bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of NO Modulation on Cardiac Arrhythmias in the Rat Isolated Heart
Authors: Pabla Ravinder , Curtis Michael J.
Published: June 2012

Abstract
It has been proposed that NO may function as an endogenous cardioprotectant. We have investigated whether modulation of NO levels (detected in coronary effluent by chemiluminescence) by a blocker of its synthesis, by supplementation of its precursor, and by administration of an NO donor can influence reperfusion arrhythmias in the isolated rat heart. Rat hearts were perfused with modified Krebs’ solution and subjected to 5, 35, or 60 minutes of left regional ischemia followed by 10 minutes of reperfusion.
N
G
-Nitro-
l
-arginine methyl ester (L-NAME), which blocks NO synthase, increased the incidence of reperfusion-induced ventricular fibrillation (VF) from 5% in the control condition to 35% after 60 minutes of ischemia (n=20,
P
<.05). The profibrillatory effect of L-NAME was prevented in hearts coperfused with 1 or 10 mmol/L
l
-arginine (an NO precursor) but persisted in hearts coperfused with
d
-arginine (1 mmol/L). L-NAME did not increase VF susceptibility in hearts reperfused after 5 or 35 minutes of ischemia. L-NAME caused sinus bradycardia (264±10 versus 309±5 bpm in control groups,
P
<.05) and reduced coronary flow before ischemia (6.2±0.6 versus 9.2±0.6 mL · min
−1
· g
−1
tissue in controls,
P
<.05). L-NAME reduced coronary effluent NO levels after 60 minutes of ischemia; during the first minute of reperfusion, values were reduced from 1457±422 to 812±228 pmol · min
−1
· g
−1
(
P
<.05). This effect was prevented by coperfusion with
l
-arginine (10 344±1730 pmol · min
−1
· g
−1
,
P
<.05). Qualitatively similar changes occurred with other durations of ischemia, but the effects of L-NAME were not significant. L-NAME had no effect on QT interval; eg, values after 5 minutes of ischemia were 76±7 and 85±5 milliseconds in control and L-NAME–treated groups, respectively (n=20,
P
=NS). The NO donor sodium nitroprusside (10 μmol/L) significantly increased coronary flow 1 minute before ischemia (15.4±1.1 versus 9.2±0.6 mL · min
−1
· g
−1
tissue and coronary effluent NO levels (from 1122±122 to 4093±1466 pmol · min
−1
· g
−1
,
P
<.05). Sodium nitroprusside prevented the proarrhythmic effect of L-NAME and maintained coronary effluent NO levels during reperfusion. NO appears to function as an endogenous cardioprotectant antifibrillatory factor in rat heart during reperfusion following sustained ischemia.
Brain ‘Ouabain’ Mediates Sympathetic Hyperactivity in Congestive Heart Failure
Authors: Leenen Frans H. H. , Huang Bing S. , Yu Huilian , Yuan Baoxue
Published: June 2012

Abstract
In congestive heart failure (CHF), endogenous compounds with ouabainlike activity (OLA) may contribute to the maintenance of the circulatory homeostasis by peripheral as well as central effects. In the present study, we assessed changes in peripheral (plasma and left ventricle) and central (pituitary, hypothalamus, pons, and cortex) OLA in two animal models of CHF and determined whether brain OLA mediates sympathetic hyperactivity in CHF. Cardiomyopathic hamsters with their controls were studied at 9 months of age for tissue OLA. Rats were studied 4 weeks after acute coronary artery ligation for tissue OLA and sympathetic activity. In both models, left ventricular end-diastolic pressure was markedly increased. CHF was associated with significant increases in both plasma and tissue OLA in both models. In the brain, the most marked (twofold to threefold) increases occurred in the hypothalamus. In vitro, all OLA measured could be blocked by antibody Fab fragments (Digibind). Conscious rats with CHF showed elevated plasma catecholamines and enhanced responses of mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) to air stress and to intracerebroventricular (ICV) injection of the α
2
-adrenergic receptor agonist guanabenz compared with sham-operated rats. ICV administration of the Fab fragments did not change resting RSNA or responses to air stress at 1 hour. However, 18 hours after injection of the Fab fragments, resting RSNA levels had significantly decreased compared with the control values, and plasma catecholamine levels had decreased to control values. Moreover, the magnitudes of the increases or decreases in MAP, HR, and RSNA to air stress or ICV guanabenz had markedly decreased as well and were now similar to those observed in sham-operated control animals. The present results show that the development of CHF in two animal models is associated with marked increases in both peripheral and brain OLA. Brain OLA appears to mediate the increase in resting sympathetic tone and enhanced sympathoexcitatory responses to stress.
Selectivity of Connexin-Specific Gap Junctions Does Not Correlate With Channel Conductance
Authors: Veenstra Richard D. , Wang Hong-Zang , Beblo Dolores A. , Chilton Mark G. , Harris Andrew L. , Beyer Eric C. , Brink Peter R.
Published: June 2012

