Bioaccumulation and molecular effects of sediment-bound metals in zebrafish embryos.

Predicting the bioavailability and effects of metals in sediments is of major concern in context with sediment risk assessment. This study aimed to investigate the bioavailability and molecular effects of metals spiked into riverine sediments to zebrafish (Danio rerio) embryos. Embryos were exposed to a natural and an artificial sediment spiked with cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) individually or as a mixture at concentrations ranging from 150 to 3000 mg/kg dry weight (dw) over 48 h, and uptake of metals was determined. Furthermore, transcript abundances of the metallothioneins MT1 and MT2, the metal-responsive element-binding transcription factor (MTF) and the genes sod1, hsp70 and hsp90α1 were measured as indicators of metal-induced or general cellular stress. D. rerio embryos accumulated metals from sediments at concentrations up to 100 times greater than those spiked to the sediment with the greatest bioaccumulation factor (BAF) for Cu from artificial sediment (275.4 ± 41.9 (SD)). Embryos accumulated greater concentrations of all metals from artificial than from natural sediment, and accumulation was greater when embryos were exposed to individual metals than when they were exposed to the mixture. Exposure of embryos to Zn or the mixture exhibited up to 30-fold greater transcript abundances of MT1, MT2 and hsp70 compared to controls which is related to significant uptake of Zn from the sediment. Further changes in transcript abundances could not be related to a significant uptake of metals from sediments. These studies reveal that metals from spiked sediments are bioavailable to D. rerio embryos directly exposed to sediments and that the induction of specific genes can be used as biomarkers for the exposure of early life stages of zebrafish to metal-contaminated sediments.

Exposure study on chemosensory effects of epsilon-caprolactam in the low concentration range.

OBJECTIVE: Aim of the study was to examine possible chemosensory effects of epsilon-caprolactam in the low concentration range relevant to indoor environmental conditions. METHODS: Twenty healthy subjects (10 male, 10 female) aged from 21 to 38 years were exposed for 6 h, respectively, to 0, 0.15, 0.5 and 5 mg/m3 epsilon-caprolactam vapours in a randomized and double-blind method. As a measure of trigeminal stimulation of the eye, blink frequency was video-recorded four times per day and evaluated by using a new semi-automatic, computer-assisted method compared to baseline recording and manual counting. Digital slit lamp photographs were taken at the same time to examine conjunctival hyperaemia. A standardized ophthalmologic grading scale was used to measure redness of the eyes objectively. Active anterior rhinomanometry compared nasal resistance before and after exposure. Subjective ratings of discomfort and mental orientation were assessed using the German version of the Swedish Performance Evaluation system (SPES). As a measure of personality traits, positive and negative affectivity was determined (PANAS). RESULTS: Six hour exposures to epsilon-caprolactam revealed no significant dose-response relationship concerning blink frequency, nasal resistance and redness of the bulbar conjunctiva. Subjective ratings of discomfort (sum scores) significantly increased only at the highest concentration of 5 mg/m3. However, the increase in discomfort was only moderate, ranging between "not at all" and "somewhat". Significant increases of the subjective detection of malodour (subscore) already occurred at 0.15 mg/m3, showing no adaptation over time. Irritation of the eyes or upper airways was not reported. CONCLUSIONS: Exposure to epsilon-caprolactam vapour did not elicit any acute health effects in a concentration range up to 0.5 mg/m3. Even at the highest concentration of 5 mg/m3, we could only find a slight increase in subjective symptoms, mainly due to an unincisive increase of perception of malodour.

Subchronic alpha- and beta-adrenergic regulation of cardiac gap junction protein expression.

