bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of oxidant stress on endothelium-derived relaxing factor-induced and nitrovasodilator-induced cGMP accumulation in vascular cells in culture.
Authors: Marczin N , Ryan U S , Catravas J D
Published: June 2012

The effects of hydrogen peroxide (H2O2) on the action of basally produced endothelium-derived relaxing factor (EDRF) were investigated by measuring cGMP accumulation in single and cocultures of calf pulmonary artery endothelial cells (CPAEs) and rabbit pulmonary artery smooth muscle cells (RPASMs) as a model for determining the contribution of EDRF dysfunction to altered vascular tone and reactivity frequently associated with oxidant-induced vascular injury. Higher cGMP levels in long-term cocultures (20.4 +/- 1.8 pmol/mg protein/15 min) than in single-cell cultures (CPAE, 9.6 +/- 0.9 pmol/mg protein/15 min; RPASM, 3.7 +/- 0.2 pmol/mg protein/15 min), and CPAE-induced increases (fivefold) in intracellular RPASM cGMP content in short-term cocultures suggest basal release of EDRF. Basal generation and release of an L-arginine-derived endothelial labile factor accounted for the increases in cGMP, since the response was completely blocked by pretreatment of CPAEs with NG-monomethyl L-arginine. Pretreatment of long-term cocultures with H2O2 for 30 minutes resulted in a dose-dependent (0.5-2 mM) decrease in cGMP formation (49-79%). To determine the effects of H2O2 on EDRF synthesis, transport, and RPASM responsiveness, CPAEs or RPASMs were selectively pretreated with H2O2 before establishment of short-term cocultures. In cocultures of H2O2-pretreated CPAEs with untreated RPASMs, RPASM cGMP levels were reduced, suggesting a decrease in EDRF production rather than deterioration of EDRF during transport, because cGMP levels were unaffected by posttreatment with oxygen radical scavengers during coculture. Pretreatment of RPASMs with H2O2 attenuated the untreated CPAE-induced, the putative EDRF S-nitroso-L-cysteine-induced, or the nitroprusside-induced increases in RPASM cGMP levels. This attenuation was prevented by pretreatment with either dimethylthiourea, deferoxamine, or dithiothreitol, suggesting a mechanism of H2O2 action involving iron-catalyzed formation of intracellular hydroxyl radicals and their attack on cellular thiols. H2O2 diminution of cGMP accumulation was not associated with lytic cell injury in the experimental time frame, because morphology and 51Cr release from prelabeled RPASMs and CPAEs were unchanged.
Influence of platelet-vessel wall interactions on leukocyte rolling in vivo.
Authors: oude Egbrink M G , Tangelder G J , Slaaf D W , Reneman R S
Published: June 2012

The influence of platelet-vessel wall interactions on leukocyte rolling was investigated in rabbit mesenteric venules (diameter, 21-40 microns) using intravital videomicroscopy. Puncture of the wall with glass micropipettes (tip, 6-8 microns) evoked the formation of a thrombus in all venules. In most vessels, emboli were produced as well. The rolling of leukocytes (i.e., their movement along the vessel wall at a velocity clearly lower than that of the other blood cells) was quantitated simultaneously in vessel segments upstream and downstream from a thrombus up to 10 minutes after puncture. During embolization the number of rolling leukocytes decreased significantly from the upstream to the downstream vessel segment (median decrease, 45%; p less than or equal to 0.001). It was still decreased by approximately 50% after embolization had stopped, indicating that the decrease in leukocyte rolling was not caused by inclusion of leukocytes in the emboli. In venules without embolization, leukocyte rolling did not change systematically, indicating that fluid dynamic changes induced by the thrombus do not influence leukocyte rolling. Inhibition of prostaglandin formation with aspirin (100 mg/kg) almost completely abolished the influence of the thromboembolic reaction on leukocyte rolling, but blockade of thromboxane A2 receptors with sulotroban (30 mg/kg) had no effect. In conclusion, this is the first report on a functional interaction in vivo, at a site of vessel wall injury, between platelets, vascular cells, and leukocytes. The findings suggest that substances produced by activated platelets and/or damaged vascular cells diminish leukocyte rolling. The identity of these substances is not yet clear, but the present study indicates that prostaglandins other than thromboxane A2 are involved.
Indexing tricarboxylic acid cycle flux in intact hearts by carbon-13 nuclear magnetic resonance.
Authors: Weiss R G , Gloth S T , Kalil-Filho R , Chacko V P , Stern M D , Gerstenblith G
Published: June 2012

