation, dithionite causes edema and loss of vascular reactivity in the lung by generating superoxide anion and hydrogen peroxide. Hypoxia elicited by dithionite is not equivalent to authentic hypoxia because of the obligatory associated generation of AOS. Dithionite usage should not be substituted for authentic hypoxia in studies of O
2
sensing.
Osmotic Compression of Single Cardiac Myocytes Eliminates the Reduction in Ca
2+
Sensitivity of Tension at Short Sarcomere Length
Authors: McDonald Kerry S. , Moss Richard L.
Published: June 2012

Abstract
According to the Frank-Starling relation, cardiac output varies as a function of end-diastolic volume of the ventricle. The cellular basis of the relation is thought to involve length-dependent variations in Ca
2+
sensitivity of tension; ie, as sarcomere length is increased in cardiac muscle, Ca
2+
sensitivity of tension also increases. One possible explanation for this effect is that the decrease in myocyte diameter as muscle length is increased reduces the lateral spacing between thick and thin filaments, thereby increasing the likelihood of cross-bridge interaction with actin. To examine this idea, we measured the effects of osmotic compression of single skinned cardiac myocytes on Ca
2+
sensitivity of tension. Single myocytes from rat enzymatically digested ventricles were attached to a force transducer and piezoelectric translator, and tension-pCa relations were subsequently characterized at short sarcomere length (SL), at the same short SL in the presence of 2.5% dextran, and at long SL. The pCa (−log[Ca
2+
]) for half-maximal tension (ie, pCa
50
) increased from 5.54±0.09 to 5.65±0.10 (n=7, mean±SD,
P
<.001) as SL was increased from ≈1.85 to ≈2.25 μm. Osmotic compression of myocytes at short length also increased Ca
2+
sensitivity of tension, shifting tension-pCa relations to [Ca
2+
] levels similar to those observed at long length (pCa
50
, 5.68±0.11). These results support the idea that the length dependence of Ca
2+
sensitivity of tension in cardiac muscle arises in large part from the changes in interfilament lattice spacing that accompany changes in SL.
Measurement of Platelet-Activating Factor in a Canine Model of Coronary Thrombosis and in Endarterectomy Samples From Patients With Advanced Coronary Artery Disease
Authors: Mueller Howard W. , Haught Courtney A. , McNatt Janice M. , Cui Kexin , Gaskell Simon J. , Johnston Dennis A. , Willerson James T.
Published: June 2012

Abstract
Platelet-activating factor (PAF, 1-
O
-alkyl-2-acetyl-
sn
-glycero-3-phosphocholine) is a potent phospholipid mediator of numerous inflammatory and thrombotic responses. The purpose of this study was to determine if PAF synthesis is elevated in damaged coronary arteries after a sustained period of cyclic flow variation (CFV), a phenomenon caused by alternating periods of thrombosis and reperfusion at sites of endothelial injury. Cyclic flow was established and maintained in the left anterior descending coronary arteries (LADs) of 10 dogs. After 8 hours of CFV, the section of damaged LAD containing the thrombus and control sections of the circumflex artery, carotid artery, and saphenous vein was excised, and the total lipids were extracted. The PAF was then purified by silica column chromatography and high-performance liquid chromatography and assayed by both a rabbit platelet bioassay and a PAF radioimmunoassay. With the platelet bioassay, PAF levels of 8.9±4.0 (range, 4.8 to 15.5) pg/mg wet wt were found in the damaged LADs from the 10 dogs. This PAF bioactivity was completely inhibited by a PAF receptor antagonist. When the radioimmunoassay was used, slightly higher PAF levels of 16.3±12.9 (range, 4.5 to 41.8) pg/mg wet wt were observed in the LADs. Overall, these PAF levels were 3- to 64-fold higher than in the control vessels when either assay method was used. Although increases in PAF were observed in the damaged LADs, measurements of PAF in blood samples taken from the LAD and the aorta (control) failed to demonstrate any site-specific increase of PAF in the blood. In related experiments, PAF was also measured in 23 endarterectomy samples taken from the coronary arteries of 16 patients with severe atherosclerosis. The PAF levels in these samples were highly variable (2.9±2.2 [range, 0.3 to 8.5] pg/mg wet wt) and showed no correlation with tissue mass, suggesting that PAF is affected by factors other than the simple presence of atherosclerotic tissue in the vessel. These findings provide direct evidence that PAF is synthesized locally at the site of endothelial injury during thrombosis and that PAF accumulates in the atherosclerotic plaque of some patients with advanced coronary artery disease.
Angiotensin II Partly Mediates Mechanical Stress–Induced Cardiac Hypertrophy
Authors: Yamazaki Tsutomu , Komuro Issei , Kudoh Sumiyo , Zou Yunzeng , Shiojima Ichiro , Mizuno Takehiko , Takano Hiroyuki , Hiroi Yukio , Ueki Kohjiro , Tobe Kazuyuki , Kadowaki Takashi , Nagai Ryozo , Yazaki Yoshio
Published: June 2012