Abstract
Connexins form a variety of gap junction channels that vary in their developmental and tissue-specific levels of expression, modulation of gating by transjunctional voltage and posttranslational modification, and unitary channel conductance (γ
j
). Despite a 10-fold variation in γ
j
, whether connexin-specific channels possess distinct ionic and molecular permeabilities is presently unknown. A major assumption of the conventional model for a gap junction channel pore is that γ
j
is determined primarily by pore diameter. Hence, molecular size permeability limits should increase and ionic selectivity should decrease with increasing channel γ
j
(and pore diameter). Equimolar ion substitution of 120 mmol/L KCl for potassium glutamate was used to determine the unitary conductance ratios for rat connexin40 and connexin43, chicken connexin43 and connexin45, and human connexin37 channels functionally expressed in communication-deficient mouse neuroblastoma (N2A) cells. Comparison of experimental and predicted conductance ratios based on the aqueous mobilities of all ions according to the Goldman-Hodgkin-Katz current equation was used to determine relative anion-to-cation permeability ratios. Direct correlation of junctional conductance with dye transfer of two fluorescein-derivatives (2 mmol/L 6-carboxyfluorescein or 2′,7′-dichlorofluorescein) was also performed. Both approaches revealed a range of selectivities and permeabilities for all five different connexins that was independent of channel conductance. These results are not consistent with the conventional simple aqueous pore model of a gap junction channel and suggest a new model for connexin channel conductance and permselectivity based on electrostatic interactions. Divergent conductance and permeability properties are features of other classes of ion channels (eg, Na
+
and K
+
channels), implying similar mechanisms for selectivity.
Synergistic Actions of Glucagon and Miniglucagon on Ca
2+
Mobilization in Cardiac Cells
Authors: Sauvadet Anne , Rohn Troy , Pecker Françoise , Pavoine Catherine
Published: June 2012

Abstract
It has been recently shown that the physiological processing of glucagon into its C-terminal (19-29) fragment, miniglucagon, by cardiac cells was essential for the contractile positive inotropic effect of the hormone. However, the mechanisms underlying the effects of miniglucagon remained undetermined. In the present study, we assessed the effects of miniglucagon on Ca
2+
homeostasis in embryonic chick ventricular myocytes. In quiescent cells, short-term applications of 0.1 nmol/L miniglucagon markedly increased the accumulation of
45
Ca into intracellular compartments resistant to digitonin lysis and sensitive to caffeine. Ca
2+
accumulation into the sarcoplasmic reticular (SR) store was further attested by fura 2 imaging studies on quiescent or prestimulated cells: miniglucagon potentiated Ca
2+
release from the SR compartment triggered by caffeine and evoked a rise in cytosolic Ca
2+
when applied on cells pretreated with 1 μmol/L thapsigargin, a specific inhibitor of the SR Ca
2+
pump. Glucagon alone produced a small cytosolic Ca
2+
signal that was considerably amplified by miniglucagon. The action of glucagon was mimicked by 8-bromo-cAMP and was blocked by isradipine, suggesting that it relied on the activation of L-type Ca
2+
channels, via phosphorylation. We conclude that the combined actions of miniglucagon and glucagon on Ca
2+
accumulation into SR stores and Ca
2+
release from the same stores are likely to support the positive inotropic effect elicited in vivo by glucagon on heart contraction.
Adenosine A
1
Stimulation Activates δ-Protein Kinase C in Rat Ventricular Myocytes
Authors: Henry Patrick , Demolombe Sophie , Pucéat Michel , Escande Denis
Published: June 2012

Abstract
By making use of immunoblotting and immunocytochemical analysis, we explored whether stimulation of adenosine A
1
receptors would promote the activation of δ-protein kinase C (δ-PKC) immunolabeled with a polyclonal antibody. Immunoblot analysis of Triton X-100–soluble cell membrane and cytosolic fractions revealed the presence of a specific 75-kD band reactive to the δ-PKC polyclonal antibody. In freshly isolated rat cardiac myocytes, 28% of the total immunoreactive δ-PKC was associated with the membrane fraction, whereas 72% was associated with the soluble fraction. Under stimulation with the tumor-promoting phorbol 12-myristate 13-acetate (PMA, 500 nmol/L) used as a positive control, δ-PKC translocated to the cell membrane, with the membrane fraction representing 88% and the cytosolic fraction representing 12% of the total immunoreactive δ-PKC. Transverse optical sections performed with confocal laser microscopy showed that immunostaining with anti–δ-PKC antibody was distributed in the cytosol of unstimulated cells but accumulated in the cell membrane under PMA stimulation. In the membrane fraction of cells pretreated with adenosine (100 μmol/L) or with the adenosine A
1
agonist (−)-
N
6
-(2-phenylisopropyl)-adenosine (R-PIA, 1 μmol/L), the 75-kD band corresponding to δ-PKC increased by 57% and 66%, respectively, when compared with nonstimulated cells processed under the same experimental conditions. In cells exposed to either of the purine agonists, specific fluorescence staining decorated the cell membrane, a pattern that was not observed in control cells. Activation of membrane δ-PKC produced either by adenosine itself or by its analogue R-PIA was fully antagonized by the specific A
1
antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 μmol/L). From these data, we conclude that adenosine A
1
stimulation activates δ-PKC in freshly isolated rat ventricular myocytes.
Two Components of Delayed Rectifier Current in Canine Atrium and Ventricle
Authors: Gintant Gary A.
Published: June 2012

Abstract
Because the number and characteristics of delayed rectifier K
+
current (I
K
) components vary between species, the role of each component in the action potential and modulation by class III agents is uncertain. To address these issues, I
K
was assessed in adult isolated canine ventricular and atrial myocytes by using whole-cell and perforated-patch techniques. I
K
components were characterized by using two complementary approaches: a kinetic approach (based on biexponential fits to deactivating tail currents) and a pharmacological approach (using the methanesulfonanilide compound E-4031). In ventricular myocytes, two exponential tail current components were distinguished; these components differed in the voltage and time dependence of activation and the effect of lower [K
+
]
o
. Both kinetic components contributed equally to peak tail current amplitude (measured at −35 mV) after a single 300-ms pulse to 5 mV, simulating an action potential. By use of E-4031, rapidly and slowly activating components of I
K
(I
Kr
and I
Ks
, respectively) that were analogous to tail components described kinetically were identified. The activation kinetics and rectification properties of canine I
Kr
and I
Ks
are qualitatively similar to those described previously for guinea pigs. In contrast, canine I
Kr
and I
Ks
deactivation kinetics differed markedly from those found in guinea pigs, with canine I
Kr
deactivating slowly (time constant τ, 2 to 3 s near −35 mV) and I
Ks
deactivating rapidly (τ, 150 ms near −35 mV and decreasing to 30 ms near −85 mV). E-4031 elicited reverse rate-dependent effects (greater drug-induced prolongation of the action potential at slower stimulation rates); this effect is inconsistent with the hypothesis attributing reverse rate dependence to incomplete I
Ks
deactivation during rapid stimulation (due to rapid deactivation of canine I
Ks
). Two I
K
components with characteristics comparable to those found in ventricular myocytes were also observed in atrial myocytes. In conclusion, (1) I
Kr
- and I
Ks
-like components of I
K
are present in canine atrial and ventricular myocytes, with deactivation kinetics strikingly different from those found in guinea pigs, and (2) the rapid deactivation kinetics of canine I
Ks
do not support its role in reverse rate dependence with class III agents in this species.
Reduced Gene Expression of Vascular Endothelial NO Synthase and Cyclooxygenase-1 in Heart Failure
Authors: Smith Carolyn J. , Sun Dong , Hoegler Carl , Roth Barrie S. , Zhang Xiaoping , Zhao Gong , Xu Xiao-Bin , Kobari Yukage , Pritchard Kirkwood , Sessa William C. , Hintze Thomas H.
Published: June 2012

