bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in prostaglandin metabolism in cultured aortic and pulmonary arterial endothelial cells exposed to acute and chronic hypoxia.
Authors: Farber H W , Barnett H F
Published: June 2012

In vivo, a marked difference in blood oxygen tension exists between the pulmonary artery and the aorta. Responses of vascular endothelial cells from these vessels to changes in ambient oxygen might be influenced by the oxygen tension to which they are continuously exposed in vivo or by their anatomic site. To explore this hypothesis, we initially studied the production of the cyclooxygenase metabolites prostacyclin and thromboxane in bovine aortic and main pulmonary arterial endothelial cells grown in 21% O2 and exposed to different degrees of acute hypoxia over a wide range of times. We found that short-term hypoxia (3% or 0% O2) rapidly and transiently activates the cyclooxygenase pathway in both cell types, with a more rapid response in bovine aortic endothelial cells. To determine whether culture in an oxygen tension similar to that to which main pulmonary arterial endothelial cells are exposed in vivo alters this response, we evaluated these cyclooxygenase metabolites in bovine aortic and main pulmonary arterial endothelial cells cultured long-term in 3% O2, both at baseline and after exposure to acute anoxia (0% O2). In both cell types, we found a decrease in prostacyclin and thromboxane synthesis at baseline and evidence of an increase in the Vmax of thromboxane synthetase following stimulation with exogenous arachidonic acid. In chronically hypoxic cells exposed to acute anoxia, there were marked differences in enzyme activity compared with that in endothelial cells maintained in 21% O2 with differences depending on the origin of the endothelial cells. In bovine aortic endothelial cells, production of neither cyclooxygenase metabolite increased; in bovine main pulmonary arterial endothelial cells, only thromboxane production increased, suggesting isolated activation of the cyclooxygenase-thromboxane synthetase pathway. These studies demonstrate that acute and chronic hypoxia have profound effects on endothelial cell cyclooxygenase metabolism and that these effects depend on the duration and degree of the hypoxic exposure and the vascular bed from which the endothelial cells are derived.
Cardiomyopathy of the aging human heart. Myocyte loss and reactive cellular hypertrophy.
Authors: Olivetti G , Melissari M , Capasso J M , Anversa P
Published: June 2012

To determine the effects of aging on the human myocardium, 67 hearts were obtained from individuals who died from causes other than cardiovascular disease. The age interval examined was 17-90 years. Regression analysis demonstrated that the aging process was characterized by a loss of 38 million and 14 million myocyte nuclei/yr in the left and right ventricular myocardium, respectively. This loss in muscle mass was accompanied by a progressive increase in myocyte cell volume per nucleus in both ventricles. Left ventricular myocytes enlarged by 110 microns3/yr, whereas right ventricular myocytes increased by 118 microns3/yr, resulting in a preservation of ventricular wall thickness. However, the cellular hypertrophic response was unable to maintain normal cardiac mass. Left and right ventricular weights decreased by 0.70 and 0.21 g/yr, respectively. In conclusion, loss of cells and enlargement of the remaining myocytes may represent the structural basis for the reduced compensatory capacity of the aged heart and together may contribute to the development of myocardial dysfunction and failure in the elderly.
Reduction of canine myocardial infarct size by a diffusible reactive oxygen metabolite scavenger. Efficacy of dimethylthiourea given at the onset of reperfusion.
Authors: Carrea F P , Lesnefsky E J , Repine J E , Shikes R H , Horwitz L D
Published: June 2012

A number of scavengers of reactive oxygen metabolites reduce myocardial injury when given before ischemia and reperfusion, but few, if any, have proven to be effective when given near the onset of reperfusion. This is particularly true when infarct size is measured after at least 48 hours of reperfusion, when the full extent of myocardial damage has become apparent. Dimethylthiourea (DMTU) is an extremely diffusible, potent scavenger of hydroxyl radical, hydrogen peroxide, and hypochlorous acid, with a long half-life of 43 hours. Sixteen chloralose-anesthetized dogs underwent 90 minutes of left anterior descending coronary artery (LAD) occlusion followed by 48 hours of reperfusion. Collateral flow was measured by radioactive microspheres. Infarct size and risk area were measured by a postmortem dual-perfusion technique using triphenyl tetrazolium chloride and Evan's blue dye. In eight dogs, therapy with DMTU (500 mg/kg i.v.) was given during the last 15 minutes of ischemia and the first 15 minutes of reperfusion. In eight control dogs, the same volume of 0.9% saline was given during the last 15 minutes of ischemia through the first 15 minutes of reperfusion. Infarct size as a percent of risk area was reduced in the DMTU-treated group compared with the saline-treated controls (DMTU = 42 +/- 4% versus saline = 59 +/- 4%, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of increasing degrees of ischemic injury on myocardial oxidative metabolism early after reperfusion in isolated rat hearts.
Authors: Görge G , Chatelain P , Schaper J , Lerch R
Published: June 2012