Although the tricarboxylic acid (TCA) cycle is the prime means of carbon metabolism for energy generation in normal myocardium, the noninvasive quantification of TCA cycle flux in intact cardiac tissues is difficult. A novel approach for estimating citric acid cycle flux using 13C nuclear magnetic resonance (NMR) is presented and evaluated experimentally by comparison with measured myocardial oxygen consumption over a wide range of cardiac contractile function in intact, beating rat hearts. Continuous series of 13C NMR spectra, obtained after the introduction of [2-13C]acetate as substrate, quantified the time course of 13C appearance in the carbon positions of myocardial glutamate, which are sequentially enriched via citric acid cycle metabolism. A TCA cycle flux parameter was calculated using the premise that TCA cycle flux is inversely proportional to the time difference between 13C appearance in the C-4 and C-2 positions of glutamate (glutamate delta t50 [minutes]), which are enriched in subsequent "turns" of the TCA cycle. This TCA cycle flux parameter, termed KT, correlated strongly with myocardial oxygen consumption over a range of developed pressures in hearts perfused with 5 mM acetate (r = 0.98, p less than 0.001), as well as in separate studies in hearts perfused with 5 mM glucose and 0.5-0.8 mM acetate (r = 0.94, p less than 0.001). Results of numerical modeling of 13C glutamate kinetics suggest that this TCA cycle flux parameter, KT, is relatively insensitive to changes in metabolite pool sizes that could occur during metabolism of other substrates or during conditions of altered oxygen availability. Additional studies in separate hearts indicated that the time course of 13C appearance in citrate, which is predominantly mitochondrial in the rat heart, is similar to that in glutamate, further supporting the premise that the described 13C NMR parameters reflect mitochondrial citric acid cycle activity in intact cardiac tissues.
"Tissue need" and limb collateral arterial growth. Skeletal contractile power and perfusion during collateral development in the rat.
Authors: Paskins-Hurlburt A J , Hollenberg N K
Published: June 2012

Among the factors that might influence collateral arterial growth after arterial occlusion, the capacity to deliver blood flow in relation to metabolic need and work performance are obvious candidates. In this study in rats after superficial femoral artery ligation, we assessed collateral arterial growth (by arteriography), basal and peak limb blood flow during acetylcholine-induced vasodilation (by electronic drop counting), pressure-flow relations, and contractile power of the gastrocnemius muscle (force transduction during sciatic nerve stimulation) at intervals over 3 months after superficial femoral artery ligation. Basal and peak blood flow and muscle contractile power were clearly reduced 1 week after ligation but had returned to normal by 3 weeks. Major collateral arterial growth, however, progressed between 3 weeks and 3 months. The limb perfusion pressure-blood flow relation was still altered at 3 weeks, with blunting of the normal autoregulation, and became more normal by 3 months after superficial femoral artery ligation. Collateral arterial growth continues after blood flow adequate to maintain work performance has been restored and may reflect a response to more subtle abnormalities involving distal pressure delivery, evident in altered pressure-flow relations.
Acute resetting in two functionally different types of carotid baroreceptors.
Authors: Seagard J L , Gallenberg L A , Hopp F A , Dean C
Published: June 2012

The presence of two types of carotid sinus baroreceptors, as characterized by two different stimulus-response curves in an earlier study, suggests that each type may play a different role in the regulation of blood pressure. The discontinuous hyperbolic curve of the type I baroreceptors, marked by higher firing rates and greater sensitivity than the sigmoidal curve of type II baroreceptors, suggests that these baroreceptors would contribute more to the buffering of arterial pressure changes than the "tonically" active type II baroreceptors, which fired over greater pressure ranges and generally had spontaneous subthreshold discharge. The firing characteristics of type II baroreceptors suggest that these receptors would contribute more to regulation of tonic, baseline levels of arterial pressure. If this functional differentiation exists, the acute resetting characteristics of the two types of baroreceptors could be different. Resetting is defined as a shift in the response curve of a baroreceptor, marked by shifts in pressure threshold, in the same direction as the change in pressure to which it is exposed. Type I baroreceptors would be more likely to reset in response to a sustained acute change in pressure, since their primary role would be to prevent the initial change in pressure. However, type II baroreceptors would not reset to the acute change in pressure, since their primary role would be to maintain consistent information on the level of existing pressure. Therefore, this study was performed to examine the acute resetting ability of both types of baroreceptors by using a vascularly isolated carotid sinus preparation in the dog.(ABSTRACT TRUNCATED AT 250 WORDS)
Time course of changes in intracellular K+, Na+, and pH of subendocardial Purkinje cells during the first 24 hours after coronary occlusion.
Authors: Kline R P , Hanna M S , Dresdner K P , Wit A L
Published: June 2012

We investigated the basis for the alterations in the intracellular potassium and sodium activity occurring in subendocardial Purkinje fibers surviving in 24-hour infarcts by examining ion activities in these Purkinje fibers removed from infarcting hearts at earlier times. Specifically, we examined intracellular potassium activity, sodium activity, and pH at 1 and 3 hours after ligation of the left anterior descending coronary artery, and we correlated the changes in ion activity with changes in maximum diastolic potential. We tested various mechanistic hypotheses relating to how the ion activity changes develop and how they affect membrane potential. We found that intracellular sodium activity in tissue removed 1 hour after ligation was on average already maximally elevated by a factor of 2 over control (19.2 +/- 2.0 mM [mean +/- SEM] versus 9.4 +/- 0.4 mM). Potassium activity diminished progressively over the first 24 hours (from normal of 112.0 +/- 2.7 to 61.6 +/- 2.8 mM), although half of the decrease occurred during the first hour (to 86.8 +/- 4.1 mM). Intracellular pH did not change at either 1 or 3 hours. Whereas maximum diastolic potential depolarization exceeded the calculated depolarization of the potassium equilibrium potential by a factor of 2 in 24-hour infarcts, the depolarization at 1 and 3 hours could be more nearly attributed to the loss of potassium. The change in the dependence of maximum diastolic potential on potassium equilibrium potential may be due to changes in membrane conductance caused by ionic or biochemical factors. The changes in ion activity continuously develop during the first day after ligation and may be due to multiple factors and mechanisms.
Nuclear magnetic resonance evaluation of metabolic and respiratory support of work load in intact rabbit hearts.
Authors: Lewandowski E D
Published: June 2012

