Maintained contractile reserve in a transgenic mouse model of myocardial stunning

Cardiac excitation-contraction (E-C) coupling is impaired at the myofilament level in the reversible postischemic dysfunction known as "stunned" myocardium. We characterized tension development and calcium cycling in intact isolated trabeculae from transgenic (TG) mice expressing the major proteolytic degradation fragment of troponin I (TnI) found in stunned myocardium (TnI(1-193)) and determined the ATPase activity of myofibrils extracted from TG and non-TG mouse hearts. The phenotype of these mice at baseline recapitulates that of stunning. Here, we address the question of whether contractile reserve is preserved in these mice, as it is in genuine stunned myocardium. During twitch contractions, calcium cycling was normal, whereas tension was greatly reduced, compared with non-TG controls. A decrease in maximum Ca2+-activated tension and Ca2+ desensitization of the myofilaments accounted for this contractile dysfunction. The decrease in maximum tension was paralleled by an equivalent decrease in maximum Ca2+-activated myofibrillar ATPase activity. Exposure to high calcium or isoproterenol recruited a sizable contractile reserve in TG muscles, which was proportionately similar to that in control muscles but scaled downward in amplitude. These results suggest that calcium regulatory pathways and beta-adrenergic signal transduction remain intact in isolated trabeculae from stunned TG mice, further recapitulating key features of genuine stunned myocardium.     

Myocardial Ca-sequestration failure and compensatory increase in Ca-ATPase with congestive cardiomyopathy: kinetic characterization by a homogenate microassay using real-time ratiometric indo-1 spectrofluorometry

A novel, simple, rapid and reproducible microassay is used for kinetic analysis of Ca-sequestration by homogenates of myocardium of turkeys with furazolidone-induced congestive cardiomyopathy. The assay monitors Ca in real-time using dual-emission ratiometric spectrofluorometry and the Ca-indicator dye indo-1. Using this assay and isolated SR studies we make several novel findings regarding the mechanism of SR failure in furazolidone cardiomyopathy.Qualitative differences in Ca-sequestration were not detected between groups. However, compared to controls the furazolidone treatment resulted in: 1) 50% depression in maximal activities (1.54 ± 0.36 vs 0.73 ± 0.12 µM/sec); 2) 2-fold increases in post-sequestration concentrations of ionized Ca(79 ± 23 vs 141 ±13 nmol Ca/L homogenate); 3) 2-fold increases in Ca half-life (415 vs 790 msec); and 4) 25% increased passive Ca-binding capacity of homogenates. The Ca-ATPase specific activity of isolated sarcoplasmic reticulum was 60% increased in congestive cardiomyopathy (543 ± 140 vs 873 ± 108 nmol ATP hydrolyzed/min/mg membrane protein) although membrane yield was 20% decreased (0.79 ± 0.09 vs 0.63 ± 0.03 mg/g heart). The increased ATPase and decreased Ca-uptake activities in combination with the occurrence of 36% cardiac hypertrophy and 19% decreased body weights resulted in estimates of the relative energy cost to the animal for myocardial Ca transport being 5.5-fold increased with cardiomyopathy (20.5 vs 111 nmol ATP hydrolyzed per µM decrease of sarcoplasmic free Ca/kg body weight).These data indicate that congestive cardiomyopathy is associated with markedly increased permeability of sarcoplasmic reticulum to Ca and compensatorily increased Ca-ATPase activity. Accelerated energy consumption due to the increased energy cost of Ca transport and increased time of myocyte activation are predicted to predispose the myocardium to fatigue and irreversible failure.     