Abstract
Endothelium-dependent responses are depressed in coronary and peripheral blood vessels after the onset of pacing-induced heart failure in dogs and heart failure of various etiologies in humans. The present study was designed to examine whether these responses were due to decreases in the expression of endothelial cell NO synthase (ecNOS) and cyclooxygenase-1 (COX-1). After 1 month of left ventricular pacing, 8 mongrel dogs were monitored for heart failure as defined by clinical signs and left ventricular end diastolic pressures >25 mm Hg. Total RNA and protein were isolated from endothelial cells scraped from the thoracic aorta and analyzed by Northern and Western blotting, respectively. Blots probed with
32
P-labeled cDNAs for ecNOS and COX-1 were quantified densitometrically, and results were normalized against GAPDH or von Willebrand factor (vWF). In arbitrary units, the ratios of ecNOS to GAPDH were 2.66±0.77 (mean±SEM, n=17) and 1.12±0.37 (n=6), and the ratios of COX-1 to GAPDH were 1.52±0.52 and 0.56±0.15 before and after heart failure, respectively. These represent 56% to 64% (
P
<.05) reductions in ecNOS and COX-1 gene expression. There was no change in the ratios of either COX-1 or ecNOS to vWF. There was also a marked reduction in ecNOS protein after heart failure, estimated at 70%. A marked reduction in nitrite production, a measure of enzyme activity, from thoracic aortas in response to stimulation by either acetylcholine or bradykinin also occurred. To determine whether ecNOS and COX-1 could be independently regulated, an orally active NO-releasing agent, CAS 936, was given to 7 normal dogs for 7 days, and aortic ecNOS and COX-1 mRNAs were analyzed. The ratio of ecNOS to GAPDH was depressed by 52% (
P
<.05) in aortas from these dogs, whereas the ratio of COX-1 to GAPDH was unchanged. Similar results were found when data were normalized to vWF. These results suggest that at least two endothelial vasodilator gene products are reduced in heart failure, as opposed to a selective defect in NO synthase gene expression.
Nitric Oxide Attenuates Neutrophil-Mediated Myocardial Contractile Dysfunction After Ischemia and Reperfusion
Authors: Pabla Ravinder , Buda Andrew J. , Flynn David M. , Blessé Steven A. , Shin Alice M. , Curtis Michael J. , Lefer David J.
Published: June 2012

Abstract
With the knowledge of NO as an antiadhesion molecule, we performed studies to investigate the effects of NO on postischemic polymorphonuclear leukocyte (PMN)–mediated myocardial contractile dysfunction. Studies were performed with isolated perfused rat hearts subjected to 20 minutes of global ischemia and 45 minutes of reperfusion. Human PMNs (50 million) were infused over the first 5 minutes of reperfusion, and the recovery of left ventricular function was compared with baseline values. Infusion of PMNs alone (n=10) led to a 61% reduction in left ventricular developed pressure (LVDP) and a 57% reduction in the pressure-rate product (PRP) at 45 minutes of reperfusion. Infusion of an NO donor, CAS-754 (n=9), resulted in 80.2±6.7% recovery of LVDP and 77.0±8.6% recovery of PRP. Treatment with
l
-arginine (2.5 mmol/L, n=10) resulted in a similar improvement in the postischemic contractile state of the heart. In contrast,
N
G
-nitro-
l
-arginine methyl ester (L-NAME) treatment (250 μmol/L, n=10) resulted in an exacerbation of contractile dysfunction, as evidenced by a 93% reduction in LVDP at 45 minutes of reperfusion and a 91% reduction in PRP. The deleterious effects of L-NAME were prevented by
l
-arginine coperfusion. We failed to observe any cardioprotective effects when NO or
l
-arginine was administered to hearts subjected to 25 minutes of ischemia and 45 minutes of reperfusion in the absence of PMNs. In conclusion, PMN-mediated myocardial contractile dysfunction is attenuated by NO and exacerbated by blockade of NO synthesis.
Low Increase in cGMP Induced by Organic Nitrates and Nitrovasodilators Improves Contractile Response of Rat Ventricular Myocytes
Authors: Kojda Georg , Kottenberg Karin , Nix Petra , Schlüter Klaus Dieter , Piper Hans Michael , Noack Eike
Published: June 2012