Abstract
We have previously shown that mechanical stress induces activation of protein kinases and increases in specific gene expression and protein synthesis in cardiac myocytes, all of which are similar to those evoked by humoral factors such as growth factors and hormones. Many lines of evidence have suggested that angiotensin II (Ang II) plays a vital role in cardiac hypertrophy, and it has been reported that secretion of Ang II from cultured cardiac myocytes was induced by mechanical stretch. To examine the role of Ang II in mechanical stress–induced cardiac hypertrophy, we stretched neonatal rat cardiac myocytes in the absence or presence of the Ang II receptor antagonists saralasin (an antagonist of both type 1 and type 2 receptors), CV-11974 (a type 1 receptor–specific antagonist), and PD123319 (a type 2 receptor–specific antagonist). Stretching cardiac myocytes by 20% using deformable silicone dishes rapidly increased the activities of mitogen-activated protein (MAP) kinase kinase activators and MAP kinases. Both saralasin and CV-11974 partially inhibited the stretch-induced increases in the activities of both kinases, whereas PD123319 showed no inhibitory effects. Stretching cardiac myocytes increased amino acid incorporation, which was also inhibited by approximately 70% with the pretreatment by saralasin or CV-11974. When the culture medium conditioned by stretching cardiocytes was transferred to nonstretched cardiac myocytes, the increase in MAP kinase activity was observed, and this increase was completely suppressed by saralasin or CV-11974. These results suggest that Ang II plays an important role in mechanical stress–induced cardiac hypertrophy and that there are also other (possibly nonsecretory) factors to induce hypertrophic responses.
Control of Carotid Vasomotor Tone by Local Renin–Angiotensin System in Normotensive and Spontaneously Hypertensive Rats
Authors: Caputo Lidia , Tedgui Alain , Lévy Bernard I.
Published: June 2012