Loss of heart phospholipid arachidonic acid without phospholipid peroxidation in anaesthetized rats rewarmed after prolonged deep hypothermia

Hypothermia–rewarming of the heart results in contractile dysfunction under in vitro as well as in vivo conditions. Increase in reactive oxygen species (ROS), lipid peroxidation and calcium overload are proposed mechanisms. In the first protocol of this study, the effect of putative phospholipase and calcium channel modulator mepacrine during deep hypothermia (4 h 14 °C) plus rewarming was tested in an isolated perfused rat heart model previously reported not to involve increase in lipid peroxides. Contractile function was measured under isovolumetric conditions using an intra-ventricular balloon connected to a transducer and recording system. Mepacrine completely reversed hypothermia–rewarming induced contractile failure in this model (LV dP/dt_max: 3236 ± 517 vs. 1058 ± 185 mm Hg/s in untreated hearts). In the second part of the study, lipid peroxidation of the heart was examined in vivo in anesthetized rats subjected to 4 h of deep hypothermia followed by rewarming. In this model recovery of heart function judged by cardiac output is decreased whereas blood pressure and heart rate recover fully. Peroxy conjugated diene isomers of unsaturated fatty acids were measured in heart phospholipids. The composition of the non-esterified fatty acids and the phospholipid fatty acid pool was examined in order to reveal signs of membrane remodeling. The results demonstrated no significant changes in phospholipid peroxidation after rewarming (91.07 ± 5.23 vs. 88.63 ± 7.73 nmol/g dry wt. in control). There was significant relative reduction in the content of arachidonic acid in the phospholipid fraction (29.55 ± 1.65 vs. 24.76 ± 1.48%). There was marked decrease in non-esterified fatty acids in myocardial tissue (1992 ± 291 vs. 1069 ± 189 nmol/g dry wt.), but a significant relative increase in arachidonic acid (20:4) in this fraction (3.46 ± 0.42 vs. 4.99 ± 0.30%). In conclusion, rewarming from deep hypothermia is not associated with increased phospholipid peroxidation. There is, however, a significant remodeling of the phospholipid fraction of myocardial lipids in vivo probably as a result of receptor or calcium stimulated phospholipase activity. Calcium or calcium stimulated phospholipase activity could contribute to posthypothermic contractile dysfunction.     

Complex I syndrome in myocardial stunning and the effect of adenosine

Isolated rabbit hearts were exposed to ischemia (I; 15 min) and reperfusion (R; 5-30 min) in a model of stunned myocardium. I/R decreased left-ventricle O₂ consumption (46%) and malate-glutamate-supported mitochondrial state 3 respiration (32%). Activity of complex I was 28% lower after I/R. The pattern observed for the decline in complex I activity was also observed for the reduction in mitochondrial nitric oxide synthase (mtNOS) biochemical (28%) and functional (50%) activities, in accordance with the reported physical and functional interactions between complex I and mtNOS. Malate-glutamate-supported state 4 H₂O₂ production was increased by 78% after I/R. Rabbit heart Mn-SOD concentration in the mitochondrial matrix (7.4 ± 0.7 μM) was not modified by I/R. Mitochondrial phospholipid oxidation products were increased by 42%, whereas protein oxidation was only slightly increased. I/R produced a marked (70%) enhancement in tyrosine nitration of the mitochondrial proteins. Adenosine attenuated postischemic ventricular dysfunction and protected the heart from the declines in O₂ consumption and in complex I and mtNOS activities and from the enhancement of mitochondrial phospholipid oxidation. Rabbit myocardial stunning is associated with a condition of dysfunctional mitochondria named “complex I syndrome.” The beneficial effect of adenosine could be attributed to a better regulation of intracellular cardiomyocyte Ca²⁺ concentration.     

Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systems

Brief periods of myocardial ischemia prior to timely reperfusion result in prolonged, yet reversible, contractile dysfunction of the myocardium, or "myocardial stunning". It has been hypothesized that the delayed recovery of contractile function in stunned myocardium reflects damage to one or a few key sarcomeric proteins. However, damage to such proteins does not explain observed physiological alterations to myocardial oxygen consumption and ATP requirements observed following myocardial stunning, and therefore the impact of alterations to additional functional groups is unresolved. We utilized two-dimensional gel electrophoresis and mass spectrometry to identify changes to the protein profiles in whole cell, cytosolic- and myofilament-enriched subcellular fractions from isolated, perfused rabbit hearts following 15 min or 60 min low-flow (1 mL/min) ischemia. Comparative gel analysis revealed 53 protein spot differences (> 1.5-fold difference in visible abundance) in reperfused myocardium. The majority of changes were observed to proteins from four functional groups: (i) the sarcomere and cytoskeleton, notably myosin light chain-2 and troponin C; (ii) redox regulation, in particular several components of the NADH ubiquinone oxidoreductase complex; (iii) energy metabolism, encompassing creatine kinase; and (iv) the stress response. Protein differences appeared to be the result of isoelectric point shifts most probably resulting from chemical modifications, and molecular mass shifts resulting from proteolytic or physical fragmentation. This is consistent with our hypothesis that the time course for the onset of injury associated with myocardial stunning is too brief to be mediated by large changes to gene/protein expression, but rather that more subtle, rapid and potentially transient changes are occurring to the proteome. The physical manifestation of stunned myocardium is therefore the likely result of the summed functional impairment resulting from these multiple changes, rather than a result of damage to a single key protein.     

Physiological and biochemical adrenergic regulation of the stunned myocardium

A dual approach was employed to study -adrenergic receptor signal transduction in post ischemic (stunned) myocardium, examining physiological interventions in awake, chronically instrumented pigs and biochemical, cellular mechanisms in sarcolemmal preparations from the stunned hearts using the contralateral non-ischemic zone as a control. Ten min of coronary artery occlusion (CAO) and 30 min coronary artery reperfusion (CAR) resulted in depressed posterior wall-thickening (myocardial stunning). Isoproterenol increased transmural wall thickening more in stunned myocardium than in non-ischemic myocardium. In contrast, the responses of wall thickening to forskolin, actually decreased during stunning compared with control. NKH 477, a water soluble forskolin derivative, that does not activate cardiac nerves, increased wall thickening in non-ischemic tissue similarly to the effects on stunned myocardium. Increasing cardiac neural tone reflexly with inferior venal caval occlusion (IVCO) elicited similar results to forskolin, i.e., stunned myocardium responded with less of an increase in wall thickening as compared with non-ischemic myocardium. -adrenergic receptor density, as determined with ¹²⁵I-cyanopindolol binding, was significantly increased in stunned subendocardium and subepicardium compared with respective values in non-ischemic myocardium. There were no differences in the response of adenylyl cyclase to isoproterenol in stunned and non-ischemic myocardium. The enhanced responsiveness of the -adrenergic receptor to isoproterenol stimulation in stunned myocardium corresponded to the increase in -adrenergic receptor density. The combination of enhanced responses to isoproterenol, and decreased responses to forskolin and to IVCO and preserved responsiveness to NKH 477, suggest that stunned myocardium is characterized by transient sympathetic neural stunning. The enhanced sensitivity to -adrenergic receptor stimulation has important clinical implications, both in terms of therapy of stunned myocardium and detection of stunned and /or hibernating myocardium, i.e., low dose dobutamine echocardiography.     

Cardiodepressive effect elicited by the essential oil of Alpinia speciosa is related to L-type Ca current blockade