Pre-steady-state 13C nuclear magnetic resonance (NMR) spectra can provide a nondestructive probe of metabolic events associated with the physiology of intact organs. Therefore, the relation between phosphorylation state and intermediary metabolism in rabbit hearts, oxidizing [2-13C]acetate, was examined with a combination of 31P and 13C NMR. Multiple enrichment of the tissue glutamate pool with 13C as an index of metabolic turnover within the tricarboxylic acid cycle was readily observed as a function of work load. Dynamic changes in pre-steady-state 13C spectra evolved according to work load and correlated closely to respiratory rate in rabbit hearts perfused 1) under normal conditions (n = 7), 2) at basal metabolic rates (20 mM KCl arrest, n = 5), 3) and at heightened contractile state (10(-7) M isoproterenol, n = 7). The ratio of signal intensity arising from the secondary labeling sites within glutamate (C-2 and C-3) to that of the initial labeling site (C-4) reached steady state within 8.5 minutes in isoproterenol-treated hearts versus 18.5 minutes in control hearts. Work load did not affect glutamate concentration or fractional enrichment at the C-4 position, although an unlabeled fraction of glutamate persisted. Arrested hearts displayed slowed evolution of steady-state 13C enrichment with increased contributions from anaplerotic sources for tricarboxylic acid intermediate formation (32%) as compared with control (9%). Thus, the response of mitochondrial dehydrogenase activity to the demands of cardiac performance is likely to influence the recruitment of anabolic sources supplying the tricarboxylic acid cycle.(ABSTRACT TRUNCATED AT 250 WORDS)
In-plane myocardial wall stress is not the primary determinant of coronary systolic flow impediment. A study in the isolated, perfused dog septum.
Authors: Resar J R , Livingston J Z , Yin F C
Published: June 2012

The hypothesis that ventricular in-plane tensile wall stresses are the major determinant of systolic coronary flow was investigated in this study. We measured coronary artery inflow in the maximally vasodilated bed of the isolated beating septum (n = 10) during two modes of contraction characterized by markedly different levels of developed in-plane stress. An increase in contractility was induced by changing from the control steady-state pacing state to a postextrasystolic potentiated state induced by a modified rapid pacing protocol. Over a range of increments of passive stretch, the systolic flow impediment versus the diastolic wall strain was described by an inverse linear relation. Despite the differences in developed in-plane wall stresses between the two modes of contraction (p less than 0.001), the slope and intercept of these relations in both the control and potentiated states were not different for the low versus high developed stress modes. The systolic flow impediment versus diastolic wall strain relation for the potentiated beats, compared with the control beats, was characterized by an increase in the intercept in both the low developed stress beats (p less than 0.05) and the high developed stress beats (p less than 0.05). These data indicate that the impediment to coronary flow during systole is not primarily determined by systolic myocardial in-plane tensile wall stresses but rather by the contractile state of the muscle.
Bilateral atrial appendectomy abolishes increased plasma atrial natriuretic peptide release and blunts sodium and water excretion during volume loading in conscious dogs.
Authors: Stewart J M , Dean R , Brown M , Diasparra D , Zeballos G A , Schustek M , Gewitz M H , Thompson C I , Hintze T H
Published: June 2012

The atrial appendages contain most of the atrial natriuretic factor (ANF) in the mammalian heart, and atrial appendage mechanical function predicts ANF secretion during volume loading. To demonstrate the crucial role of the atrial appendages in ANF release, we first measured hemodynamics and changes in plasma ANF after injection of 1,000 ml i.v. normal saline in conscious dogs and again after bilateral atrial appendectomy; we next measured changes in renal function using infusions of atriopeptin 24 to achieve plasma levels corresponding to levels achieved during volume loading; and we lastly measured renal function during acute volume expansion and also after atrial appendectomy. Plasma ANF increased from 65 +/- 11 to 246 +/- 54 pg/ml after volume loading but did not increase after atrial appendectomy. Atrial appendectomy did not alter the tachycardia or hemodynamic effects of volume loading. Infusion of 10 ng/kg/min atriopeptin 24 increased plasma ANF from 50 +/- 9 to 234 +/- 54 pg/ml, increased urine output 34 +/- 10%, and increased sodium excretion 62 +/- 10% in dogs with intact atrial appendages. Renal function was compared in dogs before atrial appendectomy: 20, 40, and 60 minutes after volume loading, urine flow rate increased by 5.9 +/- 0.5, 6.9 +/- 0.4, and 4.4 +/- 0.8 ml/min, while sodium excretion increased by 717 +/- 60, 839 +/- 84, and 582 +/- 57 mueq/min. After atrial appendectomy urine flow rate increased 2.1 +/- 0.7, 2.7 +/- 0.7, and 2.0 +/- 0.6 ml/min, and sodium excretion increased only by 327 +/- 110, 324 +/- 77, and 340 +/- 92 mueq/min (p less than 0.01) during volume loading.(ABSTRACT TRUNCATED AT 250 WORDS)
Contractile proteins in myocardial cells are regulated by factor(s) released by blood vessels.
Authors: McClellan G , Weisberg A , Kato N S , Ramaciotti C , Sharkey A , Winegrad S
Published: June 2012