Gap junction channels are essential for intercellular electrical communication in the heart. The most important cardiac gap junction proteins are connexin43 (predominantly) (Cx43), connexin40 (Cx40), and in early developmental stages connexin45. Since catecholamines play an important role in cardiac (patho)physiology, we wanted to elucidate whether catecholamines may affect expression of Cx43 and Cx40. Cultured neonatal rat cardiomyocytes were exposed for 24 h to increasing concentrations of noradrenaline (1-10000 nM) (physiological agonist at alpha and beta-adrenoceptors), resulting in significantly increased Cx43-expression, while Cx40 was unaffected. In further experiments cells were incubated with either phenylephrine (alpha-adrenergic agonist) or isoproterenol (beta-adrenergic agonist) (0.1-1000 nM) for 24 h. Both catecholamines lead to a concentration-dependent increase in Cx43 protein and mRNA expression (EC50: 10-20 nM). Inhibition experiments showed that the phenylephrine effect was transduced via PKC, while the isoproterenol effect was mediated by PKA. Dual whole-cell voltage clamp demonstrated that increased Cx43-expression was accompanied by significant increases in gap junction current. In additional in vivo experiments, adult rats were subjected to 24-h infusion of isoproterenol or phenylephrine showing again significant increase in Cx43 but not Cx40. Adrenergic stimulation of cardiomyocytes can enhance Cx43 expression thereby increasing cellular coupling, indicating a possible role for catecholamines in the regulation of cardiac gap junction expression in cardiac disease.

Norepinephrine-induced interleukin-6 increase in rat hearts: differential signal transduction in myocytes and non-myocytes.

Continuous i.v. infusion of norepinephrine in rats has been shown to induce early interleukin (IL)-6 mRNA expression in the left ventricle (LV) which was followed by hypertrophy and fibrosis. In this study, two approaches were used. In the first, NE (0.1 mg/kg per hour) was infused i.v. in rats for several time periods, and freshly obtained ventricular myocardium was dissociated into myocyte (MC) and non-myocyte (NMC) fractions. Second, isolated adult MCs and fibroblasts were treated with NE (10 microM). NE infusion (4 h, in vivo) caused an 11-fold increase in IL-6 mRNA in both cell populations. In vitro treatment of isolated adult MCs for 2 h and of fibroblasts for 1 h with NE induced a 3.5- and 23-fold maximum increase, respectively, in IL-6 mRNA. After in vivo NE treatment, the expression of the mRNA of the transcriptional factor of IL-6, C/EBP-beta, was elevated earlier (after 45 min of NE infusion) than IL-6 mRNA (after 4 h) and was seen in MCs and NMCs. The mRNAs of both receptors of IL-6, the soluble IL6R and gp130, were increased subsequently to IL-6 mRNA. Gp130 was elevated after 24 h and, like IL6R, predominantly in NMCs. In contrast, the IL6R protein and the downstream regulator STAT3 were increased only in MCs after 24 h of NE infusion. The mRNA of C/EBP-delta, which is regulated by STAT3, was elevated only in myocytes.

Serum depletion induces cell loss of rat cardiac fibroblasts and increased expression of extracellular matrix proteins in surviving cells.

OBJECTIVE: Since reduced nutrient supply is one component of ischemia, we have studied the effect of serum depletion on the survival of fibroblasts isolated from adult rat hearts and on the expression and degradation of extracellular matrix (ECM) proteins. Furthermore, we measured the role of the cAMP-dependent pathway in these processes. METHODS: Isolated cardiac fibroblasts were grown to confluency in 10% serum containing medium. Serum was then removed and cell number was measured by use of a Coulter Counter. The activity of the cAMP response element binding protein (CREB) was investigated by Western blotting and subsequent use of the specific antibody which binds to the active form of the protein. The expression of colligin, collagen I and III, matrix metalloproteinases 2 (MMP-2), and tissue inhibitor of matrix metalloproteinase 2 (TIMP-2) was examined by ribonuclease protection assay (RPA) and Western blotting. Zymographic measurements were done to investigate gelatinase activity of MMP-2. RESULTS: Serum withdrawal caused the death of 36% of the cells during the first 8 h. CREB was strongly phosphorylated 5 min after serum removal. Activation persisted up to 8 h and decreased thereafter. The mRNA abundance of colligin, collagen I and III, MMP-2, and TIMP-2 started to increase after 5 and 10 h, respectively, reaching a maximum after 20-30 h and decreasing thereafter. Protein levels of collagen I, collagen III, colligin and TIMP-2 were higher after 24 h until up to 96 h. MMP-2 zymographic activity was elevated 15-fold after 72 h. Application of the protein kinase A (PKA) blocker RpcAMPS suppressed the increase in phosphorylation of CREB. The increase in collagen III and MMP-2 mRNA abundance and elevation of collagen I and III, and TIMP-2 protein was diminished by RpcAMPS. The rise of colligin protein was completely suppressed. The increase in MMP-2 zymographic activity was also attenuated. RpcAMPS improved survival rate from 56 to 84%. CONCLUSIONS: Serum depletion led to cell death of isolated cardiac fibroblasts. Survival was associated with the increase in the expression of various ECM proteins. The transcription factor CREB was activated after serum removal. Inhibition of PKA improved the serum depletion induced decrease in the survival rate. The increase in collagen I, collagen III, MMP-2, TIMP-2, and colligin evoked by serum depletion was also diminished by PKA inhibition.