Abstract
Whether organic nitrates are bioactivated to NO in cardiac muscle cells and may thus directly affect cardiac contractile function has remained an open question. Therefore, we determined the effects of the organic nitrates glyceryl trinitrate (100 μmol/L), pentaerythritol tetranitrate (10 μmol/L), and isosorbide-5-mononitrate on electrically stimulated contractile response (CR) and cAMP and cGMP content of isolated adult rat ventricular cardiomyocytes compared with different concentrations of the spontaneous NO donors
S
-nitroso-
N
-acetyl-d,l-penicillamine (SNAP) and 2,2-diethyl-1-hydroxy-1-nitroso-hydrazine (DEA/NO). A high concentration of spontaneous NO donors (100 μmol/L) caused a large increase in cGMP content that was accompanied by a decrease in CR to 73.8±6.7% (SNAP) and 80.9±6.1% (DEA/NO) of the control values. Inhibition of cGMP-dependent protein kinase by 10 μmol/L KT 5822 converted this effect into a pronounced improvement of CR (163.5±14.0%). By contrast, the organic nitrates caused a small but significant increase in cGMP, which was accompanied by an increase in cAMP and CR identical to that induced by 10 nmol/L isoprenaline (141.6±6.4%). A similar effect was observed with a low concentration (1 μmol/L) of SNAP and DEA/NO. All increases in CR induced by nitrates were abolished after inhibition of cAMP-dependent protein kinase by Rp-cAMPS (10 μmol/L). The positive contractile effect of isoprenaline was enhanced by 1 μmol/L SNAP. This effect was also demonstrated in isolated rat papillary muscles. These results indicate that in cardiac muscle (1) organic nitrates are bioactivated to NO; (2) this results in a moderate increase in cGMP, which causes an improved CR by increasing cAMP and activating cAMP-dependent protein kinase; and (3) a large increase in cGMP, produced by high doses of NO donors, reduces CR because of the activation of cGMP-dependent protein kinase.
Nitric Oxide Reversibly Inhibits the Migration of Cultured Vascular Smooth Muscle Cells
Authors: Sarkar Rajabrata , Meinberg Eric G. , Stanley James C. , Gordon David , Clinton Webb R.
Published: June 2012

Abstract
Augmentation of nitric oxide (NO) production in vivo decreases lesions in a variety of models of arterial injury, and inhibition of NO synthase exacerbates experimental intimal lesions. Both vascular smooth muscle cell (VSMC) proliferation and migration contribute to lesion formation. Although NO inhibits VSMC proliferation, its effects on VSMC migration are unknown. To test the hypothesis that NO inhibits VSMC migration independent of inhibition of proliferation, we examined migration of rat aortic VSMCs after wounding of a confluent culture in the presence of chemical donors of NO. Hydroxyurea was used to eliminate any confounding effect of NO on proliferation. Three NO donors, diethylamine NONOate, spermine NONOate, and
S
-nitrosoglutathione, exhibited concentration-dependent inhibition of both number of migrating VSMCs and maximal distance migrated. Inhibition of migration was also seen with 8-Br-cGMP, suggesting that activation of guanylate cyclase may play a role in mediating the antimigratory effects of NO. Migration resumed after removal of NO donors, as evidenced by an increase in distance migrated. Measurement of VSMC protein synthesis and mitochondrial respiration indicated that inhibition of migration by NO donors was not due to metabolic cytostasis. These findings indicate that NO reversibly inhibits VSMC migration independent of proliferation or cytotoxicity, a novel mechanism by which both endogenous and pharmacological NO may alter vascular pathology.
Expression of Endothelin-1, Endothelin-3, Endothelin-Converting Enzyme-1, and Endothelin-A and Endothelin-B Receptor mRNA After Angioplasty-Induced Neointimal Formation in the Rat
Authors: Wang Xinkang , Douglas Stephen A. , Louden Calvert , Vickery-Clark Lynne M. , Feuerstein Giora Z. , Ohlstein Eliot H.
Published: June 2012

Abstract
Endothelins (ETs) are potent vasoconstrictors known to play a role in tissue remodeling after vascular wall injury. The molecular mechanisms for the expression and functions of ETs and their receptors after carotid artery angioplasty are not fully understood. Using quantitative reverse transcription and polymerase chain reaction, the present study demonstrates the temporal mRNA expression of ET-converting enzyme-1 (ECE-1), preproET-1, preproET-3, and both ET
A
and ET
B
receptors after rat carotid artery balloon angioplasty. A significant increase in ECE-1 mRNA was observed at 6 hours (1.8-fold increase over control,
P
<.01) and 24 hours (1.7-fold increase,
P
<.01) in carotid arteries after angioplasty. In contrast, a significant increase in preproET-1 mRNA levels was not observed until 3 days (1.9-fold increase,
P
<.05) and 7 days (2.1-fold increase,
P
<.05). A similarly delayed increase in preproET-3 mRNA was observed at 7 days (2.8-fold increase,
P
<.05) and 14 days (2.6-fold increase,
P
<.05) after angioplasty. A parallel but marked increase in ET
A
and ET
B
receptor mRNAs compared with preproET-1 and -3 messages was observed after angioplasty. The levels of ET
A
receptor mRNA were elevated 29.3-fold (
P
<.001) and 24.3-fold (
P
<.01) at 3 and 7 days, respectively, after angioplasty. The increase in ET
B
receptor mRNA occurred slightly earlier than the increase in ET
A
receptor mRNA, showing 15.1-fold increase at 1 day (
P
<.001) and 11.3-fold increase at 3 days (
P
<.01) after angioplasty. Immunohistochemical studies using anti-ET antibodies demonstrated a corresponding increase in ET immunoactivity, which was distributed mainly in the neointimal cells 14 days after angioplasty. The increases in ECE-1, ET-1, and ET-3 and their receptor expression after balloon angioplasty suggest that these proteins play an active role in the pathogenesis of neointimal formation.
Cell DistensionInduced Increase of the Delayed Rectifier K
+
Current in Guinea Pig Ventricular Myocytes
Authors: Wang Zhuren , Mitsuiye Tamotsu , Noma Akinori
Published: June 2012

