bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of density of sympathetic varicosities and their closeness to smooth muscle cells in rabbit middle cerebral and ear arteries and their branches.
Authors: Dodge J T , Bevan R D , Bevan J A
Published: June 2012

The density and nerve varicosity-smooth muscle cell separation of rabbit cerebral and ear arterial beds were compared. The rabbit middle cerebral artery and three of its successive branches and a comparable-sized ear artery and two branches were perfusion-fixed for electron microscopy and analyzed by quantitative morphometric procedures. The purpose was to determine if there are structural correlates to previously observed differences in the sympathetic control of these two vascular systems. The in vitro contractile response of isolated artery segments to electrical field stimulation of their intramural nerves is considerably less in cerebral arteries compared with the similar-sized ear arteries. Furthermore, in the cerebral but not the ear circulation, there is progressive diminution of the neurogenic response with successive branching. Although the total varicosity densities of the major ear and brain arteries studied are similar, and this parameter stays fairly constant with successive branching of the ear, it falls off considerably in the cerebral vessels. There is a significant difference in densities between the two vascular beds when "bare" varicosities located < 1 micron from the medial smooth muscle are compared. The second-order branch of the ear artery has an average of 18 bare varicosities per 500-micron circumference, and the corresponding cerebral vessel has only 2.8 bare varicosities per 500-micron circumference. The mean bare varicosity-smooth muscle cell separation (mean +/- SEM) is significantly (P < .05) less in the ear (1.18 +/- 0.06 microns) than in the cerebral arteries (4.95 +/- 0.23 microns). This is true of all vessels studied. Fifty-nine percent of the bare varicosities in the ear arteries are < 1 micron from the smooth muscle cells, and 1.2% are more distant than 5 microns. These values for cerebral vessels are 9.5% and 37%, respectively. In the ear vessels, 25% of the bare varicosities make close neuromuscular contact (within 500 nm of the smooth muscle), whereas only 3% do so in cerebral vessels; in cerebral compared with ear vessels, the percentage becomes significantly less with branching. These structural features of brain vessels, taken together with the lower sensitivity to and the diminished capacity to respond to norepinephrine, probably account for their weak neurogenic control. The results indicate that the cerebral circulation of the rabbit receives a sympathetic innervation that is relatively ineffective in altering cerebrovascular tone.
Wave-front curvature as a cause of slow conduction and block in isolated cardiac muscle.
Authors: Cabo C , Pertsov A M , Baxter W T , Davidenko J M , Gray R A , Jalife J
Published: June 2012

We have investigated the role of wave-front curvature on propagation by following the wave front that was diffracted through a narrow isthmus created in a two-dimensional ionic model (Luo-Rudy) of ventricular muscle and in a thin (0.5-mm) sheet of sheep ventricular epicardial muscle. The electrical activity in the experimental preparations was imaged by using a high-resolution video camera that monitored the changes in fluorescence of the potentiometric dye di-4-ANEPPS on the surface of the tissue. Isthmuses were created both parallel and perpendicular to the fiber orientation. In both numerical and biological experiments, when a planar wave front reached the isthmus, it was diffracted to an elliptical wave front whose pronounced curvature was very similar to that of a wave front initiated by point stimulation. In addition, the velocity of propagation was reduced in relation to that of the original planar wave. Furthermore, as shown by the numerical results, wave-front curvature changed as a function of the distance from the isthmus. Such changes in local curvature were accompanied by corresponding changes in velocity of propagation. In the model, the critical isthmus width was 200 microns for longitudinal propagation and 600 microns for transverse propagation of a single planar wave initiated proximal to the isthmus. In the experiments, propagation depended on the width of the isthmus for a fixed stimulation frequency. Propagation through an isthmus of fixed width was rate dependent both along and across fibers. Thus, the critical isthmus width for propagation was estimated in both directions for different frequencies of stimulation. In the longitudinal direction, for cycle lengths between 200 and 500 milliseconds, the critical width was < 1 mm; for 150 milliseconds, it was estimated to be between 1.3 and 2 mm; and for the maximum frequency of stimulation (117 +/- 15 milliseconds), it was > 2.5 mm. In the transverse direction, critical width was between 1.78 and 2.32 mm for a basic cycle length of 200 milliseconds. It increased to values between 2.46 and 3.53 mm for a basic cycle length of 150 milliseconds. The overall results demonstrate that the curvature of the wave front plays an important role in propagation in two-dimensional cardiac muscle and that changes in curvature may cause slow conduction or block.
Surface staining and cytotoxic activity of heat-shock protein 60 antibody in stressed aortic endothelial cells.
Authors: Xu Q , Schett G , Seitz C S , Hu Y , Gupta R S , Wick G
Published: June 2012

Heat-shock protein (hsp) expression can be induced by high temperature, exposure to cytokines or oxygen radicals, ischemia, hemodynamic overload, or viral infections. To determine whether surface expression of hsp60 occurs in aortic endothelial cells stressed by high temperature or cytokines, cells from rat aortas were cultivated and stained with several types of monoclonal antibodies against hsp60. Other antibodies, eg, those against intercellular adhesion molecule-1 (ICAM-1), or immune response-associated antigens were also used as controls. Positive staining of endothelial cells on the surface and in the cytoplasm was observed after pretreatment of the cells with cytokine-containing medium, tumor necrosis factor-alpha (TNF-alpha), or interleukin-1 alpha and labeling with a specific monoclonal antibody against hsp60 (II-13). Fluorescence-activated cell sorter analyses showed that over 80% of living endothelial cells stressed by cytokine-containing medium, by TNF-alpha, or at 42 degrees C, but not by interleukin-1 alpha, were positively surface stained with this antibody. Increased intensity of immunostaining with antibodies to ICAM-1 and immune response-associated antigen was also seen on the cytokine-stressed endothelial cells. Furthermore, when TNF-alpha stimulated endothelial cells labeled with 51Cr were incubated with antibody II-13 in the presence of complement, significant lysis occurred. In summary, endothelial cells stressed by high temperature or certain cytokines, eg, TNF-alpha, express hsp60 in the cytoplasm and on their surfaces, and these cells were susceptible to complement-dependent lysis by hsp60-specific antibody. These observations may be significant for elucidating the mechanisms of the involvement of immune reactions to hsp65/60 in initiating atherosclerosis.
Allosteric enhancer PD 81,723 acts by novel mechanism to potentiate cardiac actions of adenosine.
Authors: Kollias-Baker C , Ruble J , Dennis D , Bruns R F , Linden J , Belardinelli L
Published: June 2012