Cardiac remodeling after long term norepinephrine treatment in rats.

OBJECTIVE: In this study we have tested the hypothesis that degradation of collagen by matrix metalloproteinase 2 (MMP-2) precedes the deposition of extracellular matrix (ECM) after long term norepinephrine (NE) treatment. METHODS: Female Sprague-Dawley rats received continuous i.v. infusion of NE (0.1 mg/kg.h) for 1, 2, 3, 4 and 14 days. Heart function and weight as well as expression of cardiac colligin and of collagen I and III were examined. Furthermore, we have assessed the degradation pathway of collagen by measuring the mRNA and activity of myocardial MMP-2 and tissue inhibitor of metalloproteinase 2 (TIMP-2) as well as the protein level of TIMP-2. RESULTS: NE induced hypertrophy predominantly of the left ventricle (LV) in a time-dependent manner. It increased the mRNAs of colligin, collagen I and III, and of MMP-2 and TIMP-2 as well as MMP-2 activity in two phases: In the initial phase, at 3 and 4 days, the mRNA of colligin and of collagen I and III was elevated predominantly in the LV, MMP-2 and TIMP-2 mRNA, as well as TIMP-2 protein and MMP-activity were increased in both ventricles. The second phase, after 14 days, was characterized by a less pronounced increase in colligin, collagen I and III and in MMP-2 activity which occurred exclusively in the LV. Finally, long-term treatment with NE induced a 37% increase in interstitial fibrosis which was shown to occur exclusively in the LV after 14 days. CONCLUSION: NE treatment induced fibrosis exclusively in the LV which was associated with hypertrophy predominantly of the LV. The elevated MMP-2 activity seems to be necessary for the ECM to adapt to the enlargement of myocytes and to reduce overproduction of collagen.

Perfusion of isolated organs and the first heart-lung machine.

In 1885, Max von Frey (1852-1932), while working in Carl Ludwig's Physiological Institute in Leipzig, Germany, designed an apparatus that had criteria characteristic of a heart-lung machine. With this device, he perfused the entire lower extremity of dogs, and took measurements of oxygen consumption, and carbon dioxide and lactate production. In 1935, another type of perfusion apparatus was constructed by Charles A Lindbergh (1902-1973). This device was the result of cooperation with Alexis Carrel (1873-1944) who was a pioneer of experimental organ transplantation. Using Lindbergh's pulsating device, organs such as thyroid, ovary, suprarenal gland, spleen, heart and kidney from fowls and cats were perfused with an oxygenated medium, and were maintained under sterile conditions. Beginning in 1934, John H Gibbon (1903-1973) developed and tested a heart-lung machine to institute cardiopulmonary bypass in cats during experimental occlusion of the pulmonary artery. In 1953, he performed the first successful open-heart operation in a patient using a heart-lung machine. This included elements that were similar to those used by von Frey - ie, the oxygenator and the pumps for continuous circulation of blood. A comparison of the three experimental devices revealed the following: the application for experimental purposes preceded clinical use; the development shifted from Europe to the United States, and was achieved by people who were not specialists; and the intention to build such a device was first purely scientific interest, but later shifted to the care for and treatment of patients with heart and circulatory defects by open-heart surgery.

Transient pleural effusion in norepinephrine-stimulated rats.