Abstract
Single ventricular myocytes of guinea pig heart were distended by applying a positive pressure of 5 to 20 mm Hg in the pipette during the whole-cell voltage clamp. The amplitude of delayed rectifier K
+
current (I
K
) was increased by ≈1.5 times, whereas the inward rectifier K
+
current was scarcely affected. The increase of I
K
was reversible by applying a negative pressure of −10 to −30 mm Hg accompanied by shrinkage of the inflated cell. This response of I
K
was largely attributed to the E-4031–insensitive component of I
K
. The fully activated current amplitude, measured using long-lasting depolarizing pulses (>30 seconds) to +60 mV, was increased by the cell distension. The activation time course of I
K
during the long pulse consisted of more than three exponential components, and the slowest time constant was decreased by the distension from control 20.2±7.7 seconds (n=4) to 7.6±1.6 seconds (n=5). We failed to detect an involvement of microtubules or microfilaments, protein kinase C, and Ca
2+
in the inflation-mediated increase of I
K
.
Activation of Histamine H
3
-Receptors Inhibits Carrier-Mediated Norepinephrine Release During Protracted Myocardial Ischemia
Authors: Imamura Michiaki , Lander Harry M. , Levi Roberto
Published: June 2012

Abstract
We previously showed that prejunctional histamine H
3
-receptors downregulate norepinephrine exocytosis, which is markedly enhanced in early myocardial ischemia. In the present study, we investigated whether H
3
-receptors modulate nonexocytotic norepinephrine release during protracted myocardial ischemia. In this setting, decreased pH
i
in sympathetic nerve endings sequentially leads to a compensatory activation of the Na
+
-H
+
antiporter (NHE), accumulation of intracellular Na
+
, reversal of the neuronal uptake of norepinephrine, and thus carrier-mediated release of norepinephrine. Accordingly, norepinephrine overflow from isolated guinea pig hearts undergoing 20-minute global ischemia and 45-minute reperfusion was attenuated ≈80% by desipramine (10 nmol/L) and 70% by 5-(
N
-ethyl-
N
-isopropyl)-amiloride (EIPA, 10 μmol/L), inhibitors of norepinephrine uptake and NHE, respectively. The H
3
-receptor agonist imetit (0.1 μmol/L) decreased carrier-mediated norepinephrine release by ≈50%. This effect was blocked by the H
3
-receptor antagonist thioperamide (0.3 μmol/L), indicating that H
3
-receptor activation inhibits carrier-mediated norepinephrine release. At lower concentrations, imetit (10 nmol/L) or EIPA (3 μmol/L) did not inhibit carrier-mediated norepinephrine release. However, a 25% inhibition occurred with imetit (10 nmol/L) and EIPA (3 μmol/L) combined. This synergism suggests an association between H
3
-receptors and NHE. Conceivably, activation of H
3
-receptors may lead to inhibition of NHE. In fact, α
2
-adrenoceptor activation, which is known to stimulate NHE, enhanced norepinephrine release, whereas α
2
-adrenoceptor blockade attenuated it. Furthermore, activation of adenosine A
1
-receptors markedly attenuated norepinephrine release, whereas their inhibition potentiated it. Because norepinephrine release directly correlated with the severity of reperfusion arrhythmia and imetit reduced the incidence of ventricular fibrillation by 50%, our findings with H
3
-receptor agonists may further the development of novel pharmacological means to reduce reperfusion arrhythmias in the clinical setting.
Extracellular ATP Inhibits Adrenergic Agonist–Induced Hypertrophy of Neonatal Cardiac Myocytes
Authors: Zheng Jing-Sheng , Boluyt Marvin O. , Long Xilin , O’Neill Lydia , Lakatta Edward G. , Crow Michael T.
Published: June 2012

Abstract
We have previously shown that extracellular ATP, like norepinephrine (NE) and many other hypertrophyinducing agents, increases expression of the immediate-early genes c-
fos
and
junB
in cultured neonatal cardiac myocytes but that the intracellular signaling pathways activated by ATP and responsible for these changes differ from those stimulated by NE. Furthermore, whereas NE increases incorporation of [
14
C]phenylalanine (
14
C-Phe) and cell size in neonatal cardiomyocytes, ATP does not. Since ATP is coreleased with NE from sympathetic nerve endings in the heart, we investigated whether ATP could modulate cardiac hypertrophy induced by adrenergic agonists, such as NE. We report in the present study that extracellular ATP inhibited the increase in incorporation of
14
C-Phe into cellular protein and the increase in cell size in neonatal rat cardiac myocytes that was induced by NE, phenylephrine (PE), basic fibroblast growth factor, or endothelin-1. This inhibition was dose dependent, occurred predominantly through P
2
purinergic receptors, and was observed even when cells were treated with ATP for as little as 1 hour before the addition of the hypertrophy-inducing agent. ATP also selectively affected changes in gene expression associated with hypertrophy. It prevented PE-stimulated increases in atrial natriuretic factor and myosin light chain-2 mRNA levels, while appearing to augment basal and PE-stimulated skeletal α-actin mRNA levels. ATP alone increased sarcoplasmic reticulum Ca
2+
-ATPase mRNA levels but had no effect when added with PE. ATP did not significantly affect the level of the constitutively expressed mRNA for GAPDH. Neither the PE-stimulated increase in immediate-early gene expression nor the initial induction of mitogen-activated protein kinase activity by PE was inhibited by ATP. These results demonstrate that extracellular ATP can inhibit hypertrophic growth of neonatal cardiac myocytes and differentially alter the changes in gene expression that accompany hypertrophy.
Evidence for the Presence of a Proteinase-Activated Receptor Distinct From the Thrombin Receptor in Vascular Endothelial Cells
Authors: Hwa Joyce J. , Ghibaudi Lorraine , Williams Patricia , Chintala Madhu , Zhang Rumin , Chatterjee Meeta , Sybertz Edmund
Published: June 2012