The 2-amino-3-benzoylthiophene derivative PD 81,723 is an allosteric enhancer of agonist binding to brain A1 adenosine receptors. One aim of this study was to characterize and contrast the effects of PD 81,723 on the A1 receptor-mediated negative dromotropic and A2a receptor-mediated vasodilatory actions of adenosine and of a nonmetabolizable and unselective N6-(3-pentyl)adenosine derivative. A second aim was to determine the mechanism of action of PD 81,723. In guinea pig isolated hearts, PD 81,723 potentiated the adenosine and the N6-(3-pentyl)adenosine derivative-induced prolongations of the stimulus-to-His bundle (S-H) interval in a concentration-dependent manner. PD 81,723 (30 mumol/L) decreased the EC50 value for adenosine to prolong the S-H interval by ninefold from 7.4 +/- 1.2 to 0.8 +/- 0.1 mumol/L but did not increase the content of adenosine in cardiac effluent. PD 81,723 (30 mumol/L) increased the specific binding of the A1 agonist [3H]cyclohexyladenosine ([3H]CHA) to human atrial and guinea pig atrial and brain membranes by 38%, 78%, and 300%, respectively. PD 81,723 also increased the fraction of A1 receptors in the high-affinity binding state by an average of 56 +/- 13%. The dissociation rate of [3H]CHA from guinea pig brain membranes was decreased in the presence of PD 81,723 (10 mumol/L) from 0.55 +/- 0.01/min to 0.35 +/- 0.01/min. PD 81,723 did not alter the binding of the A1 antagonist [3H]cyclopentyldipropylxanthine to guinea pig brain membranes. The IC50 values for 5'-guanylylimidodiphosphate to reduce specific binding of [3H]CHA to guinea pig cardiac and brain membranes were increased from 1.5 +/- 0.2 and 2.0 +/- 0.2 mumol/L in the absence of PD 81,723 to 10 +/- 3.3 and 18 +/- 0.5 mumol/L, respectively, in the presence of PD 81,723 (30 mumol/L). PD 81,723 did not potentiate the coronary vasodilatory actions of the N6-(3-pentyl)adenosine derivative. Specific binding of the A2a agonist [3H]CGS 21680 to brain membranes and the nucleoside transporter ligand [3H]nitrobenzylthioinosine to cardiac membranes was unchanged in the presence of PD 81,723. The results suggest that PD 81,723 specifically potentiates the action of adenosine on A1 receptors by stabilizing receptor-G protein interactions in the presence of agonists.
Antibody-Mediated Imbalance of Myocardial Energy Metabolism
Authors: Schultheiß Heinz-Peter , Schulze Karsten , Schauer Rolf , Witzenbichler Bernhard , Strauer Bodo Eckehard
Published: June 2012

Abstract
The ADP-ATP carrier of the inner mitochondrial membrane is an autoantigen in myocarditis and dilated cardiomyopathy. Sera of patients with these diseases contain carrier-specific autoantibodies that inhibit the transmembrane nucleotide transport on isolated mitochondria. Guinea pigs immunized with the isolated ADP-ATP carrier protein also generate specific carrier-inactivating antibodies. In this study, we measured the cardiac function of guinea pigs immunized with the ADP-ATP carrier by determining the external heart work (EHW) of their isolated perfused spontaneously beating hearts stimulated by 4.0 mmol/L calcium and aortic ligature. Further, the electrogenic transport activity of the ADP-ATP carrier was estimated by calculating the cytosolic-mitochondrial difference of the phosphorylation potential of ATP [ΔG(cyt-mit)] in the freeze-clamped isolated hearts by nonaqueous fractionation. The EHW of immunized guinea pigs was seen to be reduced by 54% (
P
<.005) compared with nonimmunized control guinea pigs, and ΔG(cyt-mit) declined from 4.9 kJ/mol ATP in nonimmunized control hearts to 2.3 kJ/mol ATP in the hearts of the immunized guinea pigs (
P
<.005). The decisive result of this study, however, is the close relation observed between the magnitude of reduction of ΔG(cyt-mit) and the size of the decrease in EHW (
r
=.87). Therefore, it seems plausible that antibody-mediated carrier dysfunction (creating the observed imbalance in myocardial energy metabolism) is responsible for the impairment of cardiac function. Our data support the hypothesis that immunopathic mechanisms in myocarditis and dilated cardiomyopathy can trigger subsequent heart failure. The underlying pathophysiological reason seems to be a metabolic disorder initiated by the antibody-mediated inactivation of the ADP-ATP carrier.
Na
+
-Ca
2+
Exchange in Intact Endothelium of Rabbit Cardiac Valve
Authors: Li Li , van Breemen Cornelis
Published: June 2012