This study was undertaken to elucidate the effect of the essential oil from Alpinia speciosa (EOAs) on cardiac contractility and the underlying mechanisms. The essential oil was obtained from Alpinia speciosa leaves and flowers and the oil was analyzed by GC-MS method. Chemical analysis revealed the presence of at least 18 components. Terpinen-4-ol and 1,8-cineole corresponded to 38% and 18% of the crude oil, respectively. The experiments were conducted on spontaneously-beating right atria and on electrically stimulated left atria isolated from adult rats. The effect of EOAs on the isometric contractions and cardiac frequency in vitro was examined. EOAs decreased rat left atrial force of contraction with an EC₅₀ of 292.2 ± 75.7 μg/ml. Nifedipine, a well known L-type Ca²⁺ blocker, inhibited in a concentration-dependent manner left atrial force of contraction with an EC₅₀ of 12.1 ± 3.5 μg/ml. Sinus rhythm was diminished by EOAs with an EC₅₀ of 595.4 ± 56.2 μg/ml. Whole-cell L-type Ca²⁺ currents were recorded by using the patch-clamp technique. EOAs at 25 μg/ml decreased I_Ca,L by 32.6 ± 9.2% and at 250 μg/ml it decreased by 89.3 ± 7.4%. Thus, inhibition of L-type Ca²⁺ channels is involved in the cardiodepressive effect elicited by the essential oil of Alpinia speciosa in rat heart.     

Stoichiometry of electrocatalytic cycle of cytochrome P450 2B4

Stoichiometry of the electrocatalytical cycle of cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized cytochrome P450 2B4 were used as the operating electrode (at the potential E^0′ = −450 mV) and electrodes, modified with cytochrome c (E^0′ = −50 mV) or Prussian Blue (E^0′ = 0), as measuring electrodes (for H₂O₂) and Clark-type electrode (for O₂). Benzphetamine N-demethylation rate was 17 ± 3 nmol/nmol of enzyme/min, peroxide production was 4.8 ± 0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3 ± 0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by Р450 2В4 was 19.4 ± 0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8 ± 0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed.     

Is stunning prevented by ischemic preconditioning?

In a model of global ischemia in the isolated perfused rat heart, a 20-min ischemic period followed by 30 min of reperfusion induces a decrease in isovolumic developed pressure (LVDP) and +dP/dt_max to 61 ± 6% and 61 ± 7% of baseline, respectively. Left ventricular end-diastolic pressure (LVEDP) increases to 36 ± 4 mmHg at the end of the reperfusion period. No significant necrotic area as assessed by triphenyltetrazolium chloride (TTC) was detected at the end of the reperfusion period. By an immunohistochemical method using antiactin monoclonal antibodies 10.8 ± 1.9% of unstained cells were detected in the stunned hearts and 10.3 ± 1.2% in control hearts. Preceding the ischemic episode with a cycle of 5 min of ischemia followed by 10 min of reperfusion (ischemic preconditioning) protected contractile function. LVDP and +dP/dt_max now stabilized at 89 ± 5% and 94 ± 5% of baseline respectively. LVEDP was 20 ± 2 mmHg at the end of the reperfusion period. The protection of contractile dysfunction after 20 min of ischemia was achieved also by early reperfusion of low Ca²⁺-low pH perfusate. With this intervention LVDP stabilized at 87 ± 5% of baseline. LVEDP was 12 ± 2 mmHg at the end of the reperfusion period. A positive inotropic intervention induced by a modified postextrasystolic potentiation protocol at the end of the reperfusion period increases LVDP to levels higher than baseline in the stunned hearts. However, these values were less than those obtained in control hearts. Ischemic preconditioning significantly increased the maximal inotropic response. Therefore, ischemic preconditioning diminishes the contractile dysfunction of early stunning.     

Use of Spin Traps in Intact Animals Undergoing Myocardial Ischemia/Reperfusion: A New Approach to Assessing the Role of Oxygen Radicals in Myocardial “Stunning”