Abstract
The thrombin receptor was the first cloned G protein–coupled receptor reported to be activated by proteolytic cleavage of its extracellular amino terminus. A second proteinase-activated receptor (PAR-2) was cloned recently and expressed in
Xenopus laevis
oocytes. PAR-2 was activated by trypsin and by a peptide (SLIGRL) derived from the new amino terminus. Since PAR-2 mRNA was detected in highly vascularized organs, we compared the physiological functions of the thrombin receptor and PAR-2 in vascular endothelium. Thrombin and trypsin both elicited endothelium-dependent relaxations in prostaglandin F
2α
(PGF
2α
)–contracted strips of porcine coronary artery. Whereas high doses of both thrombin or trypsin (10 U/mL) caused homologous desensitization, trypsin caused further relaxation of thrombin-desensitized tissues. Thrombin and PAR-2–derived peptides (SFLLRN and SLIGRL) both induced endothelium-dependent relaxations in PGF
2α
-contracted porcine coronary arteries. SFLLRN or SLIGRL (30 μmol/L) also showed homologous desensitization but not cross desensitization. In the presence of the NO synthase inhibitor
N
G
-monomethyl-
l
-arginine (1 mmol/L), both SFLLRN- and SLIGRL-induced relaxations were partially inhibited. SFLLRN elicited weak contraction in coronary arteries without endothelium, whereas SLIGRL had no effect. Intravenous injection of SFLLRN (1 mg/kg, bolus) into anesthetized rats elicited a transient depressor response followed by pronounced pressor response. In contrast, intravenous administration of SLIGRL (1 mg/kg, bolus) produced only a marked depressor response. Consistent with the in vivo data, SFLLRN contracted the endothelium-rubbed rat aortic rings and aggregated human platelets in vitro, whereas SLIGRL had no effect. The finding that both trypsin and SLIGRL induced endothelium-dependent relaxations indicates the presence of PAR-2 on endothelial cells. In addition, both trypsin and SLIGRL elicited relaxations in thrombin- or SFLLRN-desensitized tissue, suggesting that PAR-2 is distinct from thrombin receptor in vascular endothelium. The lack of PAR-2–mediated platelet aggregation or smooth muscle contraction suggested it might not share the pathogenic properties associated with the thrombin receptor in the vasculature.
Removal of Arg
141
From the α Chain of Human Hemoglobin by Carboxypeptidases N and M
Authors: Rajko Igić Bruno Michel , Leray Valérie , Deddish Peter A. , Erdös Ervin G.
Published: June 2012

Abstract
Both human plasma carboxypeptidase N (CPN) and membrane-bound carboxypeptidase M (CPM) released the C-terminal arginine (α-Arg
141
) of the α chain of human adult hemoglobin. An arginase contamination present in the hemoglobin preparation, which converted the released arginine to ornithine, was removed by gel filtration. CPM was about 20 times more efficient than CPN or its active subunit in hydrolyzing oxyhemoglobin and cleaved oxyhemoglobin twice as fast as deoxyhemoglobin. The hydrolysis of the peptide bond of α-Arg
141
accelerated the dissociation rate of the tetramer deoxy-des-α-Arg
141
hemoglobin to dimers 2500-fold over that of deoxyhemoglobin, as measured by haptoglobin binding. Moreover, the dissociation of the deoxy-des-α-Arg
141
hemoglobin tetramer to dimers was not affected by 2,3-diphosphoglyceric acid. Des-α-Arg
141
hemoglobin had a higher oxygen affinity (P
50
, 5.51 mm Hg; control, 19.94 mm Hg [P
50
is the partial pressure of oxygen that gives 50% of the saturation of hemoglobin]) and a lower apparent cooperativity (Hill coefficient: n, 1.02; control, 2.24) than unhydrolyzed hemoglobin. After hemoglobin was incubated in human plasma, its oxygen-binding parameters, the P
50
, and the Hill coefficient decreased drastically due to cleavage by CPN. In the perfused rat heart, des-α-Arg
141
hemoglobin was a more effective coronary vasoconstrictor than hemoglobin, possibly because it dissociated to dimers in the coronary vascular bed. A covalently cross-linked hemoglobin was less active than native hemoglobin. The coronary vasoconstriction was caused by multiple factors, including interference with vasodilation by nitric oxide and eicosanoids. Thus, the hydrolysis of hemoglobin by CPM and CPN demonstrated the contribution of the α-Arg
141
residue to sustaining the tetrameric structure of hemoglobin and its normal oxygen affinity and vasoactivity.
Reentrant Wave Fronts in Wiggers’ Stage II Ventricular Fibrillation
Authors: Lee John J. , Kamjoo Kamyar , Hough Dustan , Hwang Chun , Fan Wei , Fishbein Michael C. , Bonometti Claudio , Ikeda Takanori , Karagueuzian Hrayr S. , Chen Peng-Sheng
Published: June 2012