Abstract
A new method of measuring cytoplasmic free Ca
2+
([Ca
2+
]
i
) of individual intact cardiovascular endothelial cells by using imaging fluorescence microscopy was designed. Application of agonist to the aortic or pulmonary valve of the rabbit triggered an increase in [Ca
2+
]
i
, which depended on the existence of endothelium on the surface of the valve. Under resting conditions, sudden reversal of the Na
+
gradient by substituting external Na
+
with
N
-methyl
d
-glucamine (NMDG) resulted in a [Ca
2+
]
i
spike, which then returned toward the resting level. Increasing intracellular Na
+
concentration ([Na
+
]
i
) by application of ouabain or monensin induced a sustained [Ca
2+
]
i
increase. Na
+
substitution by NMDG during the agonist- or monensin-induced [Ca
2+
]
i
increase gave rise to a further [Ca
2+
]
i
spike, which subsequently declined to a level higher than that before removal of external Na
+
. A selective inhibitor of Na
+
-Ca
2+
exchange, 3′,4′-dichlorobenzamyl (DCB), abolished the transient [Ca
2+
]
i
increase induced by Na
+
substitution, and Mg
2+
, an inorganic inhibitor of Na
+
-Ca
2+
exchanger, markedly reduced this transient [Ca
2+
]
i
increase. On the other hand, the selective Na
+
-H
+
exchanger blocker 5-(
N,N
-hexamethylene)amiloride (HMA) did not abolish the transient [Ca
2+
]
i
increase caused by Na
+
substitution. In summary, decreasing the Na
+
gradient of the endothelial cells through either receptor stimulation (agonist), Na
+
-K
+
pump inhibition (ouabain), pretreatment with Na
+
ionophore (monensin), or reversing the Na
+
gradient through Na
+
substitution (NMDG) all increased [Ca
2+
]
i
. This raised [Ca
2+
]
i
was antagonized by agents such as DCB or Mg
2+
, which are thought to inhibit Na
+
-Ca
2+
exchange, but not by HMA, an inhibitor of Na
+
-H
+
exchange. Taken together, these results strongly imply the presence of Na
+
-Ca
2+
exchange as a viable mechanism for Ca
2+
transport in intact cardiovascular endothelium and that the Ca
2+
entry component is enhanced when [Na
+
]
i
is elevated.
Long-term High Osmolality Activates Na
+
-H
+
Exchange and Protein Kinase C in Aortic Smooth Muscle Cells
Authors: Soleimani Manoocher , Singh Gurinder , Dominguez Jesus H. , Howard Randy L.
Published: June 2012

Abstract
The effect of long-term exposure to hypertonic medium on Na
+
-H
+
exchange activity was studied in cultured vascular smooth muscle (VSM) cells by using a combination of
22
Na
+
influx and pH measurement with the pH-sensitive dye BCECF. Incubation of VSM cells in high-osmolality medium (510 mOsm/L) for 48 hours significantly increased the acid-stimulated
22
Na
+
influx (control, 3.16±0.41 nmol/mg protein per minute; high osmolality, 6.40±0.66 nmol/mg protein per minute;
P
<.01) and Na
+
-dependent pH
i
recovery (control, 0.29±0.06 pH/min; high osmolality, 0.65±0.13 pH/min;
P
<.03). Activation of Na
+
-H
+
exchange was osmolality dependent and reached maximal stimulation at ≈700 mOsm/L. Na
+
-H
+
exchanger stimulation was independent of serum in the culture media. Na
+
-H
+
exchanger isoform (NHE-1) mRNA in VSM cells cultured in high-osmolality medium was unchanged from that in VSM cells cultured in control medium, indicating an absence of transcriptional regulation by high osmolality. Long-term high osmolality significantly increased protein kinase C (PKC) activity in cultured VSM cells, as assessed by phosphorylation of a PKC-specific substrate (control, 20.9±2.1 pmol phosphorylation/mg protein per minute; high osmolality, 33.6±2.9 pmol phosphorylation/mg protein per minute;
P
<.01). Downregulation of PKC by preincubation of VSM cells with 0.1 μmol/L phorbol 12-myristate 13-acetate (PMA) prevented osmolality-induced stimulation of the Na
+
-H
+
exchanger (control plus PMA, 0.27±0.05 pH/min; high osmolality plus PMA, 0.33±0.08 pH/min;
P
>.05). These results indicate that long-term exposure to hypertonic medium stimulates Na
+
-H
+
exchange activity in cultured VSM cells and that this effect is independent of antiporter gene expression regulation. The results further demonstrate that the stimulatory effect of osmolality on Na
+
-H
+
exchanger is mediated via posttranslational modification of the Na
+
-H
+
exchanger by chronic PKC activation. The Na
+
-H
+
exchanger may be involved in VSM cell volume regulation in long-term high osmolality.
Endothelium-Derived Nitric Oxide Plays a Larger Role in Pulmonary Veins Than in Arteries of Newborn Lambs
Authors: Gao Yuansheng , Zhou Haiyan , Raj J. Usha
Published: June 2012

Abstract
In perinatal lungs, veins contribute substantially to total pulmonary vascular resistance. The present study was designed to determine the role of endothelium-derived nitric oxide (EDNO) in modulating the responsiveness of pulmonary veins and arteries in newborn lambs. Fourth-order pulmonary arterial and venous rings of newborn lambs were suspended in organ chambers filled with modified Krebs-Ringer bicarbonate solution (95% O
2
/5% CO
2
, 37°C), and their isometric force was measured. Nitro-
l
-arginine had no effect on the resting tension of pulmonary arteries but caused an endothelium-dependent contraction of pulmonary veins. During contraction to endothelin-1 or U46619, acetylcholine, bradykinin, and calcium ionophore A23187 induced larger endothelium-dependent relaxation in pulmonary veins than in arteries. The endothelium-dependent relaxation of pulmonary vessels was abolished by nitro-
l
-arginine. In vessels without endothelium, nitric oxide induced significantly greater relaxation in pulmonary veins than in arteries. All vessels relaxed similarly to 8-bromo-cGMP. Radioimmunoassay showed that the basal level of intracellular cGMP of pulmonary veins with endothelium was higher than that of arteries with endothelium. Acetylcholine, bradykinin, and calcium ionophore A23187 induced a significantly larger endothelium-dependent increase in the intracellular content of cGMP in pulmonary veins than in arteries. In vessels without endothelium, nitric oxide induced a larger increase in cGMP content in pulmonary veins than in arteries. The present study suggests that EDNO may play a larger role in modulation of pulmonary venous than arterial reactivity, which may be mainly due to a difference in the activity of soluble guanylate cyclase in vascular smooth muscle.
Diminished Ca
2+
Sensitivity of Skinned Cardiac Muscle Contractility Coincident With Troponin T–Band Shifts in the Diabetic Rat
Authors: Akella Árvind Babu , Ding Xiao-Ling , Cheng Rendi , Gulati Jagdish
Published: June 2012