Transient pleural effusions occurred in rats receiving continuous intravenous infusion of norepinephrine (NE, 0.1 mg/kg/h). We hypothesized that these pleural effusions result from a NE-induced increase in right ventricular systolic pressure (RVSP) and total peripheral resistance (TPR). NE was administered over time intervals between 20 min and 72 h. It induced an immediate doubling in RVSP whereas LVSP remained at the control level. TPR increased with a delay of 6 h. At this time, pleural effusions occurred in NE-treated animals, reached their maximum after 8h and disappeared after 24 h of NE stimulation. Combining NE with the alpha-blocker prazosin normalized TPR and prevented pleural effusions. Therefore, we interpret the pleural effusion as a consequence of pulmonary venous congestion, mainly caused by an increased TPR. LV hypertrophy which developed after 24 h of NE stimulation is considered to compensate for the hemodynamic disturbance due to the NE-induced elevation in TPR. This is reflected in the disappearance of pleural effusion.

Changes in extracellular matrix and in transforming growth factor beta isoforms after coronary artery ligation in rats.

Extensive myocardial remodeling occurs after transmural myocardial infarction (MI). The infarcted myocardium is being replaced by scar tissue after gradual resorption of the necrotic tissue. The remodeling process involves both synthesis and degradation of collagens as major components of the extracellular matrix (ECM). In the present study we have analyzed the time-dependent changes of the processes related to this fibrosis in the infarct area and in the non-infarcted left ventricle (LV) six hours to 82 days after occlusion of the left anterior descending coronary artery (LAD) in rats. We also examined whether changes occurred in the expression pattern of the transforming growth factor (TGF) beta isoforms, since this cytokine is known as powerful inductor of fibrosis. Elevation in colligin expression preceded the pronounced increase in mRNA expression of both type I and type III collagen after MI from day three onwards. The maximal increase in colligin protein in the infarct area coincided with the most pronounced expression of collagen I and collagen III mRNA expression. Also, the expression and activity of matrix metalloproteinases (MMPs) and of tissue inhibitor of matrix metalloproteinase (TIMP)-2 mRNA were increased predominantly in the infarct area. TGF beta(1)and TGF-beta(2)expression increased within the first days after MI, whereas TGF-beta(3)expression was elevated predominantly in the infarct area. This pronounced increase in TGF-beta(3)persisted up to 82 days and correlated positively with the parameters of ECM metabolism. Thus, the scar formation is an ongoing dynamic process in which TGF-beta(3)seems to play an active role in the complex ventricular remodeling.

Catecholamines stimulate interleukin-6 synthesis in rat cardiac fibroblasts.

Proinflammatory cytokines have been implicated in the pathophysiology of different heart diseases. Recent evidence suggests that interleukin-6 (IL--6) may play a role in mechanisms leading to cardiac hypertrophy. In addition, catecholamines are known to induce cardiac hypertrophy. In the present study, we examined whether cardiac fibroblasts may be a potential source of IL--6 production in the rat heart and whether catecholamines can modulate the IL--6 synthesis. Only a small amount of IL--6 mRNA was detected in unstimulated rat cardiac fibroblasts. However, a 50-fold increase of IL--6 mRNA was found after stimulation with norepinephrine (NE). Addition of carvedilol, a alpha- and beta-adrenergic receptor antagonist, prevented almost completely the NE-induced synthesis of IL--6 mRNA. Phenylephrine, an alpha-adrenergic agonist, and isoproterenol, a beta-adrenergic agonist, also induced an increase in IL--6. However, the stimulation via beta-receptors led to a more pronounced elevation. These data show that NE increases IL--6 expression in rat cardiac fibroblasts and that IL--6 may play an important autocrine/paracrine role in cardiac disease states associated with hypertrophy.

Comitogenic effect of catecholamines on rat cardiac fibroblasts in culture.