Abstract
To investigate the relation between the tissue renin–angiotensin system (RAS) and the local vasomotor tone of large arteries, we used in vitro isolated carotid arteries from 14-week-old Wistar-Kyoto rats (WKY; n=80) and spontaneously hypertensive rats (SHR; n=80). Diameters were measured with the use of an ultrasonic echo-tracking system (12 MHz) under flow (2 mL/min) (F+) or no-flow (Fo) conditions, with intact endothelium (Endo+) or after endothelium removal (Endo−). The role of tissue RAS was assessed by incubating isolated carotid arteries with an angiotensin-converting enzyme inhibitor (ACE I; lisinopril, 10
−6
mol/L) or with a specific antagonist of angiotensin II AT
1
receptors (AT
1A
; losartan, 10
−6
mol/L). In addition, maximal dilation of carotid arteries was measured after poisoning with KCN (100 mg/L). In all experiments, KCN significantly increased carotid diameters (WKY, 23±0.9%; SHR, 19±0.8%;
P
<.001 versus control conditions). In intact carotid arteries, flow caused significant dilation in WKY (7±0.5%,
P
<.001) but had no effect in SHR. In the presence or absence of flow, ACE I and AT
1A
induced similar dilations in both strains, and a specific antagonist of bradykinin B
2
receptors (Hoe 140, 10
−7
mol/L) had no effect on ACE I–induced dilation. After endothelium removal, carotid artery diameters were significantly increased (
P
<.001) in both strains, although more in SHR (13±0.8%) than in WKY (8±1.1%) (
P
<.001). Also, flow did not modify the diameter of deendothelialized vessels and ACE I had no effect in either strain. In contrast, AT
1A
increased diameters further (WKY, 4±0.4%; SHR, 3±0.6%;
P
<.01). The results of the present study suggest that in in vitro isolated intact carotid artery, (1) the endothelium exerts a basal vasoconstrictor effect that is larger in SHR than in WKY, which could participate in the relative insensitivity to flow in SHR; (2) in the absence of exogenous substrate the vessel wall is able to produce angiotensin II, which is active locally on the vascular tone; and (3) inhibition of angiotensin II formation, not degradation of bradykinin, appears to be responsible for the local relaxing effect of ACE inhibition.
Differential Phospholamban Gene Expression in Murine Cardiac Compartments
Authors: Koss Kimberly L. , Ponniah Sathivel , Jones W. Keith , Grupp Ingrid L. , Kranias Evangelia G.
Published: June 2012

Abstract
Phospholamban, the regulator of the Ca
2+
pump in cardiac sarcoplasmic reticulum, is differentially expressed between murine atrial and ventricular muscles. Quantitative analyses of RNA isolated from atrial flaps and ventricular apices indicated that the phospholamban gene transcript copy number is 2.5-fold higher in the ventricle compared with the atrium of the FVB/N mouse and 6-fold higher in the ventricle compared with the atrium of the B6D2/F1 mouse strain. These findings were corroborated by in situ hybridization studies of cardiopulmonary sections from both murine strains, and phospholamban transcripts were also observed in pulmonary myocardia of both strains. Analyses of phospholamban transcript levels relative to α-myosin heavy chain (α-MHC) revealed a 3-fold higher phospholamban abundance in the ventricle compared with the atrium of the FVB/N murine strain. However, the relative mRNA level of Ca
2+
-ATPase (ratio of sarcoplasmic reticulum Ca
2+
-ATPase [SERCA2] to α-MHC) in the ventricle was 80% of that in the atrium. Consequently, the relative ratio of phospholamban to SERCA2 mRNA was 4.2-fold lower in the atrium than in the ventricle. The lower transcript ratio of phospholamban to SERCA2 in the atrium was associated with significantly shortened times to half-relaxation (17.40±0.71 milliseconds for atrium versus 30.58±2.04 milliseconds for ventricle), assessed in isolated superfused cardiac tissue preparations recorded at maximum length tension. Contraction times, measured as times to peak tension, were also significantly shortened in atrial muscle (27.36±0.82 milliseconds) compared with ventricular muscle (44.60±2.55 milliseconds), assessed in the same tissue preparations. These findings suggest that phospholamban gene expression is differentially regulated in murine atrial and ventricular muscles and that this differential expression may be associated with differences in the contractile parameters of these cardiac compartments.
Biphasic Defibrillation Waveforms Reduce Shock-Induced Response Duration Dispersion Between Low and High Shock Intensities
Authors: Tovar Oscar H. , Jones Janice L.
Published: June 2012