Abstract
We have measured the apparent Ca
2+
sensitivities of force development in skinned cardiac trabeculae at different sarcomere lengths together with shifts in troponin (Tn) T subunits on specimens from the same hearts and drawn insights into the pathogenesis of myocardial dysfunction in the diabetic rat. The Ca
2+
-force relations were measured at a long (2.4-μm) and a short (1.9-μm) sarcomere length. In disease, compared with the control condition, the apparent Ca
2+
sensitivity was greatly diminished at a sarcomere length of 1.9 μm but not affected at all at the long length (2.4 μm). We also examined the alterations in contractile regulatory proteins TnT and TnI by both sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blots. The TnI band was largely unperturbed, but major changes were discerned in TnT. The normal rat heart indicated two major bands (TnT1 and TnT2) and a faint third band (TnT3); in the diabetic rat heart, there was a significant shift in intensity from TnT1 to TnT3. Since myosin isozyme shifts also accompany diabetes in the rat, we used a prototypical hypothyroid rat as well to evaluate the myosin influence in the length-induced effects on Ca
2+
sensitivity. Myosin shifts during hypothyroidism were unaccompanied by significant changes in TnT, and there were also no length-dependent modifications in Ca
2+
sensitivity. The findings raise the possibility that diabetic Ca
2+
-sensitivity changes in the myocardium are coupled with TnT alterations. A plausible explanation is offered whereby these TnT alterations modify the length dependence of Ca
2+
sensitivity.
MCI-154 Increases Ca
2+
Sensitivity of Reconstituted Thin Filament
Authors: Sata Masataka , Sugiura Seiryo , Yamashita Hiroshi , Fujita Hideo , Momomura Shin-ichi , Serizawa Takashi
Published: June 2012

Abstract
MCI-154 (6-[4-(4′-pyridylamino)phenyl]-4,5-dihydro-3(2
H
)pyridazinone hydrochloride trihydrate) is a potent novel cardiotonic agent whose positive inotropism is shown to be mainly based on an increase in Ca
2+
sensitivity of the contractile apparatus. To elucidate the exact mechanism through which this drug acts, we investigated the movement of the reconstituted thin filament on a myosin layer in vitro. Cardiac thin filaments were reconstituted from actin and tropomyosin-troponin complex purified from rat cardiac acetone powder separately. Double staining of the filament showed that tropomyosin-troponin complex was integrated along actin filament homogeneously. Thin filaments thus prepared were fluorescently labeled and made to slide on rat cardiac myosin fixed on a glass coverslip while varying the [Ca
2+
] of the medium (control, pH 7.2 at 25°C). When [Ca
2+
] was low, the filaments showed only brownian motion. However, above a certain level of [Ca
2+
] (the threshold [Ca
2+
]), the filaments started to slide, and the velocity increased, reaching the maximum velocity within a very narrow range of [Ca
2+
]. The regulation was completely abolished by using simple actin filaments without tropomyosin-troponin complex, demonstrating that the regulatory proteins are responsible for this Ca
2+
regulation of the movement of the reconstituted thin filament. Under the control condition, addition of MCI-154 shifted the threshold [Ca
2+
] to a lower level (sensitization) in a concentration-related manner. And 10
−4
mol/L of MCI-154 reversed the desensitization effect induced by either acidosis (pH 6.8), low temperature (15°C), or the addition of inorganic phosphate (10 mmol/L). However, the maximum sliding velocity was not affected by the drug under any condition. In conclusion, MCI-154 directly sensitized the contractile apparatus under not only physiological but also pathophysiological conditions. This in vitro motility assay technique using reconstituted thin filaments is a useful tool for studying the mechanism of action of Ca
2+
sensitizers.
Effects of Doxorubicin on Excitation-Contraction Coupling in Guinea Pig Ventricular Myocardium
Authors: Wang Yong-Xiao , Korth Michael
Published: June 2012

Abstract
Doxorubicin, an anticancer drug, was recently shown to release Ca
2+
from cardiac sarcoplasmic reticulum (SR) by increasing the open probability of Ca
2+
release channels. In the present study, we investigated the effects of doxorubicin on excitation-contraction coupling of guinea pig heart preparations. In papillary muscles contracting at 0.5 Hz, 100 μmol/L doxorubicin produced within 3 hours the following effects: it increased the force of contraction by 269.3±19.8% (n=6) and prolonged the time to peak force by 75.1±8.7% (n=6), relaxation time by 54.7±8.7% (n=6), and action potential duration (APD) at 90% repolarization (APD
90
) by 38.6±2.9% (n=3). Despite its positive inotropic effect, doxorubicin depressed the early contraction component by increasing the latency between stimulus and the onset of force development. In single myocytes, 100 μmol/L doxorubicin prolonged APD
90
by 62.1% (n=18) and blocked time-dependent delayed rectifier K
+
current (I
K
) by 44% (n=9). Ca
2+
inward current and inward rectifier K
+
current were not affected by doxorubicin. Ca
2+
transients elicited in myocytes loaded with the fluorescent Ca
2+
indicator fura 2 were strongly suppressed by doxorubicin in their initial rising phase. Thereafter, doxorubicin produced a delayed rise in intracellular Ca
2+
, which reached a late peak exceeding that of the control peak by 52±8% (n=5). The results suggest that doxorubicin decreases Ca
2+
-induced Ca
2+
release from cardiac SR, probably by increasing the SR Ca
2+
leak. On the other hand, prolongation of APD due to inhibition of I
K
allows more Ca
2+
to enter the cell. After being only temporarily buffered by the SR, Ca
2+
may accumulate in the cytosol as long as depolarization is maintained and lead to a more complete activation of contractile proteins.
Protein Kinase A Does Not Alter Economy of Force Maintenance in Skinned Rat Cardiac Trabeculae
Authors: de Tombe Pieter P. , Stienen G.J.M.
Published: June 2012