OBJECTIVE: We studied the ability of norepinephrine and of other catecholamines to affect the proliferation of cardiac fibroblasts isolated from adult rat hearts. Furthermore, we investigated the possible adrenergic receptor involved in this process. METHODS: Norepinephrine (NE), phenylephrine (PE), isoproterenol (ISO), forskolin (FO), epidermal growth factor (EGF), platelet-derived growth factor AA (PDGF-AA) and specific inhibitors of the alpha(1)-, alpha(2)-, beta(1)- and beta(2)-adrenoceptors and of the protein kinase A (PKA) were applied to cardiac fibroblasts in culture. Cell number was measured by use of a Coulter Counter. Activation of the cAMP response element binding protein (CREB) was measured by Western blotting and subsequent use of a phospho-specific antibody. Activation of the p42- and the p44-mitogen activated protein kinase (p42/p44(MAPK)) was assessed by detection of phosphorylation shifts and by incorporation of 32P-labelled phosphate into myelin basic protein. RESULTS: Fibroblasts isolated from hearts of adult rats were grown in 10% serum-containing media which induced an increase in cell number by 94%. After 48 h, treatment with 10 microM NE caused an even greater increase in cell number by 222%, i.e. another 128% (comitogenic effect). In contrast, NE alone had no effect on the growth of serum-deprived cells. EGF and PDGF-AA did not replace serum as the basic mitogen. After addition of NE to proliferating cells under serum conditions, there was a rapid, time-dependent significant activation of the p42/p44(MAPK) and of CREB for up to 60 and 120 min, respectively. In both cases, the maximum of activation was reached after 5 min. Application of FO (0.1-20 microM) caused a strong activation of CREB, while no increase in the phosphorylation of the p42/p44(MAPK) was detected. Treatment with 20 microM FO led to an identical increase in cell number as application of NE. Specific blockade of PKA with RpcAMPS prevented the activation of CREB and also the comitogenic effect of FO as well as of NE. The alpha- and beta-adrenergic receptor blocker carvedilol (10 microM) normalized all NE-induced effects. Prazosin and yohimbine, inhibitors of alpha(1)- and alpha(2)-adrenoceptor activation, respectively, did not influence the NE-evoked increase in cell number. In contrast, the non-selective beta-adrenoceptor blocker propranolol (1 microM) completely suppressed the comitogenic effect of NE. A similar effect was obtained with the specific beta(2)-adrenoceptor blocker ICI 118,551 (5 microM), while the beta(1)-adrenoceptor blocker metoprolol did not influence the increase in cell number. CONCLUSIONS: NE elicits a comitogenic effect on cultured rat cardiac fibroblasts which is prevented by beta(2)-adrenergic blockade. The activation of CREB contributes to the increase in proliferation. The p42/p44(MAPK) which was also found to be activated by NE might as well be involved in the regulation of the comitogenic effect.

Habituation and laterality of orienting processes as reflected by slow negative waves.

The study is concerned with the question of whether the orienting wave (O-wave), a slow potential shift of the event-related brain potential, is a component of the orienting response (OR). As habituation is supposed to be the most important characteristic of the OR, we focussed particularly on any habituating aspect of the O-wave. Results suggest that its bilateral distribution over midfrontal areas might constitute such a link relating the O-wave to orienting activity. Hemispheric asymmetry linearly decreased its right-sided predominance in response to repeated presentations of an initially novel auditory stimulus. A similar, concomitant diminution of the skin conductance response (SCR) occurred. Both, the O-wave and the SCR varied, moreover, with signal value and electrodermal lability which are known to relate to the OR. Therefore we suggest the O-wave comprise features typical of a component of the OR. A hypothesis is put forward according to which the O-wave and its laterality are dependent on the ascending noradrenergic locus coeruleus system.

[Frequency and mean amplitude of spontaneous electrodermal fluctuations are not interchangeable indicators of psychological processes]. / Frequenz und mittlere Amplitude spontaner elektrodermaler Fluktuationen sind keine austauschbaren Indikatoren psychischer Prozesse.

The study was concerned with the question of whether the frequency and the mean amplitude of spontaneous electrodermal fluctuations convey the same information about mental processes. In order to examine this, the content and the personal relevance of induced imaginations was varied experimentally. The experimental manipulations followed a 2 x 2 factorial design for independent groups (content of cells: 24 subjects each). Changes in electrodermal activity were measured as differences between the imagination and a resting phase. Results revealed that the two variables are not exchangeable indicators of mental processes. Although the two variables correlated significantly, they responded differently to the experimental manipulations. The imagination of death-related contents increased the frequency of spontaneous electrodermal fluctuations more than the imagination of neutral contents. High personal relevance enhanced the mean amplitude of these spontaneous fluctuations regardless of the imagined content. Therefore it was concluded that the two variables have something in common but, on the whole, do not depend on identical factors. Frequency of spontaneous electrodermal fluctuations seems to be a reflection of covert orienting processes, whereas their mean amplitude seems to reflect the activity of an appraisal system incorporating goals and past experiences. However, both variables, seem to be, in the same way, dependent on a kind of basal activation or vigilance.