The present investigation studied the effect of increasing severities of ischemic injury on recovery of oxidative metabolism after reperfusion in isolated rat hearts perfused retrogradely with erythrocyte-containing medium. Hearts subjected to 60 minutes of low-flow ischemia (5% of control perfusion) exhibited delayed but sustained recovery of left ventricular pressure development during reperfusion and preservation of ultrastructure delineated with electron microscopy. Immediately after reperfusion, myocardial oxygen consumption returned to control values, well before left ventricular pressure development recovered. Early after reperfusion release of 14CO2 from [1-14C]palmitate was reduced (-53%, p less than 0.01). Conversely, release of 14CO2 from [U-14C]glucose was increased (+131%, p less than 0.05). After 60 minutes of reperfusion 14CO2 release had completely returned to normal for both labeled substrates. Pulse-labeling experiments indicated that during transient depression of [1-14C]palmitate oxidation more tracer was incorporated into myocardial lipid esters, primarily triglycerides. In contrast to hearts subjected to low-flow ischemia, hearts subjected to 60 minutes of no-flow ischemia exhibited poor recovery of contractile function during the reperfusion period. Electron microscopic examination of reperfused hearts showed advanced myocyte damage consistent with irreversible injury. Interestingly, myocardial oxygen consumption in this group also recovered to control values. The substrate pattern during the early reperfusion period was similar to that of hearts subjected to low-flow ischemia. After 120 minutes of no-flow ischemia, recovery of oxidative metabolism was virtually absent. The results indicate a pronounced dissociation between recovery of oxidative metabolism and of contractile function in reperfused myocardium. The oxidative metabolic rate was disproportionately high compared with contractile function, not only in reversibly "stunned" hearts, but also in severely damaged hearts exhibiting signs of irreversible injury.
Hirudin and nitrates inhibit the thrombin-induced release of endothelin from the intact porcine aorta.
Authors: Boulanger C M , Lüscher T F
Published: June 2012

In intact porcine aorta, endothelium-derived nitric oxide released on thrombin stimulation inhibits the concomitant production of endothelin. Experiments were designed to examine the effect of hirudin (which inactivates thrombin) and the nitrovasodilators nitroglycerin and 3-morpholinosydnonimine on the spontaneous and thrombin-stimulated release of endothelin in intact blood vessels. Endothelin was detected by radioimmunoassay in the incubating medium of intact porcine aortas with endothelium. The spontaneous release of endothelin was not affected by hirudin (0.1 micrograms/ml) but that induced by thrombin (4 units/ml) was prevented. Nitroglycerin (10(-5) M) and the active metabolite of molsidomine, 3-morpholinosydnonimine (10(-5) M), did not modify the basal production of endothelin from the intima of intact porcine aortas. However, the nitrates fully inhibited the release of the peptide induced by thrombin (4 units/ml). The inhibitory effects of both 3-morpholinosydnonimine and nitroglycerin on the thrombin-stimulated release of endothelin were abolished in the presence of an inhibitor of soluble guanylate cyclase, methylene blue (10(-5) M). Thus, the thrombin-stimulated release of endothelin is inhibited by inactivation of thrombin with hirudin or by agents that mimic the effect of endothelium-derived nitric oxide. In contrast, the spontaneous production of endothelin is not modulated by the drugs.
Myocardial beta-adrenergic adenylate cyclase complex in a canine model of chagasic cardiomyopathy.
Authors: Morris S A , Barr S , Weiss L , Tanowitz H , Wittner M , Bilezikian J P
Published: June 2012

Infection of beagles with an opossum-derived strain of Trypanosoma cruzi (Tc-O) results in features of early and chronic chagasic cardiomyopathy, that is, increases in PR interval, atrioventricular block, and frequent ventricular premature contractions, ventricular tachycardia, and decreased left ventricular ejection fraction. These signs are not observed in animals infected with a canine strain of T. cruzi (Tc-D). To understand the biochemical basis for these early cardiac effects, we examined the beta-adrenergic adenylate cyclase complex in myocardial membranes prepared from animals infected with either of the two strains. In animals infected with Tc-O (symptomatic), the maximum velocity (Vmax) decreased and concentration of agonist resulting in 50% of Vmax (Kact) increased for isoproterenol-dependent adenylate cyclase activity; in animals infected with Tc-D (asymptomatic), Vmax and Kact for isoproterenol were unchanged from control, uninfected animals. beta-Receptor density decreased by 20% in symptomatic animals with no change in affinity, whereas no differences were observed between uninfected and infected asymptomatic animals. A complex pattern of changes was apparent in the guanine nucleotide binding protein, Gs, in the setting of infection. Alterations in cholera toxin-dependent ADP-ribosylation patterns as well as immunochemical detection with anti-G alpha s antisera suggested a change in the biochemical nature of the Gs species and not necessarily a physical loss of this protein. Reconstitution of adenylate cyclase activity in cyc- membranes demonstrated a decrease in hormone-sensitive Gs activity in membranes prepared from symptomatic animals without a change in activity demonstrable in the presence of Gpp(NH)p. Collectively, the results suggest that the depression in beta-adrenergic adenylate cyclase activity associated with symptomatic infection of beagles with T. cruzi occurs primarily as a result of changes in the Gs protein complex, most likely resulting in an uncoupling of the beta-adrenergic receptor from the Gs protein.
Excess membrane cholesterol alters calcium movements, cytosolic calcium levels, and membrane fluidity in arterial smooth muscle cells.
Authors: Gleason M M , Medow M S , Tulenko T N
Published: June 2012