Numerous studies have indirectly, suggested that oxygen-derived free radicals play an important path-ogenetic role in the prolonged depression of contractile function observed in myocardium reperfused after reversible ischemia (myocardial “stunning”). In order to provide direct evidence for the oxy-radical hypothesis of stunning, we administered the spin trap, α-phenyl N-tert-butyl nitrone (PBN), to open-chest dogs undergoing a 15-min coronary artery occlusion followed by reperfusion. Plasma of local coronary venous blood was analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR signals characteristic of radical adducts of PBN appeared during ischemia and increased dramatically in the first few minutes after reperfusion. After this initial burst, the production of adducts abated but did not cease, persisting up to 3 h after reflow. The production of PBN adducts after reperfusion was inversely related to collateral flow during ischemia. PBN itself enhanced recovery of contractile function. indicating that the radicals trapped may play a pathogenetic role in myocardial stunning. Superoxide dismutase plus catalase attenuated PBN adduct production and, at the same time, improved recovery of contractile function. Antioxidant therapy given 1 min before reperfusion suppressed PBN adduct production and improved contractile recovery; however, the same therapy given 1 min after reperfusion did not suppress early radical production and did not attenuate contractile dysfunction. After i.v. administration, the elimination half-life of PBN was estimated to be approximately 4–5 h. The results demonstrate that 1) free radicals are produced in the stunned myocardium in intact animals; 2) inhibition of free radical production results in improved contractile recovery; and 3) the free radicals important in causing dysfunction are produced in the first few minutes of reperfusion. Taken together, these studies provide cogent evidence supporting the oxy-radical hypothesis of stunning in open-chest dogs. It is now critical to determine whether these results can be reproduced in conscious animal preparations.     

Determinants of contractile reserve in viable, chronically dysfunctional myocardium

There is considerable variability in the sensitivity of inotropic reserve to identify viability in chronically dysfunctional myocardium. This is partially related to the underlying pathophysiology, with more frequent contractile reserve in chronically stunned (with normal resting perfusion) than hibernating myocardium (with reduced flow). This study was undertaken to determine the physiological responses to transient and graded stimulation in chronically stunned and hibernating myocardium to define the relative roles of acute catecholamine desensitization and biphasic responses. Pigs were chronically instrumented with a fixed left anterior descending artery stenosis that resulted in chronically stunned myocardium after 2 mo. One month later, hibernating myocardium was confirmed by regional dysfunction (wall thickening, 3.2 +/- 0.3 vs. 5.5 +/- 5 mm in remote, P=0.01) with reduced resting flow (0.70 +/- 0.07 vs. 0.92 +/- 0.09 ml x min(-1) x g(-1) in remote, P=0.01) without infarction. Wall thickening in dysfunctional regions significantly increased during both graded and transient epinephrine stimulation in chronically stunned (from 3.6 +/- 0.3 to 5.6 +/- 0.5 and 4.9 +/- 0.5 mm, respectively) and hibernating myocardium (from 3.3 +/- 0.3 to 5.4 +/- 0.6 and 5.0 +/- 0.7 mm, respectively) and returned to baseline within 15 min. Although a biphasic response during graded stimulation was common, the subsequent decrement in function was small and similar in both groups (stunned, 0.7 +/- 0.2 mm; hibernating, 1.1 +/- 0.3 mm, P=0.25). We conclude that 1) the extent of contractile reserve during beta-adrenergic stimulation is similar in chronically stunned and hibernating myocardium, 2) there are no significant differences between the responses to transient compared with graded catecholamine stimulation, and 3) submaximal catecholamine stimulation does not induce additional stunning in either chronically stunned or hibernating myocardium.     

Neuregulin-1β regulates outgrowth of neurites and migration of neurofilament 200 neurons from dorsal root ganglial explants in vitro