Abstract
Recent mechanical, biochemical, and energetic experiments have suggested that catecholamines may increase the cycling rate of cross-bridges independent of changes in intracellular calcium. An increased rate of cross-bridge cycling is expected to result in decreased economy of force maintenance. The present study tested this hypothesis directly by measuring the rate of ATP consumption in skinned cardiac trabeculae as a function of steady state force. Rat cardiac trabeculae were skinned with Triton X-100. Resting sarcomere length was measured by laser diffraction, and ATP consumption was assessed by an enzyme-coupled optical technique. Force-[Ca
2+
] relations were fit to a modified Hill equation. Force dependency of the rate of ATP consumption was analyzed by multiple linear regression analysis. β-Adrenergic stimulation was mimicked by incubation of the skinned muscle preparation with the catalytic subunit of protein kinase A (PKA). Treatment with PKA (3 μg/mL, 40 minutes) induced a significant (65±23%,
P
=.01) increase in [Ca
2+
] required for half-maximal steady state force, whereas the steepness of the force-[Ca
2+
] relation was not affected. The rate of ATP consumption was linearly correlated with steady state force, regardless of PKA treatment status (
P
<.001). However, neither the slope nor the intercept was affected by PKA treatment. Hence, PKA treatment did not affect either the maximum rate of ATP consumption or the economy of force maintenance. These results suggest that β-adrenergic stimulation does not alter the rate-limiting step of cross-bridge cycling during isometric contraction in myocardium.
Decreased Adenylate Cyclase Activity and Expression of G
sα
in Human Myocardium After Orthotopic Cardiac Transplantation
Authors: Loh Evan , Barnett Joey V. , Feldman Arthur M. , Couper Gregory S. , Vatner Dorothy E. , Colucci Wilson S. , Galper Jonas B.
Published: June 2012

Abstract
We studied several aspects of guanine nucleotide–stimulated adenylate cyclase function in patients after orthotopic cardiac transplantation. In 28 patients, adenylate cyclase activity was measured in endomyocardial biopsy samples obtained just before and at monthly intervals after cardiac transplantation. In biopsies obtained ≥6 months after transplantation, basal adenylate cyclase activity was decreased by 67% (n=12;
P
<.05), GTPγS-stimulated adenylate cyclase activity was decreased by 78% (n=12;
P
<.05), Mn
+2
+forskolin–stimulated adenylate cyclase activity was decreased by 80% (n=8;
P
<.05), and Mn
+2
-stimulated adenylate cyclase activity (a measure of activity of the catalytic subunit of adenylate cyclase) was decreased by 83% (n=8,
P
<.05). Western blot analysis demonstrated that 6 months after cardiac transplantation, the level of G
sα
protein was decreased by 61±12% (n=8;
P
<.001). There was no change in the level of G
iα
as assessed by pertussis toxin–catalyzed ADP-ribosylation (n=4;
P
=NS). With the use of the quantitative polymerase chain reaction, a 50±10% (n=6;
P
<.001) reduction in the steady-state level of G
sα
mRNA was observed. There was no change in the level of mRNA for G
i-3α
. Thus, after orthotopic cardiac transplantation in humans, guanine nucleotide–stimulated adenylate cyclase activity is decreased in parallel with decreased levels of G
sα
protein and mRNA.
Interaction Between Conducted Vasodilation and Sympathetic Nerve Activation in Arterioles of Hamster Striated Muscle
Authors: Kurjiaka David T. , Segal Steven S.
Published: June 2012

Abstract
We tested the hypothesis that sympathetic nerve activity can influence the conduction of vasodilation along the arteriolar wall. Arterioles in the superfused cremaster muscle of anesthetized male hamsters (n=21, 109±4 g) were studied. Microelectrodes were positioned adjacent to the distal end of primary arterioles to stimulate sympathetic nerves throughout arteriolar networks (perivascular nerve stimulation [PNS]). Microiontophoresis micropipettes (tip outer diameter, 1 to 2 μm) filled with acetylcholine (ACh, 1 mol/L) were positioned adjacent to the wall of second-order (2A) or third-order (3A) arterioles ≈1 mm distal to their origin to induce local and conducted vasodilation; diameter responses were recorded at the micropipette tip and at vessel origins, respectively. For 2A and 3A arterioles (resting diameters, 15 to 54 and 9 to 30 μm, respectively), vasoconstriction with PNS was frequency dependent (0.5 to 32 Hz); this was attenuated by 65% (
P
<.05) with α-adrenoceptor blockade (phentolamine, 1 μmol/L). Conducted vasodilation was attenuated by >40% during 16-Hz PNS (
P
<.05); this effect was reversed by phentolamine. In a reciprocal fashion, conducted vasodilation diminished PNS-induced vasoconstriction by ≈50% (
P
<.05). Elevating oxygen (from 0% to 10%) in the superfusion solution induced vasoconstriction similar to that with 16-Hz PNS yet had no effect on conduction. Neural blockade with tetrodotoxin (1 μmol/L) eliminated PNS-induced vasoconstriction and enhanced (
P
<.05) conducted vasodilation. These findings indicate that perivascular nerves in striated muscle can influence cell-to-cell communication along the arteriolar wall both at rest and during enhanced sympathetic activity. The attenuation of sympathetic vasoconstriction by conducted vasodilation suggests a novel explanation for functional sympatholysis.
Endothelium-Dependent Contractions Are Associated With Both Augmented Expressionof Prostaglandin H Synthase-1 and Hypersensitivity to Prostaglandin H
2
in the SHR Aorta
Authors: Ge Tong , Hughes Helen , Junquero Didier C. , Wu Kenneth K. , Vanhoutte Paul M. , Boulanger Chantal M.
Published: June 2012