The relations between membrane cholesterol content, basal (unstimulated) transmembrane 45Ca2+ movements, cytosolic calcium levels, and membrane fluidity were investigated in cultured rabbit aortic smooth muscle cells (SMCs) and isolated SMC plasma membrane microsomes. SMCs were enriched with unesterified (free) cholesterol (FC) for 18-24 hours with medium containing human low density lipoprotein and FC-rich phospholipid (PL) liposomes. This procedure increased cholesterol mass without affecting PL mass, resulting in an increase in the FC/PL molar ratio compared with controls in cells (67% FC increase, p less than 0.001; 43% FC/PL ratio increase, p less than 0.01) and in SMC microsomes (52% FC increase, p less than 0.05; 43% FC/PL ratio increase, p less than 0.05). Cholesterol enrichment also increased unstimulated 45Ca2+ influx (p less than 0.001) and efflux (p less than 0.05). Cellular cholesterol content correlated in a linear fashion with these changes (influx: r = 0.722, p less than 0.01; efflux: r = 0.951, p less than 0.05). In addition, cytosolic calcium levels increased approximately 34% (p less than 0.01) with cholesterol enrichment. The cholesterol-induced increase in 45Ca2+ influx was reversible with time and demonstrated sensitivity to the channel blockers. Fluorescence anisotropy measured from 5 degrees C to 40 degrees C using the fluorophore diphenylhexatriene showed decreased membrane fluidity in microsomal membranes obtained from cholesterol-enriched SMCs compared with controls (p less than 0.02). These results suggest that the SMC plasma membrane is very sensitive to cholesterol enrichment with liposomes or human low density lipoprotein and that increases in membrane cholesterol content increase cytosolic calcium levels in SMCs, are associated with a decrease in membrane fluidity, and unmask a new, or otherwise silent, dihydropyridine-sensitive calcium channel that may be involved in altered arterial wall properties with serum hypercholesterolemia.
Defibrillation efficacy. Comparison of defibrillation threshold versus dose-response curve determination.
Authors: Jones D L , Irish W D , Klein G J
Published: June 2012

When an automatic defibrillator is implanted, it is essential to determine the efficacy of the defibrillating system accurately, while balancing the need to keep the number of fibrillation episodes to a minimum. Two methods have evolved to assess defibrillation efficacy: 1) the "defibrillation threshold," which requires few ventricular fibrillation episodes, and 2) the "dose-response curve," which requires many ventricular fibrillation episodes and relates percent success to energy. The purpose of this study was to compare these two methods directly. Twenty open-chest anesthetized pigs had triplicate defibrillation threshold determinations. To produce a dose-response curve, six shocks then were delivered at 0.5, 0.75, 1.0, 1.25, 1.5, and 2.0 times the mean defibrillation threshold, in a balanced randomized order, during separate episodes of ventricular fibrillation. The data were fitted by logistic regression, conversions of the logistic regression, and a saturable exponential and nonsaturable growth exponential. A comparison was made of the mean defibrillation threshold and the 50% point on the dose-response curve (ED50) for each model, for each animal. In addition, the reliability of each measure was assessed by comparing the coefficients of variation. There was no statistical difference between the group defibrillation threshold (6.6 +/- 0.5 J) and group ED50 values (ED50 range of the models, 5.7 +/- 1.9 to 7.0 +/- 0.9 J). However, the variability about the defibrillation threshold was less than that of the ED50 values for all mathematical models except the true logistic equation, which was virtually the same.(ABSTRACT TRUNCATED AT 250 WORDS)
Natriuretic peptides as cardiac hormones in normotensive and spontaneously hypertensive rats. The ventricle is a major site of synthesis and secretion of brain natriuretic peptide.
Authors: Ogawa Y , Nakao K , Mukoyama M , Hosoda K , Shirakami G , Arai H , Saito Y , Suga S , Jougasaki M , Imura H
Published: June 2012

To study synthesis, storage, and secretion of brain natriuretic peptide (BNP) in the heart, we have measured BNP mRNA and BNP concentrations in the hearts of Wistar-Kyoto rats and also have investigated its secretion from the isolated perfused heart. The atrium expressed the BNP gene at a high level, and a considerable amount of BNP mRNA also was present in the ventricle, which corresponded to approximately 40% of the atrial BNP mRNA concentration. When tissue weight was taken into account, the total content of BNP mRNA in the ventricle was approximately threefold larger than that in the atrium, although the atrial natriuretic peptide (ANP) mRNA content in the ventricle was only 7% of that in the atrium. By contrast, the BNP concentration in the ventricle was 4.07 +/- 0.97 pmol/g, which was less than 1% of that in the atrium (451 +/- 86 pmol/g). The basal secretory rate of BNP from the isolated perfused whole heart was 49.3 +/- 6.1 fmol/min, approximately 60% of which was maintained even after atrial removal, whereas the secretory rate of ANP was reduced to less than 5%. We also studied age-matched spontaneously hypertensive rats-stroke prone. The rank order of the BNP mRNA concentration in the hearts of these rats was left ventricle greater than right ventricle greater than right atrium = left atrium, and the total BNP mRNA content and BNP secretory rate in the ventricle were twice as large as in Wistar-Kyoto rats. These results demonstrate that BNP is a novel cardiac hormone in rats and is predominantly synthesized in and secreted from the ventricle. This is in striking contrast to ANP, which occurs mainly in the atrium. The results also suggest possible pathophysiological roles of BNP in certain cardiovascular disorders.
Myocardial glucose utilization. Failure of adenosine to alter it and inhibition by the adenosine analogue N6-(L-2-phenylisopropyl)adenosine.
Authors: Dale W E , Hale C C , Kim H D , Rovetto M J
Published: June 2012