The importance of perfusion of the coronary vasculature in the regulation of ATPase activity of myosin in rat myocardial cells has been studied. Quantitative histochemistry was used to determine the activity of the enzyme among cells in tissues that had been either perfused through the coronary system or superfused over the surface of the tissue. Enzymatic activity was measured in cryostatic sections from three different preparations: 1) hearts frozen immediately after removal from the animal; 2) isolated hearts frozen after they had been perfused through the coronary circulation; and 3) isolated papillary muscles or trabeculae that had been superfused after dissection and then frozen. ATPase activity was measured in the isolated tissues at different times after dissection. Both calcium- and actin-activated myosin ATPase activities were uniform among cells in both the ventricles of the hearts frozen immediately after dissection and those that had been perfused through the coronary system. In the superfused tissues, although calcium-activated myosin ATPase activity was uniform, actin-activated ATPase activity was not uniform for about 90 minutes after the dissection, the period required for stabilization of the contraction. The pattern of nonuniformity was complex. In all bundles the lowest enzymatic activity was found in the most superficial cells. In very thin bundles, the cells in the center had the highest activity. In the medium and thicker bundles, there were three concentric zones of actin-activated ATPase activity, the superficial zone with the lowest activity, an intermediate zone with high activity, and a central zone with lower activity. Within each zone, the activity was often greatest in myocardial cells immediately next to blood vessels even though the blood vessels had not been perfused. The transverse distribution of ATPase activity of myosin could be explained by a mechanism in which cells in blood vessels (presumably endothelium) release a substance that upregulates myosin ATPase activity, with the rate of release being related to the local oxygen tension. A downregulating substance may also be produced. The period of stabilization of the contraction coincides with the time during which the pattern of actomyosin ATPase activity is nonuniform. These data suggest that the contractile proteins are regulated by a substance produced by blood vessels in proportion to the local PO2, and possibly in relation to shear force on the vascular endothelium.
Ca2+ regulation of mechanical properties of striated muscle. Mechanistic studies using extraction and replacement of regulatory proteins.
Authors: Moss R L
Published: June 2012

Extraction of regulatory proteins from thick and thin filaments of vertebrate striated muscle has proven to be an important approach in elucidating roles of these proteins in regulating contraction and in probing specific mechanisms of activation. For some proteins, such as LC2 and C protein, extraction has been fundamental in demonstrating the importance of these proteins in modulating contraction and the kinetics of cross-bridge interaction. For other proteins, such as TnC and troponin, extraction has provided significant insight into the importance of thin-filament intermolecular cooperativity in modulating Ca2+ sensitivity of the contractile process. A combination of extraction and readdition has provided a means of introducing mutated or derivatized proteins into fibers to accomplish a variety of experimental objectives. The use of this approach is likely to grow with the need to test the functional consequences of site-specific mutations as part of studies directed to mechanisms of regulation or altered regulation in heart and skeletal muscles under normal and pathophysiological conditions. Such studies are likely to include extraction in combination with other probes of function such as flash photolysis of reaction substrates or products within the cross-bridge interaction cycle. Although extraction is a powerful approach and is likely to be extended to proteins not discussed in this review, an essential element of experimental design in studies such as these is that appropriate control experiments be done to verify that observed effects of the extraction protocol are specifically attributable to the protein that is removed.
Effects of beta-adrenergic receptor stimulation and blockade on rate-dependent atrioventricular nodal properties.
Authors: Nayebpour M , Talajic M , Nattel S
Published: June 2012

Recent work has shown that alterations in the dynamic atrioventricular (AV) nodal response to changes in heart rate can significantly modify AV nodal function. The present study was designed to evaluate the nature and potential importance of sympathetic regulation of the rate-dependent properties of the AV node. Selective stimulation protocols and mathematical formulations were used to independently quantify AV nodal recovery, facilitation, and fatigue in 12 morphine-chloralose-anesthetized dogs. Vagal effects were prevented by bilateral vagal transection and intravenous atropine, and the sinus node was crushed to allow a broader range of pacing cycle lengths. In seven dogs with sympathetic nerves intact, beta-adrenergic receptor blockade increased the recovery time constant (tau rec) for the conduction of premature test beats from 47 +/- 2 (mean +/- SEM) msec (control) to 62 +/- 1 msec (p less than 0.001), whereas isoproterenol decreased tau rec to 38 +/- 1 msec (p less than 0.001). In addition, beta-blockade increased the maximum amount of rate-dependent AV nodal fatigue from 7 +/- 1 msec (at a cycle length of 198 +/- 9 msec [control]) to 17 +/- 2 msec (p less than 0.001). In five dogs with decentralized stellate ganglia, tau rec was decreased from 71 +/- 3 msec (control) to 57 +/- 4 msec and 48 +/- 2 msec (p less than 0.001 for each) by left stellate ganglion stimulation at 5 and 10 Hz, respectively. Maximum fatigue was similarly reduced from 16 +/- 1 msec (control) to 12 +/- 2 msec (p = NS) and 8 +/- 1 msec (p less than 0.01), respectively. Stellate ganglion stimulation, isoproterenol, and beta-blockade did not alter AV nodal facilitation. A mathematical model incorporating quantitative indexes of AV nodal function accurately accounted for tachycardia-dependent increases in the atrial-His activation interval, which were enhanced by beta-adrenergic receptor blockade and reduced by isoproterenol. Furthermore, this model showed that beta-adrenergic effects were increased by increasing heart rate, with the majority of the rate-dependent action being due to changes in the time course of AV nodal recovery. We conclude that beta-adrenergic receptor stimulation alters functional properties that govern the AV nodal response to changes in heart rate. These changes in functional properties alter the ability of the AV node to conduct impulses during tachycardia and, as such, could play a major role in the ability of sympathetic stimulation to promote and beta-adrenergic receptor blockade to prevent the occurrence of AV nodal reentrant arrhythmias.
Redistribution of red blood cell flow in microcirculatory networks by hemodilution.
Authors: Pries A R , Fritzsche A , Ley K , Gaehtgens P
Published: June 2012