Abstract
Prostaglandin H
2
(PGH
2
[endoperoxide]) is an immediate product of prostaglandin H (PGH) synthase activity (cyclooxygenase) and a likely candidate to mediate endothelium-dependent contractions evoked by acetylcholine in the aorta of the spontaneously hypertensive rat (SHR). Experiments were designed to investigate whether or not endothelium-dependent contractions were associated with an increased expression of PGH synthase, an augmented acetylcholine-induced release of PGH
2
, and/or a hypersensitivity of the smooth muscle to endoperoxides in SHR aorta compared with normotensive Wistar-Kyoto (WKY) aorta. In SHR aorta, endothelium-dependent contractions to acetylcholine were abolished by tenidap (10
−8
mol/L), a preferential PGH synthase-1 inhibitor, but slightly impaired by NS-398 (10
−6
mol/L), a preferential PGH synthase-2 inhibitor. PGH synthase-1 expression, which was evaluated by both reverse transcriptase–polymerase chain reaction and Western blotting, was about twofold greater in preparations with endothelium from SHR than from WKY rats. There was no difference in PGH synthase-1 expression between preparations with and those without endothelium in both strains. In SHR but not WKY aortas, acetylcholine (10
−5
mol/L, 5 minutes) caused a significant endothelium-dependent release of PGH
2
, as measured by gas chromatography/mass spectrometry. PGH
2
evoked more potent contractions in rings without endothelium from SHR than from WKY rats, whereas the thromboxane analogue U46619 and prostaglandin F
2α
caused a comparable response in both preparations. These results show that endothelium-dependent contractions to acetylcholine in SHR aorta are associated with a greater expression of PGH synthase-1, a significant release of PGH
2
, and a hypersensitivity of the smooth muscle to the endoperoxide.
Cardiac Troponin I Phosphorylation Increases the Rate of Cardiac Muscle Relaxation
Authors: Zhang Ren , Zhao Jiaju , Mandveno Alan , Potter James D.
Published: June 2012

Abstract
Cardiac troponin (Tn) I (CTnI), compared with skeletal TnI, contains extra amino acids (32 to 33) at its amino terminus, including two adjacent serine residues. These two serine residues are believed to be phosphorylated by protein kinase A (PKA) upon stimulation of the heart by β-agonists. In this study, we found that phosphorylation of a cardiac skinned muscle preparation by PKA, mainly at CTnI, results in a decrease in the Ca
2+
sensitivity of muscle contraction. The pCa
50
decreased by ≈0.27±0.06 pCa units upon phosphorylation. To study cardiac muscle relaxation, we used diazo-2, a photolabile Ca
2+
chelator with a low Ca
2+
affinity in its intact form that is converted to a high-affinity form after photolysis. We found that the rate of cardiac muscle relaxation increased from a time of half-relaxation (t
1/2
)=110±10 milliseconds to t
1/2
=70±8 milliseconds after CTnI phosphorylation. This result demonstrates that CTnI phosphorylation can be linked with the increased rate of muscle relaxation in a relatively intact muscle preparation. Since CTnI phosphorylation has been shown previously to affect the Ca
2+
affinity and Ca
2+
off-rate of CTnC in vitro, it is likely that the faster relaxation seen here reflects faster dissociation of Ca
2+
from cardiac TnC (CTnC). Model calculations show that increased dissociation of Ca
2+
from CTnC, coupled with the faster uptake of Ca
2+
by the sarcoplasmic reticulum stimulated by PKA phosphorylation of phospholamban, can account for the faster relaxation seen in the inotropic response of the heart to catecholamines.
Calcitonin Gene-Related Peptide Mediates Acetylcholine-Induced Endothelium-Independent Vasodilation in Mesenteric Resistance Blood Vessels of the Rat
Authors: Takenaga Makoto , Kawasaki Hiromu , Wada Akihiko , Eto Tanenao
Published: June 2012

Abstract
The role of calcitonin gene-related peptide (CGRP)–containing vasodilator nerves in acetylcholine chloride (ACh)–induced vasodilation was studied in the perfused mesenteric vascular bed isolated from the rat. Bolus infusions of ACh at smaller doses (0.1 and 1 nmol) produced rapid and short-lived vasodilation. However, larger doses (10 and 100 nmol) of ACh caused a rapid and subsequent long-lasting vasodilator response in which the duration of vasodilation was prolonged in a concentration-dependent manner. Pretreatment with capsaicin (1 μmol/L for 20 minutes) significantly shortened the duration of vasodilator response to ACh but did not affect the initial rapid phase of ACh-induced vasodilation. Chemical removal of the vascular endothelium by perfusion with sodium deoxycholate (1.75 to 1.80 mg/mL) for 30 seconds and subsequent treatment with
N
ω
-nitro-
l
-arginine (100 μmol/L) to inhibit nitric oxide synthesis abolished the initial rapid vasodilator action of ACh at any given concentration. However, in the same preparation, increasing concentrations (from 1 to 1000 nmol) of ACh produced only the long-lasting vasodilator responses in a concentration-dependent manner. This long-lasting vasodilator response to ACh infusion was abolished by capsaicin pretreatment (1 μmol/L), human CGRP[8-37] (CGRP receptor antagonist, 1 μmol/L), and atropine (muscarinic ACh receptor antagonist, 1, 10, and 100 nmol/L) but not by hexamethonium (nicotinic ACh receptor antagonist, 1 and 10 μmol/L). In the preparations without endothelium, the bolus infusion of ACh (300 nmol for 30 seconds) evoked a long-lasting vasodilation and release of CGRP-like immunoreactivities into the perfusate. These results suggest that the ACh-induced vasorelaxation consists of two elements: an initial transient endothelium-dependent component and a secondary long-lasting endothelium-independent component. Moreover, ACh activates muscarinic receptors located on CGRP-containing neurons to release CGRP, which then acts at CGRP receptors on vascular smooth muscles to cause the endothelium-independent vasodilation.
Thrombin Induces the Preproendothelin-1 Gene in Endothelial Cells by a Protein Tyrosine Kinase–Linked Mechanism
Authors: Marsen Tobias A. , Simonson Michael S. , Dunn Michael J.
Published: June 2012