The effects of adenosine and the nonmetabolizable adenosine analogue N6-(L-2-phenylisopropyl)adenosine (PIA) on glucose transport or metabolism were determined in purified myocardial sarcolemmal vesicles, isolated cardiocytes, and perfused hearts. Adenosine (100 microM) did not affect hexose transport in myocytes. Also, adenosine deaminase, added to metabolize adenosine to inosine, did not alter transport of hexose into myocytes regardless of whether or not insulin was present. In contrast, PIA effectively inhibited 3-O-methyl-D-glucose uptake in myocytes even during insulin stimulation. PIA inhibited D-glucose-specific transport in both rat and bovine cardiac sarcolemmal vesicles (Ki = 26 microM at [D-glucose] = 5 mM). However, insulin did not affect glucose transport in sarcolemmal vesicles, which implies that receptor-coupled processes probably are not intact in this preparation. Thus, inhibition of PIA may not be receptor mediated. Also, PIA inhibited binding of cytochalasin B to bovine cardiac sarcolemmal vesicles, which supports the idea that PIA inhibits glucose flux by binding to the glucose transporter. To determine if adenosine altered glucose metabolism rather than transport, we measured the rate of 3H2O production from metabolism of D-[2-3H]glucose in paced rat hearts ([D-glucose] = 5.5 mM, [pyruvate] = 0.2 mM) perfused with a range of PIA or adenosine concentrations with or without 0.01 microM insulin. Adenosine (0.01-100 microM) in the presence or absence of insulin increased coronary flow but did not change glycolytic rates. Similar results were obtained with PIA (no insulin) rather than adenosine in the perfusate. However, with glucose as the only exogenous substrate, 100 microM PIA inhibited glycolysis by approximately 50%.(ABSTRACT TRUNCATED AT 250 WORDS)
Optical recordings in the rabbit heart show that defibrillation strength shocks prolong the duration of depolarization and the refractory period.
Authors: Dillon S M
Published: June 2012

The present data were obtained using the technique of optical recording with the voltage-sensitive dye WW781. This technique, unlike electrical methods, was able to provide uninterrupted recordings free of artifacts during defibrillation shocks. Optical recordings were made from sites on the ventricular epicardium of perfused rabbit hearts during electrical pacing. Continuous recordings of the electrophysiological responses of an intact heart to defibrillation threshold-strength shocks were made. It was shown that these shocks were able to stimulate normal-appearing action potentials in nonrefractory myocardium. A new and unexpected finding was that defibrillation threshold-strength shocks were also able to evoke a sustained, depolarizing response from myocardium already undergoing an action potential. This prolonged the time that the myocardium remained in the depolarized state. Prolongation of the depolarized state was accompanied by an equal prolongation of the refractory period. There was no indication that this depolarizing shock response was due to damage of the myocardium by the shock, to heterogeneous electrical responses in the optical recording area, or to the methods used in this study. It is hypothesized that these shocks were able to elicit a new action potential in already depolarized myocardium by hyperpolarizing portions of the myocardium's cellular membranes and, in so doing, to reactivate the fast sodium current. This effect, if prevalent in a fibrillating ventricle, could play a role in the defribillation process by effectively resynchronizing electrical activity.
Left ventricular function depends on previous beat ejection but not previous beat pressure load.
Authors: Slinker B K , Shroff S G , Kirkpatrick R D , Campbell K B
Published: June 2012

Previous beat contraction history, in which the performance of the left ventricle on any one beat is influenced by the mechanical events of the previous beat, may be important in the beat-to-beat regulation of left ventricular performance in the intact cardiovascular system. Prior studies of this phenomenon have established that mechanical events of the previous beat influence the function of the current beat, but it is not known whether the important mechanical influence is exerted by previous beat ejection or previous beat pressure. In addition, the magnitude of the effect of previous beat contraction history on left ventricular performance is unknown. To make these determinations, we performed experiments in six isolated rabbit left ventricle preparations buffer perfused at 30 degrees C. Left ventricular pressure and volume were controlled precisely with a servo-controlled linear motor system. After steady-state ejecting conditions were established by clamping left ventricular ejection pressure at 60% of peak isovolumic pressure, single test beats, which were pressure clamped at 40%, 60%, 80%, and 100% of peak isovolumic pressure, were introduced and followed by an isovolumic reference beat. As the level of pressure clamp decreased from 100% to 40%, developed pressure on the isovolumic beat following the single test beats increased from 139 +/- 15 (mean +/- SD) to 151 +/- 13 mm Hg. Similarly, peak positive left ventricular dP/dt increased from 1,718 +/- 209 to 1,864 +/- 181 mm Hg.sec-1 (both p less than 0.01). Multiple regression analysis showed that this increase in left ventricular function was related to previous beat ejection but not to previous beat pressure load or relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)
Concentration-dependent effects of protein kinase C-activating and -nonactivating phorbol esters on myocardial contractility, coronary resistance, energy metabolism, prostacyclin synthesis, and ultrastructure in isolated rat hearts. Effects of amiloride.
Authors: Watson J E , Karmazyn M
Published: June 2012