The effect of isovolemic hemodilution on red blood cell flow distribution was studied in complete self-contained microvessel networks of the rat mesentery. Hematocrit, diameter, and length of all vessel segments as well as the topological structure were determined in control networks (systemic hematocrit, 0.54) and after hemodilution (systemic hematocrit, 0.30). Hemodilution was performed by exchanging blood with hydroxyethyl starch (MW 450,000; 6%) or homologous plasma. With hemodilution, the decrease of microvessel hematocrit exceeded that of systemic hematocrit. The average discharge hematocrit in capillaries was 79% of systemic hematocrit in the control group and 73% with hemodilution (p less than 0.001). The heterogeneity of capillary hematocrit within the network, expressed by the coefficient of variation, increased from 0.4 to 0.7. By using the morphological and topological data of four networks, the distribution of hematocrits was also calculated using a hydrodynamic flow model. The modeling results were found to be in close agreement with the experimental data. This indicates that the observed changes can be deduced from established rheological phenomena, most of all phase separation at arteriolar bifurcations. The changes in hematocrit distribution after hemodilution are accompanied by a redistribution of red blood cell flow within the network: relative to total red blood cell flow, red blood cell flow in the distal capillaries of the network increases by about 40% at the expense of the proximal capillaries that are close to the feeding arteriole and that exhibit the highest red blood cell flow under control conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine release and high energy phosphates in intact dog hearts during norepinephrine infusion.
Authors: Gorman M W , Ning X H , He M X , Portman M A , Sparks H V
Published: June 2012

Cardiac adenosine release is thought to depend on the oxygen supply/demand ratio, and this effect may be mediated by changes in high energy phosphate concentrations. Previous studies supporting this hypothesis have been done primarily in isolated hearts. We tested this hypothesis in intact dog hearts. Anesthetized, open-chest dogs were placed in a 4.7-T magnet where 31P nuclear magnetic resonance spectra were acquired via a surface coil over the heart at 2-minute intervals (60 scans, 2-second interpulse delay). Coronary sinus flow was shunted through a flow probe and returned via a jugular vein. After a control period, intracoronary norepinephrine was infused (12 micrograms/min) for 16 minutes and plasma samples were taken every 5 minutes. The phosphocreatine/ATP peak area ratio was used as an index of high energy phosphate changes. During norepinephrine infusion, arterial pressure, heart rate, coronary sinus flow, oxygen consumption, and adenosine release all increased significantly. Adenosine release peaked at 5 minutes but remained elevated after 15 minutes. There was a transient fall in the phosphocreatine/ATP ratio (9.2 +/- 3.1%, p less than 0.05) during the first 7 minutes, but the ratio returned to control levels by 9 minutes. The oxygen supply/consumption ratio increased after 5 minutes of norepinephrine infusion and then returned to control levels. We conclude that during norepinephrine infusion in vivo, persistent adenosine release can occur with only small transient changes in high energy phosphate concentrations and with no decrease in the oxygen supply/demand ratio.
Immunolocalization and expression of functional and nonfunctional cell-to-cell channels from wild-type and mutant rat heart connexin43 cDNA.
Authors: Dunham B , Liu S , Taffet S , Trabka-Janik E , Delmar M , Petryshyn R , Zheng S , Perzova R , Vallano M L
Published: June 2012

The carboxyl terminal cytoplasmic domain of distinct gap junction proteins may play an important role in assembly of functional channels as well as differential responsiveness to pH, voltage, and intracellular second messengers. Oligonucleotide-directed site-specific mutagenesis in a paired Xenopus laevis oocyte expression system was used to examine the expression of mRNAs encoding wild-type and carboxyl terminal mutant connexin43 (Cx43) proteins. Oocytes were stripped, injected with mRNA or distilled water (dH2O), preincubated for 16-20 hours, and then paired for 5-10 hours; this process was followed by electrophysiological recording using the dual voltage-clamp technique. Initial experiments compared the relative junctional conductances (Gjs) in oocyte pairs expressing Cx43 (382 amino acid residues) and two truncated mutants lacking most or a portion of the cytoplasmic carboxyl terminal. The shortest mutant (M241) contained 240 amino acid residues and was devoid of all phosphorylatable serine residues in the cytoplasmic tail; its length approximated the length of liver connexin26. The longest mutant (M257) tested contained 256 amino acid residues, including two serine residues. Oocyte pairs expressing M241 yielded a Gj similar to that of oocytes injected with dH2O, whereas M257 yielded a Gj similar to that of oocytes injected with Cx43. Immunoprecipitation studies showed that Cx43, M257, and M241 proteins were readily detectable in oocytes injected with their respective mRNAs, indicating that the lack of Gj observed with the M241 mRNA was not due to reduced translation. Immunocytochemical studies revealed that wild-type and both truncated mutants were localized to the area of cell-to-cell contact between the paired oocytes, indicating that protein targeting to the membrane was not inhibited in oocytes injected with M241 mRNA. Oocyte pairs expressing mutants in which serine residues were replaced with nonphosphorylatable amino acids (serine codon No. 255 AGC was converted to GCC, alanine, designated as M255S----A, and serine codon No. 244 AGC was converted to GGC, glycine, designated as M244S----G) showed Gjs similar to M257, indicating that these serine residues and, by inference, their phosphorylation state are not critical for expression of functional channels. The importance of the length of the carboxyl terminus was assessed by comparing the Gjs in a series of mutants that were intermediate in length between M257 and M241. Gradual shortening of the carboxyl terminus produced a gradual reduction of Gj relative to M257. However, simple deletion of amino acid residues 241-257 from the wild-type Cx43 did not affect Gj relative to M257.(ABSTRACT TRUNCATED AT 400 WORDS)
Exclusion of cardiac myosin heavy chain and actin gene involvement in hypertrophic cardiomyopathy of several French families.
Authors: Schwartz K , Beckmann J , Dufour C , Faure L , Fougerousse F , Carrier L , Hengstenberg C , Cohen D , Vosberg H P , Sacrez A
Published: June 2012