Pop-out into memory: a retrieval mechanism that is enhanced with the recall of subject-performed tasks.

Subject-performed tasks (SPTs; i.e., carrying out the actions during study) improve free recall of action phrases without enhancing relational information. By this mechanism, items pop into a person's mind without active search, and this process especially extends the recency effect. The authors demonstrated the existence of the extended recency effect and its importance for the SPT recall advantage (Experiments 1 and 2). Carrying out the action and not semantic processing caused the effect (Experiment 3). The extended recency effect was also not a consequence of a deliberate last-in, first-out strategy (Experiment 4), and performing a difficult secondary task (an arithmetic task) during recall reduced memory performances but did not influence the extended recency effect (Experiment 5). These data support the theory that performing actions during study enhances the efficiency of an automatic pop-out mechanism in free recall.

Differential remodeling of the left and right heart after norepinephrine treatment in rats: studies on cytokines and collagen.

Continuous intravenous infusion of norepinephrine norepinephrine (NE, 0.1 mg/kg/h) induced hypertrophy of the left ventricle (LV), but not of the right ventricle (RV) in rats, although RV systolic pressure (RVSP) was much more elevated than LVSP. After NE infusion, there was a time-dependent (20 min to 72 h) expression in the mRNA of interleukin (IL)-6 and IL-1 beta. The expression of IL-6 increased markedly and reached the maximum after 4 h with an 80-fold elevation. In the RV, the expression increased only 20-fold. The mRNA of IL-1 beta increased significantly after NE stimulation only in the LV and reached the maximum after 12 h with a 12-fold elevation. After 12 h of NE infusion, colligin mRNA was elevated for the first time with further progression until 72 h. The six-fold abundance of colligin mRNA seen after 72 h was significantly higher in the LV than in the RV. A similar increase was observed on the protein level (Western blotting). The expression of collagen I and III increased significantly after 24 h only in the LV. After 72 h, the mRNA expression of collagen I was increased 16-fold and that of collagen III 10-fold. This expression was significantly higher than that in the RV. Also the elevation in atrial natriuretic peptide (ANP) mRNA started earlier and was more pronounced in the LV than in the RV. The alpha- and beta-adrenergic receptor blocker carvedilol normalized all functional parameters after 6 h and 72 h and prevented the development of LV hypertrophy that occurred after 72 h. The NE-induced increased expression of the mRNAs studied was either prevented (IL-6, IL-1 beta ) or attenuated (colligin, collagen I and III, ANP) by combined alpha- and beta-receptor blockade. The elevation of afterload which was associated with the NE effect was normalized by the calcium-channel blocker nisoldipin, but NE-induced LV hypertrophy and the increase in ANP and collagen mRNA were not affected.

Modifications of the isolated frog heart preparation in Carl Ludwig's Leipzig Physiological Institute: relevance for cardiovascular research.

Carl Ludwig was the first physiologist to systematically study isolated organs (heart, muscle, kidney, liver, lung). In his Leipzig Physiological Institute, the isolated perfused frog heart preparation was established in 1866 by Elias Cyon. This preparation was subsequently subjected to various modifications, and many important observations were made by scientists such as Joseph Coats, Henry Pickering Bowditch, Luigi Luciani, Michael Joseph Rossbach, Hugo Kronecker and Otto Frank. The influence of filling pressure on contraction amplitude, the all-or-none law of the heart, the absolute refractory period, postextrasystolic potentiation, the staircase ('Treppe') phenomenon and the dependence of heart function on oxidative metabolism were discovered. The negative chronotropic and inotropic effects of vagus nerve stimulation were also first documented, and a model to induce arrhythmias was established. The isolated frog heart preparation became a widely used standard model for teaching and for basic cardiovascular research. Sidney Ringer discovered the essential role of calcium ions for heart function. Otto Loewi discovered the chemical transduction mechanism of the vagus with acetylcholine as transmitter. In more recent times, the cyclical changes in cAMP and cGMP that occur during the cardiac cycle were first described in the frog heart by Wollenberger and associates. Thus, the isolated perfused frog heart established and modified in Carl Ludwig's Leipzig Physiological Institute led not only to the discovery of basic phenomena, but also to observations that became the basis for concepts to be developed and elaborated later. Furthermore, the isolated perfused frog heart was the starting point for the development of the isolated mammalian heart in the retrogradely perfused, nonworking mode in the heart-lung modification and in the working heart preparation.