Neuregulin-1β (NRG-1β) signaling has multiple functions in neurons. To assess NRG-1β on neurite outgrowth and neuronal migration in vitro, organotypic dorsal root ganglion (DRG) neuronal culture model was established. Neurite outgrowth and neuronal migration were evaluated using this culture model in the presence (5 nmol/L, 10 nmol/L, 20 nmol/L) or absence of NRG-1β. Neurofilament 200 (NF-200)-immunoreactive (IR) neurons were determined as the migrating neurons. The number of nerve fiber bundles extended from DRG explant increased significantly in the presence of NRG-1β (5 nmol/L, 23.0 ± 2.2, P < 0.05; 10 nmol/L, 27.0 ± 2.7, P < 0.001; 20 nmol/L, 30.8 ± 3.7, P < 0.001) as compared with that in the absence of NRG-1β (19.0 ± 2.2). The number of neurons migrating from DRG explants increased significantly in the presence of NRG-1β (5 nmol/L, 39.6 ± 5.0, P < 0.05; 10 nmol/L, 54.6 ± 6.7, P < 0.001; 20 nmol/L, 62.2 ± 5.7, P < 0.001) as compared with that in the absence of NRG-1β (31.6 ± 4.0). Moreover, the increase of the number of nerve fiber bundles and the number of migrating NF-200-IR neurons was dose-dependent for NRG-1β addition. The data in this study imply that NRG-1β promotes neurite outgrowth and neuronal migration from DRG explants in vitro.     

PKC independent inhibition of voltage gated calcium channels by volatile anesthetics in freshly isolated vascular myocytes from the aorta

In this study we used barium currents through voltage gated L-type calcium channels (recorded in freshly isolated cells with a conventional patch-clamp technique) to elucidate the cellular action mechanism for volatile anesthetics. It was found that halothane and isoflurane inhibited (dose-dependently and voltage independently) Ba²⁺ currents through voltage gated Ca²⁺ channels. Half maximal inhibitions occurred at 0.64 ± 0.07 mM and 0.86 ± 0.1 mM. The Hill slope value was 2 for both volatile anesthetics, suggesting the presence of more than one interaction site. Current inhibition by volatile anesthetics was prominent over the whole voltage range without changes in the peak of the current voltage relationship. Intracellular infusion of the GDPβS (100 μM) together with staurosporine (200 nM) did not prevent the inhibitory effect of volatile anesthetics. Unlike pharmacological Ca²⁺ channel blockers, volatile anesthetics blocked Ca²⁺ channel currents at resting membrane potentials. In other words, halothane and isoflurane induced an ‘initial block’. After the first 4–7 control pulses, the cells were left unstimulated and anesthetics were applied. The first depolarization after the pause evoked a Ca²⁺ channel current whose amplitude was reduced to 41 ± 3.4% and to 57 ± 4.2% of control values. In an analysis of the steady-state inactivation curve for voltage dependence, volatile anesthetics induced a negative shift of the 50% inactivation of the calcium channels. By contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unitary L-type Ca²⁺ channels blockade occurred under cell-attached configuration, suggesting a possible action of volatile anesthetics from within the intracellular space or from the part of the channel inside the lipid bilayer.     

Antihypertensive and endothelium-dependent vasodilator effects of aqueous extract of Cistus ladaniferus

Cistus ladaniferus L. (Cistaceae) is a medicinal plant originated from the Mediterranean region which exerts different pharmacological effects. In the present study, our goal was to examine whether the plant possessed antihypertensive properties. Aqueous extract of Cistus leaves (AEC, 500 mg/kg/day) reduced systemic blood pressure (SBP) in two animal models of hypertension, the l-NAME and renovascular two kidney-one clip (2K-1C) hypertensive rats. In the former, AEC prevented the increase in SBP when co-administered with l-NAME during four weeks (164 ± 3 mm Hg in l-NAME vs. 146 ± 1 mm Hg in l-NAME + AEC, p < 0.001). In the latter, AEC reversed the increase in SBP when administered during four weeks after installation of the hypertension (146 ± 5 mm Hg with AEC vs. 179 ± 6 mm Hg without, p < 0.05). AEC treatment also reversed the endothelial dysfunction observed in both animal models of hypertension. A direct effect on cardiac and vascular tissue was also tested by examining the contractile effects of AEC in rat isolated aortic rings and Langendorff perfused hearts. AEC (10 mg/L) had no effect on left ventricular developed pressure and heart rate in isolated perfused heart. However, AEC produced a strong relaxation of pre-contracted rat aortic rings (80 ± 2% relaxation, n = 25). When the rings were denuded from endothelium or were incubated with 1 mM Nω-nitro-l-arginine (l-NNA), the relaxant effect of AEC was lost. We conclude that C. ladaniferus possesses antihypertensive properties which are mainly due to an endothelium-dependent vasodilatory action.     