Familial hypertrophic cardiomyopathy (FHC) is characterized by idiopathic myocardial hypertrophy, which often and predominantly involves the interventricular septum. The disease is transmitted as an autosomal dominant trait, and its major risk is sudden death. It was recently demonstrated that this disease is genetically heterogeneous and that in 13 of 18 unrelated families the morbid locus, termed FHC-1, maps to chromosome 14q11-12 in and/or very near the cardiac beta-myosin heavy chain gene. We have performed linkage analysis with five chromosomal markers detecting polymorphisms in either the cardiac beta-myosin heavy chain gene or the cardiac actin gene (located on chromosome 15q) on eight families from different regions of France. We show that 1) it is possible to analyze medium-sized families by using highly informative microsatellite markers located in these genes and 2) the disease is not linked to the two contractile protein genes in any of these families. Moreover, 10-20% of chromosome 14 and 20-40% of chromosome 15 in the vicinity of the respective markers were excluded as possible locations for the morbid locus. These results provide new insights into the identification of the genes responsible for FHC.
Autoradiographic characterization of beta-adrenergic receptor subtype in the canine conduction system.
Authors: Muntz K H
Published: June 2012

It has been hypothesized, based on physiological evidence, that there is a greater proportion of beta 2-adrenergic receptors on the myocytes of the conduction system when compared with the working myocardium. The purpose of these studies was to examine beta-adrenergic receptor subtype in the conduction system of the dog by using the technique of coverslip autoradiography. Scintillation studies of [125I]pindolol binding to ventricular sections demonstrated that binding was saturable (dissociation constant of 116 pM), had the correct order of potency for a beta-receptor, and was stereoselective. Both betaxolol (beta 1-selective) and ICI-118,551 (beta 2-selective) competition curves fit a two-site model in nonlinear curve-fitting analyses (78% beta 1-receptors). Autoradiographic studies determined that the myocytes of the sinoatrial node had approximately twice as many autoradiographic grains as the surrounding atrial myocytes. The myocytes of the atrioventricular bundle had a number of grains similar to the number in surrounding septal myocytes. Autoradiographic inhibition curves with betaxolol or ICI-118,551 demonstrated that both the sinoatrial node and the atrioventricular bundle had inhibition profiles similar to the surrounding myocytes (predominantly beta 1) but unlike the inhibition profiles of arterioles (predominantly beta 2). Calculations using the dissociation constants derived from the nonlinear curve-fitting analysis and the percent specific binding in the presence of 4 x 10(-7) M betaxolol or ICI-118,551 determined that the proportion of beta 1- to beta 2-receptors was the same (70-80% beta 1) when comparing the sinoatrial node and the surrounding atrial myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Alpha B-crystallin in cardiac tissue. Association with actin and desmin filaments.
Authors: Bennardini F , Wrzosek A , Chiesi M
Published: June 2012

alpha B-Crystallin is a 20-kd peptide highly homologous to the small heat-shock proteins. This protein forms soluble homomultimeric complexes (M(r), 300-700 kd) and is very abundant in cardiac muscle cells. In vitro experiments (affinity column chromatography and binding studies with isolated proteins) have shown that alpha B-crystallin interacts directly with actin and, in particular, with desmin filaments. The immunocytochemical localization of alpha B-crystallin within the cardiomyocytes showed that the protein is distributed exclusively in the central region of the I bands (Z lines), where desmin is localized. In vitro studies have further shown that the binding affinity of alpha B-crystallin to actin and desmin filaments increases considerably at slightly acidic pH (6.5) or after a heat treatment (45 degrees C). Moreover, alpha B-crystallin was found to prevent effectively the tendency of actin filaments to form aggregates (i.e., paracrystals) at acidic pH. These in vitro data suggest a protective role of alpha B-crystallin during stress conditions such as ischemia of the heart. Crystallin could prevent the aggregation of filaments, which might occur during the acidification of the cytosol and lead eventually to irreversible structural damage.
Isolated pulmonary resistance vessels from fetal lambs. Contractile behavior and responses to indomethacin and endothelin-1.
Authors: Wang Y , Coceani F
Published: June 2012