An extensive investigation of the cardiac actions of phorbol esters and the potential role of the Na(+)-H+ exchanger in those actions was carried out using isolated rat hearts. Sixty minutes of perfusion with 10(-9) M phorbol 12-myristate 13-acetate (PMA) or 10(-8) M phorbol 12,13-dibutyrate (PDBu) produced marked cardiac dysfunction associated with depressed contractility, coronary constriction, and elevated resting tension, the latter being particularly evident with PMA. These effects were also associated with disturbances in tissue levels of energy metabolites manifested primarily by a reduction in ATP and an elevation in lactate. Furthermore, both phorbols produced a sustained stimulation of the release of 6-ketoprostaglandin F1 alpha (6-keto PGF1 alpha), the hydrolysis product of prostacyclin (prostaglandin I2). Amiloride, an inhibitor of the Na(+)-H+ exchanger, significantly attenuated the loss in contractility and elevation in coronary pressure as well as the stimulated release of 6-keto PGF1 alpha but was without effect on elevations in resting tension or on changes in energy metabolites. Increasing concentrations of PMA or PDBu 10-fold resulted in a much more rapid and severe (greater than 80% loss in contractile function after 30 minutes) effect that was nonetheless qualitatively identical to that seen with the lower concentrations of phorbol. However, the effects were not prevented by amiloride. Surprisingly, 4 alpha-phorbol 12,13-didecanoate (alpha-PDD, 10(-6) M), which does not activate protein kinase C, was found to be a potent inhibitor of cardiac function (greater than 80% loss in contractility and 50% increase in resting tension) after 30 minutes of perfusion, although these effects were not associated with changes in levels of energy metabolites or with elevations in coronary pressure. Similarly, none of the actions of this compound were attenuated by amiloride. In contrast to the sustained effects of other phorbols on 6-keto PGF1 alpha release, the effect of alpha-PDD was transient (less than 10 minutes). In all hearts studied, the marked depression in contractile function caused by all phorbol esters occurred in the absence of any ultrastructural changes. 4 alpha-Phorbol (10(-6) M), which does not activate protein kinase C, was without effect on any parameter studied. Our results demonstrate very complex effects of phorbol esters on numerous parameters of cardiac function, including an amiloride-sensitive component that occurs at low concentrations. The latter observation suggests the involvement of Na(+)-H+ exchange activation, possibly occurring as a consequence of protein kinase C stimulation, in mediation of the effects of phorbol esters at low concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)
Role of ATP-sensitive potassium channels in coronary microvascular autoregulatory responses.
Authors: Komaru T , Lamping K G , Eastham C L , Dellsperger K C
Published: June 2012

The purpose of the present study was to test the hypothesis that ATP-sensitive potassium channels mediate autoregulatory vasodilatation of coronary arterioles in vivo. Experiments were performed in 23 open-chest anesthetized dogs. Coronary arterial microvascular diameters were directly measured with fluorescence microangiography using an intravital microscope and stroboscopic epi-illumination synchronized to the cardiac cycle. A mild coronary stenosis (perfusion pressure = 60 mm Hg), a critical coronary stenosis (perfusion pressure = 40 mm Hg), and complete coronary artery occlusion were produced with an occluder around the left anterior descending coronary artery in the presence or absence of glibenclamide (10(-5) M, topically), which inhibits ATP-sensitive potassium channels, or of vehicle. During topical application of vehicle (0.01% dimethyl sulfoxide), there was dilatation of small (less than 100 microns diameter) arterioles during reductions in perfusion pressure (percent change in diameter: 6.7 +/- 1.5%, 11.7 +/- 3.5%, and 10.4 +/- 5.1% during mild stenosis, critical stenosis, and complete occlusion, respectively). In the presence of glibenclamide, arteriolar dilatations during coronary stenoses and occlusions were abolished. Glibenclamide did not affect responses of arterioles greater than 100 microns. Glibenclamide did not alter microvascular responses to nitroprusside. These data suggest that ATP-sensitive potassium channels play an important role in determining the coronary microvascular response to reductions in perfusion pressure.
Essential role of nucleotide diphosphates in nicorandil-mediated activation of cardiac ATP-sensitive K+ channel. A comparison with pinacidil and lemakalim.
Authors: Shen W K , Tung R T , Machulda M M , Kurachi Y
Published: June 2012

Vasorelaxant agents such as pinacidil, lemakalim, and nicorandil, known as K+ channel openers, can activate the ATP-sensitive K+ channel (KATP channel) in cardiac myocytes. The aim of this study was to elucidate the molecular mechanisms underlying the K+ channel opener-mediated cardiac KATP channel activation by using the inside-out patch-clamp technique in guinea pig ventricular myocytes. Effects of pinacidil, lemakalim, and nicorandil on the KATP channel were examined both before and after channel "run-down". Since nucleotide diphosphates (NDPs) could activate the channel after complete run-down, effects of the drugs on the NDP-induced channel openings were also examined. We made the following observations: 1) Pinacidil (10-100 microM) and lemakalim (300 microM) activated the KATP channel before run-down and after run-down when NDPs were present. 2) Nicorandil (30 microM-1 mM) activated the KATP channel only in the presence of NDPs regardless of the condition of the channel with respect to run-down. 3) None of these K+ channel openers activated the channel after run-down in the absence of NDPs. These observations suggest that NDP binding is essential for nicorandil-mediated activation of the KATP channel and indicate that the molecular mechanisms underlying nicorandil activation are distinct from those of pinacidil and lemakalim activation of the KATP channel. We discuss the possible interactions between the drugs and the KATP channel based on a functional channel model.
Determination of local myocardial electrical activation for activation sequence mapping. A statistical approach.
Authors: Anderson K P , Walker R , Ershler P R , Fuller M , Dustman T , Menlove R , Karwandee S V , Lux R L
Published: June 2012