Lack of the effect of superoxide dismutase and catalase on Na+,K+-ATPase activity in stunned rabbit hearts

Reactive oxygen species (ROS) have been implicated in the mechanism of postischemic contractile dysfunction, known as myocardial stunning. In this study, we examined protective effects of antioxidant enzymes, superoxide dismutase (SOD) and catalase, against ischemia/reperfusion-induced cardiac dysfunction and inhibition of Na+,K+-ATPase activity. Isolated Langendorff-perfused rabbit hearts were subjected to 15 min of global normothermic ischemia followed by 10 min reperfusion. The hearts treated with SOD plus catalase did not show significant recovery of left ventricular (LV) end-diastolic pressure compared with untreated ischemic reperfused hearts. Treatment with antioxidants had no protective effects on developed LV pressure or its maximal positive and negative first derivatives (+/-LVdP/dt). Myocardial stunning was accompanied by significant loss in sarcolemmal Na+,K+-ATPase activity and thiol group content. Inhibition of enzyme activity and oxidation of SH groups were not prevented by antioxidant enzymes. These results suggest that administration of SOD and catalase in perfusate do not protect significantly against cardiac dysfunction in stunned rabbit myocardium.     

Inclusion of calcium during isolation of high-density lipoprotein from plasma maintains antioxidant function

We investigated how inclusion of calcium during isolation of high-density lipoprotein (HDL) affected its antioxidant function. Following isolation, HDL was dialyzed against 0.154 M NaCl without or with added calcium (1 mM). HDL’s paraoxonase 1 activity was unaffected by calcium treatment (87 ± 11% of normal vs. 89 ± 16% of normal, P = 0.826). In contrast, whereas HDL dialyzed with calcium inhibited oxidation of low-density lipoprotein (LDL) by 87 ± 10%, HDL dialyzed without calcium inhibited oxidation by only 58 ± 19% (P = 0.004). Thus, inclusion of calcium during isolation is important for maintaining HDL’s antioxidant function.     

Relaxant effects of Schisandra chinensis and its major lignans on agonists-induced contraction in guinea pig ileum

In this study, the herbal extracts of Schisandra chinensis were demonstrated to inhibit the contractions induced by acetylcholine (ACh) and serotonin (5-HT) in guinea pig ileum, and the 95% ethanol extract was more effective than the aqueous extract. Analysis with High Performance Liquid Chromatography (HPLC) indicated that schisandrin, schisandrol B, schisandrin A and schisandrin B were the major lignans of Schisandra chinensis, and the ethanol extract contained higher amount of these lignans than the aqueous extract. All four lignans inhibited the contractile responses to ACh, with EC₂₀ values ranging from 2.2 ± 0.4 μM (schisandrin A) to 13.2 ± 4.7 μM (schisandrin). The effectiveness of these compounds in relaxing the 5-HT-induced contraction was observed with a similar magnitude. Receptor binding assay indicated that Schisandra lignans did not show significant antagonistic effect on muscarinic M3 receptor. In Ca²⁺-free preparations primed with ACh or KCl, schisandrin A (50 μM) attenuated the contractile responses to cumulative addition of CaCl₂ by 37%. In addition, schisandrin A also concentration-dependently inhibited ACh-induced contractions in Ca²⁺-free buffer. This study demonstrates that Schisandra chinensis exhibited relaxant effects on agonist-induced contraction in guinea pig ileum, with schisandrin, schisandrol B, schisandrin A and schisandrin B being the major active ingredients. The antispasmodic action of schisandrin A involved inhibitions on both Ca²⁺ influx through L-type Ca²⁺ channels and intracellular Ca²⁺ mobilization, rather than specific antagonism of cholinergic muscarinic receptors.     