A method was developed for recording isometric tension from isolated small arteries (mean internal diameter, 169 microns) and veins (mean internal diameter, 273 microns) of the term fetal lung and was then applied to the study of the mechanisms controlling perinatal pulmonary hemodynamics. The specific purpose was to determine whether the activity of the prostaglandin synthetic system in vessels is conditioned by the oxygen tension and the mode of action of endothelin-1. Both preparations appeared structurally intact and, after normalizing their lumen diameter to either the transmural pressure in vivo (artery) or the contractile capacity of the vessel in vitro (vein), generated force to the activating solution (5 mM Ca2+ in K+ Krebs' solution) in excess of the expected performance under physiological conditions. Treatment with indomethacin (2.8 microM) had no effect on arteries preequilibrated at low PO2 (21 +/- 1 mm Hg); however, the same treatment contracted (approximately 45% of the response to activating solution) arteries at either an intermediate (40 +/- 0.8 mm Hg) or high (70 +/- 0.9 mm Hg) PO2. Endothelin-1 contracted both arteries and veins in a concentration-dependent manner, the threshold being lower with veins (1-10 versus 10-100 pM). Endothelin-1 constriction was also seen in arteries whose tone had been raised with a thromboxane A2 analogue, whereas in thromboxane-treated veins constriction was preceded by a modest relaxation over the range of 1-1,000 pM. The findings with indomethacin lead us to infer that small pulmonary arteries are endowed with a prostaglandin-relaxing mechanism that becomes functional on raising the PO2 from fetal to neonatal levels. Endothelin-1 is a constrictor regardless of the level of intrinsic tone, suggesting a possible role of the peptide in maintaining elevated pulmonary vascular tone in the fetus.
Intraluminal flow increases vascular tone and 45Ca2+ influx in the rabbit facial vein.
Authors: Henrion D , Laher I , Bevan J A
Published: June 2012

The buccal segment of the rabbit facial vein exhibits a high level of myogenic tone in vitro that develops only in stretched vessel segments between 33 degrees and 44 degrees C. The infusion of physiological salt solution into the lumen of 2-mm-long rabbit facial vein segments induced a flow rate-dependent increase in wall tone, both in the presence (37 degrees C) and absence (30 degrees C) of myogenic tone. In calcium-free physiological solution with EGTA, neither flow nor stretch-induced tone was observed. This flow-induced contraction was associated with an increase in 45Ca2+ unidirectional influx and net uptake. These measurements correlated positively with the level of the associated constrictor responses, both in the presence or absence of myogenic tone. The mean contractile responses to flow (10 and 40 microliters/min), stretch, and histamine (1 microM) were 13%, 28%, 24%, and 33% of the tissue maximal response, respectively. When 45Ca2+ influx was expressed in relation to the force development (45Ca2+ influx per milligram), the amount of calcium entry was dependent on the stimulus. Values for 45Ca2+ influx per milligram in response to flow (10 and 40 microliters/min) and to histamine (1 microM) were not significantly different. The value was significantly lower for the response to stretch. On the other hand, 45Ca2+ net uptake, when expressed per unit force, was similar in response to flow (10 and 40 microliters/min), histamine (1 microM), and stretch.(ABSTRACT TRUNCATED AT 250 WORDS)
Time course of left ventricular function after cardiac denervation in conscious dogs.
Authors: Paré R , Parent R , Maamarbachi O , Laurier J , Lavallée M
Published: June 2012

The time course of left ventricular (LV) function was compared in normal (N) and cardiac-denervated (CD) dogs over an 8-week period after instrumentation with solid-state LV pressure gauges and three pairs of ultrasonic crystals to measure LV long and short axes and wall thickness. Baseline LV systolic, end-systolic, and end-diastolic pressures did not differ in N and CD dogs. Heart rate was higher (p less than 0.01) and LV dP/dt was lower (p less than 0.05) in CD dogs. LV short-axis shortening, shortening fraction, velocity of circumferential fiber shortening, and ejection fraction were consistently lower (p less than 0.01) in CD dogs. With angiotensin II to increase LV afterload, relations of LV short-axis shortening, shortening fraction, velocity of circumferential fiber shortening, and ejection fraction to average LV systolic wall stress were shifted downward (p less than 0.01) in CD dogs at 2, 4, and 8 weeks. Relations of LV short-axis shortening to LV end-diastolic wall stress also differed (p less than 0.01) in N and CD dogs. Ganglionic blockade abolished differences in LV function between N and CD dogs during elevated LV systolic wall stress with angiotensin II. Thus, in conscious dogs, cardiac denervation results in a sustained reduction of LV function over a wide range of ventricular loading conditions.
Developmental changes in long-opening behavior of L-type Ca2+ channels in embryonic chick heart cells.
Authors: Tohse N , Mészáros J , Sperelakis N
Published: June 2012