Abstract
Mechanisms underlying defibrillation threshold reduction with biphasic waveforms remain unclear. The interaction of local shock-induced voltage gradients, which change with distance from the shocking electrode, and the state of membrane repolarization results in different cellular responses that may influence the success of defibrillation. We used intracellular microelectrodes and S
1
S
2
pacing protocols in myocardial cell aggregates to determine the effects of shock intensity and waveform on refractory period responses during simulated fibrillation (3 s of S
1
pacing at 180-ms cycle length). We simulated defibrillation by electric field stimulation S
2
using 8-ms monophasic (MS
2
) and 4/4 biphasic (BS
2
) waveforms (65% total tilt) delivered at intensities of 1.5, 3, and 5 times S
1
diastolic threshold, or ≈2 to 7 V/cm. Responses following MS
2
varied with S
2
intensity and coupling interval (
P
<.001). When averaged over the last 10 ms of the refractory period, MS
2
produced a negligible response (8.8±1.4 ms) at 1.5 times diastolic threshold and a prolonged response (53.0±3.1 ms) at 5 times diastolic threshold (
P
<.01). In contrast, BS
2
response duration did not change significantly (
P
=NS) between 1.5 times diastolic threshold (35.1±12.6 ms) and 5 times diastolic threshold (46.2±2.7 ms). Our results suggest that biphasic waveforms not only prolong response duration at low shock intensity but reduce dispersion of refractoriness produced by differing local potential gradients generated by defibrillation shocks compared with monophasic waveforms. Preventing dispersion of refractoriness and prolonging shock-induced responses may improve biphasic waveform efficacy at low shock intensity.
Characterization of 5′ End of Human Thromboxane Receptor Gene
Authors: D’Angelo Drew D. , Davis Michael G. , Houser William A. , Eubank Jeremy J. , Ritchie Michael E. , Dorn Gerald W.
Published: June 2012

Abstract
Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5′ flanking region of the thromboxane receptor gene. The exon-intron structure of the 5′ portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5′ flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues ≈560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5′ flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into plateletlike K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5′ deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester–responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located ≈1.8 kb 5′ from the transcription initiation site. These studies are the first to determine the structure and organization of the 5′ end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2–like nuclear factor binding to upstream promoter elements. These findings strongly suggest that the mechanism for previously described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcription in platelet-progenitor cells.
Determinants of Antiarrhythmic Drug Action
Authors: Snyders Dirk J. , Yeola Sarita W.
Published: June 2012

Abstract
The molecular basis of antiarrhythmic drug action is still poorly understood. We recently reported that block of the human cardiac hKv1.5 channel by quinidine displayed similarity with internal quaternary ammonium block of squid and
Shaker
potassium channels. To gain further insight into the molecular determinants of the affinity and the stereoselectivity of antiarrhythmic drug action, we studied the effects of quinine (a diastereomer of quinidine), clofilium (a quaternary ammonium class III agent), and tetrapentylammonium (TPeA, a biophysical reference probe for the internal quaternary ammonium binding site). For all compounds, block was voltage dependent, with a steep increase over the voltage range of channel opening and a superimposed weaker voltage dependence at more positive potentials. The latter electrostatic component was similar for all drugs, consistent with a binding reaction sensing ≈20% of the transmembrane electrical field. Clofilium and TPeA displayed a higher apparent affinity (0.15 and 0.28 μmol/L, respectively), and quinine displayed a lower one (21 μmol/L) compared with quinidine (6.2 μmol/L). Block development upon depolarization was time dependent for clofilium and TPeA but slow compared with quinidine. A time-dependent component was difficult to resolve for quinine, but the time course of deactivating tail currents was slower than in the control condition. The resulting crossover phenomenon was also observed for the quaternary drugs. Compared with TPeA alone, the combined application of quinine and TPeA resulted in a reduced current that decayed slower, consistent with competition. When a bimolecular model for open-channel block was used, the apparent association rate constants for these drugs were found to be similar [range, 4.5 to 7.7 (μmol/L)
−1
· s
−1
]. The apparent dissociation rate constants for clofilium (1.9 s
−1
) and TPeA (3.6 s
−1
) were smaller compared with quinidine (34 s
−1
), whereas that for quinine was faster (≥125 s
−1
). This large range in dissociation rate constants could explain the differences between these drugs both in kinetics and affinity. The results are consistent with a general model in which these agents act as cationic open-channel blockers but with an affinity largely determined by the intrinsic stability of the drug-receptor complex. Hydrophobic interactions are most likely involved in this stabilization of binding.
Corticosterone Metabolism and Effects on Angiotensin II Receptors in Vascular Smooth Muscle
Authors: Ullian Michael E. , Walsh Lyle G.
Published: June 2012