Electrical activation sequence mapping requires accurate identification of local activation, but because extracellular recordings do not exclusively reflect local events, complex electrograms may be difficult to interpret. In such cases, the assignment of local activation is subject to error that could affect interpretation of the resulting activation maps. The purpose of this investigation was to develop an approach that would provide quantitative indexes of error in the determination of local activation. An electrode array with 64 closely spaced unipolar electrodes was used to record from the left ventricular surface during open heart surgery. Electrograms with multiple deflections were recorded from four patients with scarred myocardium; two other patients with normal myocardial function served as controls. Each of 784 deflections was scored on the basis of three features: evidence for propagation, the configuration of the bipolar signal, and the effect of changing from the chest to an average reference. Local activation was considered probable if evidence for all three features was present and improbable if none of the three features was present. Deflections that were ambiguous with respect to this standard were excluded. Of over 30 test variables analyzed, the three with the greatest power to discriminate signals due to local activation from those due to distant activity were 1) a linear combination of the extracellular potential plus the ratio of the second derivative and the extracellular potential, 2) the second derivative, and 3) the minimum (greatest negative) first derivative. For each of these variables, the threshold value providing the greatest performance was identified by the maximum quality of efficiency, an index of agreement. This statistical approach provides an objective basis for determining local activation and provides a quantitative assessment of error that could enhance interpretation of electrical activation sequence maps.
Characteristics of binding of endothelin-1 and endothelin-3 to rat hearts. Developmental changes in mechanical responses and receptor subtypes.
Authors: Ishikawa T , Li L M , Shinmi O , Kimura S , Yanagisawa M , Goto K , Masaki T
Published: June 2012

Endothelin-1 (ET-1) and endothelin-3 (ET-3) produced positive inotropic effects on electrically stimulated left atria and increased the frequency of spontaneously beating right atria of adult rats. The potency of the inotropic effect of ET-1 was greater than that of ET-3, but the potencies of the chronotropic effects of ET-1 and ET-3 were not significantly different. In the neonatal atria, ET-1 and ET-3 also induced positive inotropic and chronotropic responses. ET-1 and ET-3 showed weak or no cardiotonic effects on the adult ventricles, whereas they caused marked positive inotropy in the neonatal ventricles. The characteristics of binding sites for ET-1 and ET-3 were very similar between the atria and the ventricles of the rat neonate. Saturation and competition binding experiments have shown that neonatal cardiac membranes from both atria and ventricle have two distinct binding sites for endothelin, that is, a low-affinity and a high-affinity site. ET-1 was found to bind to the low-affinity sites with a significantly lower Kd than ET-3, whereas the estimated Kd values for ET-1 and ET-3 at the high affinity sites were similar. In contrast, the binding sites in adult atria were different from those of the ventricles: only a single binding site for both ET-1 and ET-3 was detected. Adult atrial membranes, on the other hand, had two distinct binding sites similar to those of neonatal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Experimental model of effects on normal tissue of injury current from ischemic region.
Authors: Tan R C , Osaka T , Joyner R W
Published: June 2012

An ischemic myocardial region contains cells with a depolarized resting membrane potential. This depolarization leads to an intercellular current flow between the ischemic region and the surrounding normal myocardial cells, which has been termed an "injury current." We have devised an experimental model system in which an isolated rabbit ventricular cell is electrically coupled to a model depolarized cell to evaluate the effects of this injury current on the electrical properties of a normal ventricular cell. We found that the action potential duration of the isolated cell could be reversibly altered by varying the coupling resistance such that the action potential duration was shortened by high values of coupling resistance but could be considerably prolonged by lower resistance coupling. We did not observe automaticity in the isolated cell as a consequence of coupling to the depolarized model. The changes in action potential duration were accompanied by alterations in the frequency at which the isolated cell could respond to repetitive stimuli. In addition, the depolarization of the isolated cell produced by the electrical coupling led to a significant increase in the cellular excitability. This last effect may be of particular importance in understanding the mechanisms for origination of arrhythmias in the border zone of myocardial ischemia.
Force-velocity relation and myosin light chain phosphorylation in bovine coronary arterial smooth muscle.
Authors: Miller-Hance W C , Kamm K E
Published: June 2012

We have investigated in bovine left ventricular coronary arteries the relation between the biochemical regulatory event of myosin light chain phosphorylation and the mechanical events of isometric stress and isotonic shortening, under conditions of stimulation by depolarization (65 mM KCl) or receptor occupancy (2 microM 5-hydroxytryptamine [5-HT]). At rest, levels of light chain phosphorylation were 0.07 +/- 0.01 mol phosphate/mol light chain. Maximal values were significantly different for KCl (0.42 +/- 0.02 mol phosphate/mol light chain at 1 minute) and 5-HT stimulation (0.58 +/- 0.01 mol phosphate/mol light chain at 30 seconds). Increases in light chain phosphorylation preceded isometric stress development, and values remained elevated at approximately 0.35 mol phosphate/mol light chain for up to 2 hours with both KCl and 5-HT. The sites of phosphorylation were identical for KCl and 5-HT at 2 hours. Maximal stresses for each stimulus were also maintained for 2 hours. Values of maximum velocity of shortening (Vo in muscle lengths [ML]/sec), obtained from the force-velocity relation, did not change significantly between 1 minute and 2 hours with KCl (0.070 +/- 0.008 ML/sec at 1 minute and 0.056 +/- 0.007 ML/sec at 2 hours, p greater than 0.2). However, during 5-HT stimulation, Vo declined significantly (0.053 +/- 0.006 ML/sec at 1 minute and 0.032 +/- 0.003 ML/sec at 2 hours, p less than 0.025). The relation between Vo and light chain phosphorylation was different for KCl and 5-HT, indicating that factors in addition to myosin light chain phosphorylation may modulate smooth muscle shortening velocity.
Effects of phorbol esters on pial arteriolar diameter and brain production of prostanoids in piglets.
Authors: Busija D W , Leffler C W
Published: June 2012