In the early (3-day) stage of development, long-lasting openings of the L-type Ca2+ channels (mode 2) occur in embryonic chick heart cells. Since mode-2 behavior is infrequently observed in adult heart cells of other species, in the present study, developmental change in behavior of the Ca2+ channel was examined in young (3-day) and old (17-day) embryonic chick heart cells. In the whole-cell voltage clamp, the L-type Ca2+ current carried by Ca2+ ions was smaller in amplitude and had a faster inactivation in 17-day cells than in 3-day cells. The peak current density was 8.1 +/- 0.2 microA/cm2 (mean +/- SEM, n = 5) and 5.1 +/- 0.3 microA/cm2 (n = 5) in 3-day and 17-day cells, respectively. When the charge carrier was Ba2+, the L-type Ca2+ channel current density was also smaller in 17-day cells (22.7 +/- 1.8 microA/cm2) than in 3-day cells (28.3 +/- 2.1 microA/cm2). In single-channel recordings, the mode-2 behavior was infrequent in 17-day cells compared with 3-day cells. High-open probability sweeps (with an open probability of greater than 0.25), reflecting mode-2 behavior, accounted for 20.2% and 3.7% in 3-day and 17-day cells, respectively. The ensemble-averaged currents in 17-day cells was 37% of that current in 3-day cells. In addition, decay of the averaged current appeared to be faster in 17-day cells than in 3-day cells. All data from the single-channel analysis agreed with the data from the whole-cell voltage clamp.(ABSTRACT TRUNCATED AT 250 WORDS)
Physiological hyperinsulinemia inhibits myocardial protein degradation in vivo in the canine heart.
Authors: Young L H , Dahl D M , Rauner D , Barrett E J
Published: June 2012

Myocardial protein turnover in vivo was examined in anesthetized dogs following a 16- or 36-hour fast and again during a hyperinsulinemic (2 mU/kg per minute) euglycemic clamp with or without amino acid replacement or during saline infusion. We measured myocardial phenylalanine balance and rates of protein synthesis and degradation, using the extraction of intravenously infused L-[ring-2,6-3H]phenylalanine and the dilution of its specific activity across the heart at isotopic steady state. After both a 16-hour (n = 19) and 36-hour fast (n = 10), there was net myocardial release of phenylalanine indicated by the negative balances for phenylalanine of -52 +/- 9 (p less than 0.001) and -38 +/- 9 (p less than 0.005) nmol/min, respectively. Overall, the basal rate of myocardial protein degradation was lower in the 36-hour-fasted animals (81 +/- 13 versus 121 +/- 12 nmol/min, p less than 0.05). Myocardial phenylalanine balance and rates of protein synthesis and degradation did not change during insulin and glucose infusion in the 36-hour-fasted animals (n = 10). In these animals, there was a 30-40% decline in plasma amino acid concentrations, including branched chain (p less than 0.001) and essential amino acids (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
Superoxide dismutase enhances ischemia-induced reactive hyperemic flow and adenosine release in dogs. A role of 5'-nucleotidase activity.
Authors: Kitakaze M , Hori M , Takashima S , Iwai K , Sato H , Inoue M , Kitabatake A , Kamada T
Published: June 2012

To test the hypothesis that 5'-nucleotidase activity during ischemia is attenuated by oxygen-derived free radicals, we measured ischemia-induced reactive hyperemic flow, adenosine release, and 5'-nucleotidase activity in dogs (n = 62). A 1-minute occlusion of the coronary artery caused reactive hyperemic flow (307 +/- 5 versus 92 +/- 1 ml.100 g-1.min-1 at baseline) with increased release of adenosine (14.4 +/- 1.4 versus 0.4 +/- 0.1 nmol.100 g-1.min-1 at baseline). Superoxide dismutase augmented (p less than 0.001) both peak coronary blood flow (333 +/- 6 ml.100 g-1.min-1) and repayment (436 +/- 12 versus 320 +/- 7 ml/100 g in the untreated group). Adenosine release during reperfusion was augmented (22.7 +/- 1.9 nmol.100 g-1.min-1, p less than 0.001), and 8-phenyltheophylline completely abolished the enhanced reactive hyperemia. Enzymatic assay of 5'-nucleotidase activity revealed that the administration of superoxide dismutase increases ecto-5'-nucleotidase activity in ischemic myocardium. When an inhibitor of ecto-5'-nucleotidase, alpha, beta-methyleneadenosine 5'-diphosphate, was administered, the effects of superoxide dismutase were completely abolished. Thus, we conclude that 1) the augmentation of reactive hyperemic flow caused by superoxide dismutase is attributed to the enhanced release of adenosine and 2) the enhanced release of adenosine over the untreated controls is attributed to the protection of ecto-5'-nucleotidase activity during ischemia.
Cardiac sarcoplasmic reticulum chloride channels regulated by protein kinase A.
Authors: Kawano S , Nakamura F , Tanaka T , Hiraoka M
Published: June 2012

In heart cells, several plasma membrane ion channels are targets for phosphorylation. However, it is not known whether sarcoplasmic reticulum (SR) ion channels, which are also essential in the regulation of cardiac function, are regulated by second-messenger systems. Here, we show that a Cl- channel in the cardiac SR membrane is activated by the catalytic subunit of protein kinase A (PKA). Purified cardiac heavy SR vesicles were incorporated into planar lipid bilayers. This channel spontaneously inactivated within a few minutes after the channel was incorporated into the bilayer. Mg-ATP (2-5 mM), but not the nonhydrolyzable ATP analogue 5'-adenylylimidodiphosphate, added to the cis solution prevented this spontaneous channel inactivation. After the inactivation process occurred, the catalytic subunit of PKA (with 0.05 mM Mg-ATP) reactivated this channel. These effects of Mg-ATP and PKA on the Cl- channel were prevented by an inhibitor of PKA. Thus, these results suggest that this SR Cl- channel is a novel target of PKA-dependent phosphorylation in cardiac muscle regulation.