Abstract
Thrombin stimulates synthesis and secretion of endothelin-1 (ET-1), a vasoactive peptide that triggers responses in the vascular endothelium and smooth muscle. We investigated the signal transduction pathways by which thrombin stimulates preproET-1 gene expression and ET-1 peptide secretion in macrovascular cells (human umbilical vein endothelial cells [HUVECs] and bovine pulmonary artery endothelial cells [BPAECs]) and microvascular cells (human microvascular endothelial cell line [HMEC-1]). Thrombin (4 U/mL) stimulated maximal induction of ET-1 peptide secretion and preproET-1 mRNA after 2 hours in HUVECs and BPAECs and after 1 hour in HMEC-1. A synthetic thrombin receptor activator peptide confirmed ligand-specific receptor actions to induce preproET-1 mRNA. Protein kinase C (PKC) activation by phorbol ester transiently induced preproET-1 mRNA but had no effect on ET-1 peptide synthesis. PKC inhibitors sangivamycin and calphostin C and PKC depletion failed to suppress thrombin-stimulated preproET-1 mRNA. Adenylate cyclase and cAMP-dependent protein kinase did not participate in thrombin-induced preproET-1 gene activation. Thrombin stimulated a rapid increase in phosphotyrosine-containing proteins, suggesting a role for tyrosine phosphorylation in thrombin signaling. These data demonstrate that thrombin induces the preproET-1 gene and ET-1 peptide synthesis by a PKC-independent PTK-dependent pathway in macrovascular and microvascular endothelial cells. Protein tyrosine kinase inhibitors herbimycin A and genistein blocked thrombin-stimulated preproET-1 mRNA and peptide secretion, whereas daidzein, which lacks inhibitory activity, did not suppress thrombin-induced ET-1.
Localization of Atrial Natriuretic Factor Receptors in the Mesenteric Arterial Bed
Authors: de León Héctor , Bonhomme Marie-Chantal , Thibault Gaétan , Garcia Raul
Published: June 2012

Abstract
Although receptors for atrial natriuretic factor (ANF) and angiotensin II (Ang II) have been reported in rat mesenteric arteries, both peptides induce weak biological responses. Endothelin-1 (ET-1) evokes a potent vasoconstriction in the mesenteric artery. To identify the tissue localization of ANF, Ang II, and ET-1 receptors, radioligand binding experiments with
125
I-ANF,
125
I–[Sar
1
,Ile
8
]Ang II, and
125
I–ET-1 were performed in defatted mesenteric arteries and in the surrounding adipose tissue.
125
I-ANF binding assays in adipose tissue showed a single class of high-affinity binding sites (B
max
, 420±16 fmol/mg protein;
K
d
, 343±16 pmol/L). In vascular membranes, most
125
I-ANF binding was nonspecific. The majority of receptors present in adipose tissue recognized ANF, C-type natriuretic peptide (CNP), and des-[Gln
18
,Ser
19
,Gly
20
,Leu
21
,Gly
22
]ANF-(4-23) (C-ANF) with close affinities, with C-ANF competing for >98% of the binding sites. In adipocytes, ANF and CNP stimulated cGMP generation. cGMP production by mesenteric arteries was stimulated by sodium nitroprusside but not by ANF or CNP. Autoradiographic localization of
125
I-ANF and
125
I–ET-1 showed that in the case of ANF, most specific binding occurred in adipocytes, whereas for ET-1, specific binding was present in both adipose tissue and mesenteric arteries. Cross-linking of
125
I-ANF followed by SDS-PAGE revealed two receptor species of 130 and 70 kD in adipose membranes and none in vascular tissue. Both were completely displaced by ANF, CNP, and C-ANF.
125
I–[Sar
1
,Ile
8
]Ang II binding assays in adipose tissue exhibited a single class of binding sites (B
max
, 211±4 fmol/mg protein;
K
d
, 520±10 pmol/L). In mesenteric arteries,
125
I–[Sar
1
,Ile
8
]Ang II saturation assays showed little specific binding. The profile of ligands competing for
125
I–[Sar
1
,Ile
8
]Ang II was consistent with an AT
1
receptor subtype.
125
I–ET-1 binding assays demonstrated high-affinity binding sites in both mesenteric arteries and adipose tissue (B
max
, 179±21 and 312±7 fmol/mg protein, respectively;
K
d
, 215±45 and 180±111 pmol/L, respectively), the majority corresponding to BQ-123–sensitive sites. Thus, ANF and Ang II binding sites were either absent or barely expressed in the mesenteric artery. Binding sites for ET-1 were abundantly expressed in the mesenteric artery. Adipose tissue expressed high-affinity binding sites for ANF, Ang II, and ET-1, but their biological role, if any, remains to be defined.
Regulation of Aldosterone Biosynthesis by Adrenal Renin Is Mediated Through AT
1
Receptors in Renin Transgenic Rats
Authors: Volpe Massimo , Rubattu Speranza , Gigante Bruna , Ganten Detlev , Porcellini Antonio , Russo Rosaria , Romano Michele , Enea Iolanda , Lee Min Ae , Trimarco Bruno
Published: June 2012