Abstract
The mechanisms of Wiggers’ stage II ventricular fibrillation (VF) are poorly understood. Using computerized mapping techniques, we studied the patterns of activation during Wiggers’ stage II VF in 13 open-chest dogs. In 7 of the 13 dogs, the right ventricular Purkinje fibers and adjacent subendocardial myocytes were ablated with Lugol solution. VF was induced electrically, and 3 to 5 seconds of data were obtained beginning ≈2.5 seconds after the onset of VF. Dynamic displays of the activation patterns and isochronal maps revealed the presence of reentrant wave fronts in 17 of 33 runs of VF in ablated ventricles and in 12 of 45 runs of VF in intact ventricles. The incidence of reentry was not different between the subendocardium-ablated group versus the nonablated group (1.7±1.6 versus 1.2±1.6 rotations per episode of VF,
P
=.19). There were no differences in the core size (25±19 versus 29±18 mm
2
), life span (3.4±1.1 versus 3.2±1.2 rotations), or cycle length (111±12 versus 107±8 ms) in ablated ventricles versus intact ventricles, respectively. The core was unstable as it meandered within the mapped area displacing the entire reentrant wave front. In all episodes, the reentrant wave fronts were spontaneously initiated by an interaction between two propagating wave fronts roughly perpendicular to each other. The second wave front met the tail of the first wave front 69±11 ms (range, 40 to 90 ms) after its latest activation, indicating that the interaction occurred during a vulnerable period. The reentrant wave fronts terminated spontaneously (n=7), as the result of interference by an invading wave front (n=19), or meandered off the mapped region (n=3). We conclude the following: (1) Reentrant activities with short life spans and meandering cores are present during Wiggers’ stage II VF in dogs. (2) New reentrant wave fronts are generated when one wave front interacts with another wave front during its vulnerable period. (3) The reentrant wave fronts terminate spontaneously or as the result of interference. (4) Chemical subendocardial ablation does not affect the incidence, life span, cycle length, or core size of the reentrant wave fronts.
Intracellular pH and Tyrosine Phosphorylation but Not Calcium Determine Shear Stress–Induced Nitric Oxide Production in Native Endothelial Cells
Authors: Ayajiki Kazuhide , Kindermann Markus , Hecker Markus , Fleming Ingrid , Busse Rudi
Published: June 2012

Abstract
Signaling pathways determining the shear stress–induced production of NO from endothelial cells in situ were investigated using a bioassay system in which shear stress was increased by inducing vasoconstriction in an endothelium-intact donor segment (rabbit iliac artery) while maintaining a constant luminal perfusion rate. Shear stress–induced NO production, as assessed by changes in the tone of a preconstricted endothelium-denuded detector ring, was biphasic and consisted of an initial transient (20- to 25-minute) Ca
2+
-dependent phase followed by a Ca
2+
-independent plateau phase, which was maintained as long as the donor segment remained constricted. Stretching the donor segments to their in vivo length abolished the initial phase without affecting the plateau phase of NO release. Inhibition of the Na
+
-H
+
exchanger using HOE 694 elicited an intracellular acidification, which attenuated shear stress–induced NO production. The specific protein kinase C inhibitor, Ro 31-8220, was without effect, whereas the unspecific inhibitors, staurosporine and calphostin C, abolished the shear stress–induced production of NO. Erbstatin A, a tyrosine kinase inhibitor, attenuated the shear stress–induced tyrosine phosphorylation of specific cellular proteins and abrogated the associated NO production. In summary, these data indicate that shear stress activates the NO synthase at basal levels of [Ca
2+
]
i
via a mechanotransduction cascade that involves tyrosine phosphorylation and can be modulated by changes in pH
i
. The apparent fundamental alteration of the endothelial NO synthase under shear stress that renders its maintained activation independent of an increase in [Ca
2+
]
i
is probably the consequence of a change in the enzyme microenvironment.
The α3 Isoform Protein of the Na
+
,K
+
-ATPase Is Associated With the Sites of Cardiac and Neuromuscular Impulse Transmission
Authors: Zahler Raphael , Sun Wei , Ardito T. , Zhang Zhong-ting , Kocsis Jeffery D. , Kashgarian Michael
Published: June 2012

Abstract
The α (catalytic) subunit of the Na
+
pump (Na
+
,K
+
-ATPase) has three isoforms: α1 is ubiquitous, skeletal muscle expresses predominantly α2, and α3 has been localized to specific types of neurons and, possibly, to axonal processes. The α3 isoform mRNA is also expressed in the rat cardiac conduction system. Thus, we studied rat heart and quadriceps muscles by immunohistochemistry using isoform-specific antibodies to the Na
+
pump α subunit and labeled α-bungarotoxin as a probe for the neuromuscular junction (NMJ). We found that α3 pump protein is localized to three sites important for impulse transmission: the junctional complex between cardiac myocytes, the heart conduction system, and the NMJ. Specifically, all levels of the conduction system expressed α3 immunoreactive protein, as assessed by two isoform-specific antibodies and histological conduction system markers. Specific expression at the junctional complex was confirmed by immuno-EM. Double-labeling and denervation analysis indicated that α3-positive areas in skeletal muscle were presynaptic and adjacent to postsynaptic bungarotoxin-positive regions, which had the classic morphology of NMJs. Thus, specific Na
+
,K
+
-ATPase pump isoforms may be adapted to maintenance of membrane potential and/or intracellular ion concentrations required for impulse transmission in both heart and presynaptic motor terminals contacting skeletal muscle.
Adrenergic Modulation of Ultrarapid Delayed Rectifier K
+
Current in Human Atrial Myocytes
Authors: Li Gui-Rong , Feng Jianlin , Wang Zhiguo , Fermini Bernard , Nattel Stanley
Published: June 2012

Abstract
The ultrarapid delayed rectifier K
+
current (I
Kur
) in human atrial cells appears to correspond to Kv1.5 cloned channels and to play an important role in human atrial repolarization. Kv1.5 channels have consensus sites for phosphorylation by protein kinase A and C, suggesting possible modulation by adrenergic stimulation. The present study was designed to assess the adrenergic regulation of I
Kur
in human atrial myocytes. Isoproterenol increased I
Kur
in a concentration-dependent manner, with significant effects at concentrations as low as 10 nmol/L. The effects of isoproterenol were reversible by washout or by the addition of propranolol (1 μmol/L). Isoproterenol’s effects were mimicked by the direct adenylate cyclase stimulator, forskolin, and by the membrane-permeable form of cAMP, 8-bromo cAMP. Isoproterenol had no effect on I
Kur
when the protein kinase A inhibitor peptide, PKI(6-22)amide, was included in the pipette solution; in a separate set of experiments in which isoproterenol alone increased I
Kur
by 45±9% relative to control, subsequent superfusion with isoproterenol in the presence of the protein kinase inhibitor H-7 failed to alter I
Kur
. In contrast to isoproterenol, phenylephrine (in the presence of propranolol to block β-adrenergic effects) induced a concentration-dependent inhibition of I
Kur
, with significant effects observed at concentrations as low as 10 μmol/L. The inhibitory actions of phenylephrine were reversed by the addition of prazosin and prevented by coadministration with a highly selective inhibitor of protein kinase C, bisindolylmaleimide. These results indicate that β-adrenergic stimulation enhances, whereas α-adrenergic stimulation inhibits, I
Kur
and suggest that these actions are mediated by protein kinase A and protein kinase C, respectively. The modulation of I
Kur
by adrenergic influences is a potentially novel control mechanism for human atrial repolarization and arrhythmias.
Cyclic ADP-Ribose Does Not Regulate Sarcoplasmic Reticulum Ca
2+
Release in Intact Cardiac Myocytes
Authors: Guo Xiaoqing , Laflamme Michael A. , Becker Peter L.
Published: June 2012