Inhibition of nitric oxide synthase augments the positive inotropic effect of nitric oxide donors in the rat heart.

1. In this investigation we studied the effects of nitric oxide on contractility and heart rate in normal saline-perfused rat hearts where shear stress-induced endothelial NO synthesis substantially contributes to total cardiac NO production. In addition, we sought to estimate the concentrations of exogenous NO producing inotropic effects. 2. We investigated the effects of glyceryl trinitrate (GTN), S-nitroso-d,l-penicillamine (SNAP), sodium (Z)-1-(N, N-diethylamino)diazen-1-ium-1,2-diolat (DEA/NO), and DEA/NO in the presence of the NO synthase inhibitor Nomega-nitro-L-arginine (L-NA) in constant-flow-perfused spontaneously beating rat Langendorff hearts and in rat working hearts. 3. In Langendorff hearts, GTN (10 nM to 100 microM, n = 32) induced a positive inotropic response that plateaued at 1 microM GTN with a maximal rate of increase of left ventricular pressure during ventricular contraction (+dP/dtmax) of 6. 33 +/- 2.56 % (n = 11, P < 0.5). Similarly, both spontaneous NO donors (0.1 nM to 1 microM, corresponding to approximately 0.03-0.3 microM NO) induced a positive inotropic response of 10.6 +/- 3.1 % (SNAP; n = 15, P < 0.05) and 11.5 +/- 2.7 % (DEA/NO, n = 15, P < 0. 05). 4. The positive inotropic effect of SNAP and DEA/NO progressively declined from 1 microM to 100 microM of the NO donors (corresponding to approximately 0.3-30 microM NO). 5. In the isolated working rat heart, 0.1 microM DEA/NO induced an increase of +dP/dtmax of 7.5 +/- 2.5 % (n = 9, P < 0.05). Inhibition of NO synthase by L-NA produced a 4-fold increase in this effect of DEA/NO. 6. We suggest that physiological NO concentrations support myocardial performance. In normal rat hearts the positive inotropic effect of NO appears to be almost maximally exploited by the endogenous NO production.

Levels-of-processing effects in subject-performed tasks.

In memory for subject-performed tasks (SPTs), subjects encode a list of simple action phrases (e.g., thumb through a book, knock at the door) by performing these actions during learning. In three experiments, we investigated the size of the levels-of-processing effects in SPTs as compared with those in standard verbal learning tasks (VTs). Subjects under SPT and VT conditions learned lists of action phrases in a surface or a conceptual orienting task. Under both encoding conditions, the subjects recalled fewer items with surface orienting tasks than with conceptual orienting tasks, but the levels-of-processing effects were strongly reduced in the SPT condition. In the SPT condition, items that were encoded in a surface orienting task were still substantially recalled. The items were recalled almost as well as the conceptually encoded items in the VT condition. The distinct reduction of the levels-of-processing effect is caused by the fact that, in SPT encoding even with a verbal surface orienting task, subjects process conceptual information in order to perform the denoted action. We attribute the small conceptual advantage, which remains with SPT despite the conceptual processing for performing, to the fact that items are not as well integrated into memory as they are when conceptual processing is focused on the action component, rather than on the semantic contexts. This lower integration reduces the accessibility of items in the verbal surface task, even with SPT encoding.

Determination of ethyl glucuronide, a minor metabolite of ethanol, in human serum by liquid chromatography-electrospray ionization mass spectrometry.

A rapid and sensitive determination procedure using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) has been developed for the determination of ethyl glucuronide (EtG) in human serum. Samples were precipitated with methanol, centrifuged and the supernatant was evaporated to dryness followed by reconstitution with distilled water. As mobile phase 30 mM ammonium acetate-acetonitrile (30:70, v/v) was utilized. The base peak observed at m/z 221 was the [M-H]- ion of EtG, which was detectable in satisfactory sense. The detection limit was 0.03 microg/ml in the selected ion monitoring mode. A calibration graph constructed for EtG in serum gave good linearity over the range from 0.1 to 25 microg/ml. This paper also presents the application of this LC-ESI-MS procedure to the analysis of authentic serum samples.