Abstract
The transgenic (TG) rat (mREN2)27 is characterized by overexpression of the additional mouse Ren-2
d
gene in the adrenal cortex with marked suppression of renal renin. We have previously shown that in salt-depleted TG rats enhanced activation of mineralocorticoid biosynthesis is associated with selective stimulation of adrenal renin. To investigate whether the local renin-angiotensin system regulates aldosterone biosynthesis in the adrenal cortex of TG rats, we studied the effects of the AT
1
–angiotensin subtype receptor antagonist DuP 753 on aldosterone production in 5-week-old TG rats during salt restriction. All the rats (n=56) were shifted from regular chow to a diet containing only 0.04% NaCl for 1 week. The AT
1
-receptor antagonist DuP 753 (10 mg/kg per day in drinking water) was administered to 27 of these rats during low-salt diet. Subgroups of rats were killed at 0, 4, and 7 days. Low-salt diet increased both adrenal renin activity (from 31±3 to 77±4 and 85±2 ng angiotensin I · h
−1
· mg protein
−1
at 4 and 7 days, respectively;
P
<.001) and mRNA (by 68.4±10% and 80±17% from baseline,
P
<.05). In addition, salt restriction was associated with increases of AT
1
-receptor subtype mRNA, aldosterone-synthase cytochrome P450 mRNA (by 130±56% and 227±33% at 4 and 7 days, respectively;
P
<.05), and plasma aldosterone (from 184±33 to 402±79 and 338±59 pg/mL,
P
<.05), whereas renal renin activity did not change, renin mRNA levels remained almost undetectable, and plasma renin activity showed a transient increase. In contrast, in TG rats treated with DuP 753 salt restriction was associated with increases of renal renin mRNA and renal and plasma renin activity, whereas the stimulation of aldosterone-synthase mRNA was markedly attenuated, and plasma aldosterone did not change. Separate analysis of mouse transgene and endogenous rat renin performed by RNase protection assay in 18 additional TG rats and in 18 age- and sex-matched Sprague-Dawley rats showed that the mouse transgene was prominently expressed in the adrenal glands of TG rats in all experimental conditions. In conclusion, our data show that AT
1
-receptor antagonism abolishes the adrenal renin–related stimulation of the aldosterone biosynthesis produced by salt restriction in TG rats. This indicates that in this model the adrenal renin-angiotensin system regulates the mineralocorticoid production via the AT
1
-receptor subtype.
l
-Methionine Augments Mammalian Myocardial Contraction by Sensitizing the Myofilament to Ca
2+
Authors: Kihara Yasuki , Inoko Moriaki , Sasayama Shigetake
Published: June 2012

Abstract
l
-Methionine is an essential amino acid that has been reported to have a potent positive inotropic effect on the mammalian myocardium. We studied the mechanisms of the inotropic effect in ventricular myocardium from the rabbit. In the isolated coronary-perfused whole heart,
l
-methionine in a millimolar range exerted concentration-dependent positive inotropic effects on the isovolumic left ventricle, which were associated with negative lusitropic effects (prolonged time course of relaxation). The chronotropic state and the coronary perfusion pressure were not affected. These complex effects on the isolated whole heart were not blocked by pretreatment with (μmol/L) propranolol 1, prazosin 1, carbachol 3, staurosporine 1, or [Ser
1
,Ile
8
]angiotensin II 0.1. To further study the subcellular mechanisms, isolated ventricular papillary muscles from the same species were loaded with a bioluminescent indicator, aequorin, to monitor [Ca
2+
]
i
. In the presence of 3 mmol/L
l
-methionine, the isometric tension showed a similar combination of the positive inotropic and negative lusitropic effects as observed in the whole heart. In contrast, the simultaneously recorded intracellular Ca
2+
signals did not increase in amplitude but instead decreased. The [Ca
2+
]
i
-tension relation shifted to the left compared with that obtained in response to [Ca
2+
]
o
. In saponin (250 μg/mL)–treated skinned preparations, 3 mmol/L
l
-methionine also shifted the force-pCa curve to the left by 0.16 pCa units. This is the first demonstration that an essential amino acid directly acts on the myofilaments and modulates their responsiveness to Ca
2+
, thereby producing a positive inotropic effect.
Reversible Permeabilization
Authors: Lesh Ryan E. , Somlyo Andrew P. , Owens Gary K. , Somlyo Avril V.
Published: June 2012

Abstract
Antisense oligodeoxynucleotides (ODNs) have been used to modify gene expression in vitro and are also promising therapeutic agents. Although there are numerous reports of antisense ODN–mediated changes in protein expression of cultured cells, use of these compounds to achieve antisense regulation of specific proteins in intact tissue has been limited. The aims of this study were (1) to define organ culture conditions for ileum smooth muscle that would permit long-term maintenance of force-generating capabilities and normal ultrastructure and (2) to develop a method for efficient introduction of antisense ODNs into intact tissue. Sheets of ODN-containing, reversibly permeabilized rat outer longitudinal ileum were maintained in a serum-free organ culture medium for 1 week without significant decreases in tension response to membrane depolarization or carbachol stimulation; the G protein–coupled calcium sensitization pathway was also intact after 7 days. Reversible permeabilization, a method previously used to load smooth and cardiac muscle with aequorin and heparin, was effective for loading >95% of ileum smooth muscle cells with a fluorescein-conjugated antisense ODN (5′-AAGGGCCATTTTGTT-FITC-3′). Confocal microscopy of reversibly permeabilized smooth muscle loaded with fluorescent antisense ODNs revealed intense nuclear fluorescence and less intense, homogeneous, cytoplasmic fluorescence. Internally radiolabeled ODNs (homologous to the above sequence) showed complete degradation between 4 and 16 hours after introduction into the cells. In summary, we have demonstrated methods for long-term organ culture and high-efficiency introduction of antisense ODNs into intact smooth muscle sheets. Such methods have broad potential utility for investigating many questions in smooth muscle biology. At present, however, a major limitation of this approach is the short half-life of phosphorothioated ODNs.