Abstract
It has been postulated that mineralocorticoids can bind to corticosteroid receptors in the kidney, because glucocorticoids are metabolized to inactive compounds. The present study was performed to delineate glucocorticoid metabolism by rat vascular tissue and to determine the activity of these metabolites. Vascular segments converted 25% to 30% of corticosterone (compound B), the major glucocorticoid in the rat, to 11-dehydrocorticosterone (compound A) but not to aldosterone or 6β-hydroxycorticosterone. In cultured vascular smooth muscle cells, 10% of compound B was converted to compound A, whereas >60% of compound A was converted to compound B. The 11β-hydroxysteroid dehydrogenase inhibitor carbenoxolone (1 μmol/L) completely blocked conversion in both directions. Whereas 6β-hydroxycorticosterone did not upregulate angiotensin II receptor binding (a marker for corticosteroid action in vascular smooth muscle), compound A caused concentration-dependent upregulation. Compound A was almost (75%) as effective and as potent as compound B in upregulating angiotensin II binding. Upregulation elicited by exposure to compound A persisted in the presence of 1 μmol/L carbenoxolone, which completely prevented the conversion of compound A to compound B. Compound A, even in the presence of carbenoxolone, effected other glucocorticoid actions by inhibiting cell growth and potentiating angiotensin II–stimulated inositol phosphate formation. In summary, compound B and compound A are interconverted in vascular tissue, and the latter displays significant glucocorticoid action. The concentration excess of compound B in the circulation and the activity of its metabolite compound A will make it difficult for mineralocorticoids to gain access to corticosteroid receptors in the vasculature.
cGMP and Atrial Natriuretic Factor Regulate Cell Volume of Rabbit Atrial Myocytes
Authors: Clemo Henry F. , Baumgarten Clive Marc
Published: June 2012

Abstract
Atrial natriuretic factor (ANF) reduces the volume of atrial myocytes by inhibiting Na
+
/K
+
/2Cl
−
cotransport. We determined the role of cGMP and cAMP in ANF-induced shrinkage by using digital video microscopy to measure cell volume; volumes are reported relative to control. ANF (1 μmol/L) reversibly reduced atrial cell volume from 1.0 to 0.915±0.005 (mean±SEM). This effect was mimicked by 10 μmol/L 8-bromo-cGMP (8-Br-cGMP), which decreased myocyte volume to 0.894±0.007 with an ED
50
of 0.99±0.05 μmol/L. In contrast, 100 μmol/L 8-bromo-cAMP (8-Br-cAMP) did not affect volume, and activating the cAMP pathway with 100 μmol/L 8-Br-cAMP did not alter the volume decrease caused by 8-Br-cGMP or ANF. Inhibition of Na
+
/K
+
/2Cl
−
cotransport with bumetanide (1 μmol/L) also reduced cell volume and prevented further shrinkage on subsequent exposure to 8-Br-cGMP. Similarly, 8-Br-cGMP (10 μmol/L) prevented further shrinkage by ANF. Block of Na
+
-H
+
exchange, a participant in volume regulation in other cells, did not alter the response to 8-Br-cGMP. More evidence implicating cGMP was obtained by altering its metabolism. LY83583 (10 μmol/L), a guanylate cyclase inhibitor, blocked ANF-induced cell shrinkage. Zaprinast (100 μmol/L), a cGMP-specific phosphodiesterase inhibitor, markedly potentiated the effect of a threshold concentration of ANF (0.01 μmol/L). The actions of ANF, LY83583, and zaprinast on cGMP levels were verified by radioimmunoassay. These data strongly support the idea that the cGMP cascade is the intracellular signaling pathway responsible for ANF-induced atrial cell shrinkage. ANF activates guanylate cyclase, and the subsequent rise in cGMP inhibits Na
+
/K
+
/2Cl
−
cotransport. This reduces the uptake of osmolytes, and cell shrinkage follows. In addition, LY83583 caused a small swelling and zaprinast caused a small shrinkage in the absence of other agents. This suggests that physiological levels of cGMP partially inhibit Na
+
/K
+
/2Cl
−
cotransport and help set isosmotic cell volume. It is proposed that ANF-induced elevation of atrial cGMP and the ensuing cell shrinkage serve as a negative-feedback mechanism limiting stretch-induced ANF secretion.
Differential Changes in Cardiac Phospholamban and Sarcoplasmic Reticular Ca
2+
-ATPase Protein Levels
Authors: Kiss Eva , Ball Nancy A. , Kranias Evangelia G. , Walsh Richard A.
Published: June 2012