Regional variation in adipogenesis and IGF regulatory proteins in the fetal baboon

Intrauterine growth rate is associated with body distribution in adulthood suggesting differential response of fetal fat depots to nutritional modifications. We hypothesize that there is regional differences in fetal adipogenesis, in part, due to depot-specific regulation of the availability of insulin growth factors. In near-term baboon fetuses (n = 3-5), the subcutaneous abdominal vs. omental preadipocytes had (1) more extensive lipid accumulation as assessed by BODIPY (lipid staining) to DAPI (nuclei) absorbance ratios (mean ± SEM; 0.51 ± 0.21, 0.35 ± 0.09, p < 0.05), (2) lower (p < 0.05) secretion of IGF-binding protein 4 (9.6 ± 1.2 vs. 17.4 ± 2.8 ng/ml) and its protease pregnancy associated plasma protein A (24.6 ± 1.9 vs. 39.1 ± 6.3 μIU/ml), (3) lower protein expression of IGF2 “clearance” receptor in cell lysate (0.28 ± 0.03 vs. 0.53 ± 0.02 OD U/mm², p < 0.05); all variables were intermediate in femoral preadipocytes. The regional variation of the adipogenesis and the IGF regulatory pathway set the stage for differential responsiveness of fat depots to external signals.     

Effect of myocardial stunning on thiol status,myofibrillar ATPase and troponin I proteolysis

To investigate the mechanism underlying postischemic contractile dysfunction (myocardial stunning) we examined myocardial sulfhydryl group content, myofibrillar Ca²⁺-dependent Mg²⁺-ATPase activity and protein profile after global ischemia and reperfusion. The Langerdorff-perfused rabbit hearts were subjected to 15 min normothermic ischemia followed by 10 min reperfusion and myofibrils were isolated from homogenates of left ventricular tissues. Depressed contractile function during reperfusion was accompanied by a decrease in total sulfhydryl group content. However, myofibrillar protein profile was unchanged and Western immunoblotting analysis showed no significant differences in troponin I immunoreactive bands between control and stunned hearts. Likewise, myofibrillar Mg²⁺-ATPase activity was unaltered after ischemia and reperfusion. We conclude that myocardial stunning is not caused by altered myofibrillar function and protein degradation but may be partly due to the oxidative modification of as yet undefined proteins.     

Octopaminergic modulation of the single Ca2+ channel currents in Kenyon cells isolated from the mushroom body of the cricket brain

Octopamine plays an important role in mediating reward signals in olfactory learning and memory formation in insect. However, its target molecules and signaling pathways are still unknown. In this study, we investigated the effects of octopamine on the voltage-activated Ca²⁺ channels expressed in native Kenyon cells isolated from the mushroom body of the cricket (Gryllus bimaculatus) brain. The cell-attached patch clamp recordings with 100 mM Ba²⁺ outside showed the presence of dihydropyridine (DHP) sensitive L-type Ca²⁺ channels with a single channel conductance of approximately 21 ± 2 pS (n = 12). The open probability (NPo) of single Ca²⁺ channel currents decreased by about 29 ± 7% (n = 6) by bath application of 10 μM octopamine. Octopamine-induced decrease in Po was imitated by bath application of 8-Br-cAMP, a membrane-permeable cAMP analog. Pre-treatment of Kenyon cells with the octopamine receptor antagonist phentolamine blocked the inhibitory effect of octopamine on Ca²⁺ channels. Pre-treatment of Kenyon cells with H-89, a selective inhibitor of cAMP-dependent protein kinase (PKA) attenuated the inhibitory effect of bath applied octopamine on Ca²⁺ channels. These results indicate that DHP-sensitive L-type Ca²⁺ channel is a target protein for octopamine and its modulation is mediated via cAMP and PKA-dependent signaling pathways in freshly isolated Kenyon cell in the cricket G. bimaculatus.