Cyclic ADP-ribose (cADPR), an intracellular second messenger known to mobilize Ca
2+
in sea urchin eggs, has been implicated in modulating Ca
2+
release in a variety of mammalian tissues. On the basis of studies of isolated cardiac sarcoplasmic reticulum (SR) vesicles and single SR Ca
2+
release channels, cADPR has also been proposed to be a modulator of SR Ca
2+
release in heart. In the present study, we directly examined the ability of cADPR to trigger SR Ca
2+
release and to modulate Ca
2+
-induced Ca
2+
release (CICR) in intact rat ventricular myocytes. Voltage-clamped myocytes were dialyzed with up to 100 μmol/L caged cADPR and 0.6 μmol/L calmodulin along with the Ca
2+
-sensitive dye fluo 3. A step increase in the cADPR concentration induced by flash photolysis of caged cADPR neither directly triggered SR Ca
2+
release nor modulated CICR in intact myocytes. In contrast, under similar conditions, extracellular application of caffeine (1 to 2.5 mmol/L) onto myocytes produced both effects. Under equivalent conditions, flash photolysis of caged cADPR-loaded sea urchin eggs resulted in large Ca
2+
transients. Further, the sustained presence of high cytosolic concentrations of either cADPR or its antagonist, 8-amino-cADPR, was ineffective in altering normal CICR in myocytes. These findings indicate that cADPR does not regulate SR Ca
2+
release in intact cardiac myocytes.
Shear Stress Modulates Expression of Cu/Zn Superoxide Dismutase in Human Aortic Endothelial Cells
Authors: Inoue Nobutaka , Ramasamy Santhini , Fukai Tohru , Nerem Robert M. , Harrison David G.
Published: June 2012

A major determinant of the level of cellular superoxide anion (O
2
−•
) is the dismutation of O
2
−•
to hydrogen peroxide by the enzyme superoxide dismutase (SOD). Three forms of SOD exist, but in endothelial cells, the major form outside of the mitochondria is the cytosolic copper/zinc-containing superoxide dismutase (Cu/Zn SOD). Since fluid shear stress is an important determinant of the function and structure of endothelial cells in vivo, we examined the effect of laminar shear stress on the expression of Cu/Zn SOD in cultured human aortic endothelial cells. Laminar shear stress of 0.6 to 15 dyne/cm
2
increased Cu/Zn SOD mRNA in a time- and dose-dependent manner in human aortic endothelial cells. Shear stress also increased both Cu/Zn SOD protein content and the enzyme activity. Nuclear run-on assays showed that nuclei from human aortic endothelial cells exposed to laminar shear stress had a 1.6-fold greater transcriptional activity of the Cu/Zn SOD gene compared with cells not exposed to shear, indicating that an increase in Cu/Zn SOD mRNA induced by laminar shear stress is at least in part mediated by increased transcription. In contrast, shear stress had no effect on Cu/Zn SOD mRNA levels in human aortic smooth muscle cells. These findings show that physiological levels of shear stress increase expression of Cu/Zn SOD in the endothelium. This adaptation to shear stress might augment the effect of locally produced NO
•
and thereby promote the antiatherogenic and anti-inflammatory properties of the endothelial cell.
Basic Fibroblast Growth Factor Increases Collateral Blood Flow in Rats With Femoral Arterial Ligation
Authors: Yang H.T. , Deschenes Michael R. , Ogilvie Robert W. , Terjung Ronald L.
Published: June 2012

The potential for exogenous infusion of basic fibroblast growth factor (bFGF) to increase collateral blood flow to dependent tissue was quantified in adult male rats with peripheral arterial insufficiency. Occlusion of the femoral artery at a proximal site did not infringe on resting blood flow to the distal hindlimb muscle, but did remove the blood flow reserve. Blood flow to the hindlimb muscles was measured with radiolabeled microspheres using an isolated hindlimb preparation perfused in the descending aorta (Krebs-Henseleit bicarbonate, 5% albumin medium containing red blood cells [40% hematocrit]) at 100 mm Hg. Calf muscle blood flow changed modestly (≈50%) with infusion of only the carrier (heparin/saline), increased markedly over the first 2 weeks of bFGF infusion (1 μg/d into the femoral artery), but did not change further with infusion for 4 weeks. Waiting 2 weeks after 1 week of bFGF infusion did not further increase the intermediate improvement in blood flow. The improved collateral blood flow and increased muscle capillary density likely contributed to the enhanced muscle performance observed during nerve stimulation in situ. X-ray films of arterial casts identified an expansion of upper thigh vessels that likely served as collaterals. In animals with peripheral arterial insufficiency, short-term exogenous infusion of bFGF is effective at inducing vascular expansion that is sufficient to improve the flow reserve of dependent distal tissue and enhance muscle function. This raises the expectation that a similar response in patients with peripheral arterial insufficiency would significantly improve morbidity, including the symptoms of intermittent claudication.