Abstract
The objective of this study was to elucidate the role of the sarcoplasmic reticulum (SR) in the transition from compensated pressure-overload hypertrophy (increased left ventricular [LV] mass, normal LV function, and no pulmonary congestion) to congestive heart failure (increased LV mass, depressed LV function, and pulmonary congestion). To address this issue, the descending thoracic aorta was banded for 4 and 8 weeks in adult guinea pigs, and the changes in isovolumic LV mechanics, SR Ca
2+
transport, and SR protein levels were determined and compared with age-matched sham-operated control animals. A subgroup of the 8-week banded animals manifested the congestive heart failure phenotype with diminished developed LV pressure normalized by LV mass, reduced rates of LV pressure development and relaxation, and markedly increased lung weight–to–body weight ratios. The cardiac mechanical and morphometric changes were associated with depressed protein levels of the SR Ca
2+
-ATPase (85% of the control) and phospholamban (65% of the control) assessed by quantitative immunoblotting. Resultant rates of SR Ca
2+
uptake (V
max
) and the affinity of SR Ca
2+
-ATPase for Ca
2+
(EC
50
) were significantly depressed [32±6 nmol Ca
2+
· min
−1
· mg
−1
and 0.59±0.12 (μmol/L)/L, respectively] compared with the 8-week sham-operated control animals [40±1 nmol Ca
2+
· min
−1
· mg
−1
and 0.40±0.05 (μmol/L)/L, respectively]. We conclude that this model of pressure overload–induced cardiac failure is associated with (1) diminished LV force development, rates of pressure development, and decay; (2) depressed protein expression of the Ca
2+
-cycling proteins SR Ca
2+
-ATPase and phospholamban; and (3) decreased V
max
and affinity of the SR Ca
2+
-ATPase for Ca
2+
. These findings implicate these Ca
2+
-cycling proteins in the pathogenesis of congestive heart failure.
The Mechanically Active Domain of Titin in Cardiac Muscle
Authors: Trombitás Károly , Jin Jian-Ping , Granzier Henk
Published: June 2012

Abstract
One of the main contributors to passive tension of the myocardium is titin. However, it is not exactly known what portions of this ≈1 μm-long molecule are anchored in the sarcomere (hence, are rendered inelastic) and what portions are elastic (hence, are mechanically active in developing passive tension). We assessed the length of the elastic domain of cardiac titin by ultrastructural and mechanical methods. Single cardiac myocytes were stretched by various amounts, and while in the stretched state, they were processed for immunoelectron microscopy. Several monoclonal anti-titin antibodies were used, and the locations of the titin epitopes in the sarcomere were studied as a function of sarcomere length. Only a small fraction (5% to 10%) of the ≈1000-nm-long molecule behaved elastically under physiological conditions. This mechanically active domain is located close to the A/I junction, and its contour length when unstretched is estimated at ≈50 to 100 nm. In sarcomeres that are slack (length ≈1.85 μm), the mechanically active domain is folded on top of itself, and the length of the domain reaches an elastic limit of ≈550 nm in sarcomeres that are ≈2.9 μm long.