We determined the effects of phorbol 12,13-dibutyrate (PDB), which activates protein kinase C, on pial arteriolar diameter and cerebrospinal fluid (CSF) prostanoid levels in newborn pigs. A closed cranial window was implanted, and the diameter of one pial arteriole was determined by intravital microscopy. In addition, CSF was sampled from under the window, and prostanoid levels (prostaglandin [PG] E2, 6-keto-PGF1 alpha, PGF2 alpha, and thromboxane B2) were determined by radioimmunoassay. Diameter and CSF prostanoid levels were determined during application of artificial CSF containing no drugs and during application of 10(-8), 10(-7), and 10(-6) M PDB. We also determined effects of 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), a phorbol ester that does not activate protein kinase C, and dimethyl sulfoxide, the vehicle for the phorbol esters, on pial arteriolar diameter and CSF prostanoid levels. Initial diameters were 100-200 microns. At 10(-8)-10(-6) M, PDB progressively constricted pial arterioles and increased CSF levels of prostanoids; the other phorbol ester and dimethyl sulfoxide had no such effects. Baseline arteriolar diameter was 147 +/- 17 microns (mean +/- SEM), and diameter was 140 +/- 17 microns at 10(-8) M PDB, 120 +/- 18 microns at 10(-7) M PDB (p less than 0.05), and 108 +/- 14 microns at 10(-6) M PDB (p less than 0.05) (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)
Structural and functional origins of suppressed acetylcholine vasodilation in diabetic rat intestinal arterioles.
Authors: Lash J M , Bohlen H G
Published: June 2012

This study evaluated the possible impairments to endothelium-mediated vasodilation by structural and functional properties of the intestinal arterioles in adult (20-21-week-old) rats after 8-11 days or 7-8 weeks of streptozotocin-induced diabetes. Arteriolar intravascular pressures and luminal diameters were simultaneously measured during iontophoretic application of acetylcholine, bradykinin, and nitroprusside to the outer vessel wall, and passive diameter-pressure relations were obtained during maximal vasodilation. Microvascular pressures and circumference-passive wall tension relations were similar between all diabetic and normal rats and did not appear to significantly influence vasodilation. Both acute and chronic hyperglycemia were associated with near complete suppression of acetylcholine-induced vasodilation in large arterioles, and the threshold dose for vasodilation of intermediate arterioles was approximately 10-fold higher in diabetic rats. In both diabetic groups, dilatory responses to nitroprusside were normal, and in chronically diabetic rats, the relative vasodilation in response to various doses of bradykinin was equivalent to that found in normal rats. These observations indicate that a very specific deficit of acetylcholine-induced endothelium-derived relaxing factor action rapidly develops in intestinal arterioles of diabetic rats, but the arteriolar wall mechanical properties, cGMP-mediated muscle relaxation, and endothelial release of the bradykinin-stimulated relaxing factor are not compromised after 7-8 weeks of chronic hyperglycemia.
Signal requirements for the generation of CD4+ and CD8+ T-cell responses to human allogeneic microvascular endothelium.
Authors: Pardi R , Bender J R
Published: June 2012

Microvascular endothelium has been implicated as a major target in the rejection of vascularized allografts. In an attempt to dissect the stepwise generation of the T-cell-mediated immune response to microvascular endothelial cells (ECs), we analyzed the requirements for the two major T-cell subsets, CD4+ and CD8+, in the triggering of proliferative and cytotoxic responses to allogeneic ECs in vitro. Results demonstrate that resting ECs are unable to stimulate a functional response by purified T, CD4+, and CD8+ cells in the absence of costimulatory signals. T cells and CD8+ cells develop both proliferative and cytotoxic anti-EC responses by the addition of as little as 2 units/ml interleukin-2, 10 units/ml interleukin-1, or irradiated monocytes autologous to the responder lymphocytes, whereas only autologous monocytes are capable of triggering CD4+ T-cell precursors to proliferate and become anti-EC-specific effector cytotoxic T lymphocytes. CD8+ cell-mediated anti-EC cytotoxicity is directed toward allogeneic major histocompatibility complex (MHC) class I determinants on ECs and involves recognition by the CD3/T-cell receptor complex and the CD8 molecule on the effector T cells. CD4+ cells can be driven to proliferate, produce interleukin-2, and become anti-EC-specific cytotoxic T lymphocytes despite the lack of detectable membrane MHC class II determinants on the target cells. Chloroquine inhibition experiments demonstrate that autologous monocytes/macrophages are required by CD4+ T-cell precursors for the processing of EC-derived alloantigens and their subsequent presentation in the context of self-MHC molecules. These results are in agreement with the adoptive transfer experiments in experimental allograft models and suggest that the coordinate engagement of cells of the CD4, CD8, and monocyte/macrophage series is more effective than the individual cell subsets in the
Hysteresis in the excitability of isolated guinea pig ventricular myocytes.
Authors: Lorente P , Delgado C , Delmar M , Henzel D , Jalife J
Published: June 2012

Hysteresis phenomena were demonstrated in the excitability of single, enzymatically dissociated guinea pig ventricular myocytes. Membrane potentials were recorded with patch pipettes in the whole-cell current-clamp configuration. Repetitive stimulation with depolarizing current pulses of constant cycle length and duration but varying strength led to predictable excitation (1:1) and nonexcitation (1:0) patterns depending on current strength. However, transition between patterns depended on the direction of current strength change, and stable hysteresis loops were obtained in stimulus-response pattern versus current strength plots in 31 cells. Increase of pulse duration and decrease of stimulation rate contributed to a reduction in hysteresis loop areas. In addition, at the abrupt transitions from 1:0 to 1:1 patterns, a latency adaptation phenomenon was consistently observed. Bath application of tetrodotoxin (30 microM) produced no change of hysteresis, whereas hysteresis was substantially decreased in cobalt (2 mM) superfusion experiments. Analysis of the changes in amplitude and shape of the subthreshold responses during the transitions from one stable pattern to the other suggested that activity led to an increase in membrane resistance, particularly in the voltage domain between resting and threshold potentials. We therefore modeled the dynamic behavior of the single cells, using an analytical solution aimed at calculating the recovery of activation latency as a function of diastolic membrane resistance. Numerical iteration of the analytical model equations closely reproduced the experimental hysteresis loops in both qualitative and quantitative ways. The effect of stimulation frequency on the model was similar to the experimental findings. The overall study suggests that the excitability pattern of guinea pig ventricular myocytes is responsible for hysteresis and bistabilities when current intensity is allowed to fluctuate around threshold levels.
Proliferating cell nuclear antigen in developing and adult rat cardiac muscle cells.
Authors: Marino T A , Haldar S , Williamson E C , Beaverson K , Walter R A , Marino D R , Beatty C , Lipson K E
Published: June 2012

During early development, rat cardiac muscle cells actively proliferate. Shortly after birth, division of cardiac muscle cells ceases, whereas DNA synthesis continues for approximately 2 weeks at a progressively diminishing rate. Little DNA synthesis or cell division occurs in adult cardiocytes. Thus, developing cardiac muscle cells are an ideal system in which to examine the expression of cell cycle-regulated genes during development. We chose to examine proliferating cell nuclear antigen (PCNA), a gene expressed at the G1/S phase boundary of the cell cycle. Northern blots of RNA from cardiac muscle cells from 18-day-old rat fetuses and from day 0, 5, and 14 neonatal as well as adult rat hearts revealed that the PCNA mRNA was found in cardiac muscle cells from all ages. However, because it was possible that this was a result of fibroblast PCNA gene expression, we used reverse transcription followed by polymerase chain reaction to see if it was possible to detect the message for PCNA in cardiac muscle cells from all ages. Because of the great sensitivity of this technique, RNA was recovered from 25 isolated adult cardiac muscle cells. Polymerase chain reaction amplification products for PCNA produced from the RNA isolated from these cells conclusively demonstrated that mRNA for this gene, which normally is associated with proliferating cells, is expressed in adult cardiac muscle cells that no longer divide. Furthermore, Western blot analysis demonstrated that the PCNA protein was found only in embryonic and neonatal cells and not in adult rat cardiac muscle cells. Therefore, it might be inferred from these data that PCNA might be regulated at the posttranscriptional level in adult cardiac muscle cells.
Compartmentation of cAMP in adult canine ventricular myocytes. Relation to single-cell free Ca2+ transients.
Authors: Hohl C M , Li Q A
Published: June 2012

Isolated adult canine ventricular myocytes were used to study the role of compartmentation of cAMP in the diverse functional responses to various drugs that elevate cAMP. Myocytes presented with the beta-agonist isoproterenol accumulated cAMP with a half maximally effective concentration (EC50) of 3.55 x 10(-8) M. Approximately 45% of the total cAMP was recovered in the particulate fraction of digitonin-lysed myocytes under these conditions. With phosphodiesterase inhibition (10 microM isobutylmethylxanthine), isoproterenol-stimulated cAMP production was up to 3.4-fold greater, but the proportion of total cAMP residing in the particulate fraction declined to less than 20%. Similar results were obtained with forskolin, a direct activator of adenylate cyclase. Treatment with isoproterenol shortened the duration at 50% maximum peak height (T 1/2) and increased the peak fluorescence ratio of electrically triggered single-cell free Ca2+ transients in fura-2-loaded canine myocytes. Isoproterenol dose-response curves gave EC50 values of 1.7 x 10(-9) and 4.4 x 10(-9) M for effects on T 1/2 and peak height, respectively. Alterations in peak height and T 1/2 of Ca2+ transients also showed a dose dependency on isobutylmethylxanthine and forskolin. Comparison of myocyte cAMP content with the corresponding changes in free Ca2+ transients demonstrated a close correlation between particulate cAMP and the extent of shortening or increase in peak height of the fura-2 Ca2+ transients (r = 0.92 for each). However, when these two parameters were plotted as a function of total cAMP, the resulting curves were nonlinear and divergent for each agent tested. The results support the hypothesis that differences in responses to agents that augment cAMP can be explained in part by compartmentation of cAMP. Furthermore, Ca2+ mobilization seems to be most affected by cAMP located in the particulate compartment of canine cardiac myocytes.