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Cardiac sympathetic transmitter stores are reduced in the failing heart. In this study, we proposed to investigate whether the reduction of cardiac sympathetic neurotransmitters was associated with increased interstitial norepinephrine (NE) and reactive oxygen species in congestive heart failure (CHF), using a microdialysis technique and salicylate to detect ·OH generation. Rabbits with and without rapid ventricular pacing (340 beats/min) were randomized to receive desipramine (10 mg/day) or placebo for 8 wk. Rapid pacing produced left ventricular dilation and systolic dysfunction. The failing myocardium also showed reduced tissue contents of NE and tyrosine hydroxylase protein and activity. In contrast, myocardial interstitial NE was increased in CHF (0.89 ± 0.11 ng/ml) compared with the sham-operated animals (0.26 ± 0.03 ng/ml). In addition, cardiac oxidative stress was increased in CHF animals as measured by myocardial interstitial ·OH radical, tissue oxidized glutathione, and oxidized mitochondrial DNA. Desipramine treatment produced significant NE uptake inhibition as evidence by an exaggerated pressor response and a greater increase of myocardial interstitial NE in response to intravenous NE infusion but no significant effects on cardiac function or hemodynamics in sham-operated or CHF animals. However, desipramine treatment attenuated the reductions of tissue NE and tyrosine hydroxylase protein and activity in CHF. Desipramine also prevented the reduction of tyrosine hydroxylase produced by NE in PC12 cells. Thus the reduction of cardiac sympathetic neurotransmitters is related to the increased interstitial NE and tissue oxidative stress in CHF. Also, normal neuronal uptake of NE is required for NE or its oxidized metabolites to exert their neurotoxic effects.
- oxidative stress
- microdialysis
- tyrosine hydroxylase
- norepinephrine
- ceramide
depletion of cardiac norepinephrine (NE) is one of the important salient features in human congestive heart failure (CHF) (7, 9). Prior studies (5, 7, 9, 16, 17) have shown that cardiac NE is depleted in CHF because of increased preferential cardiac release of NE, reduced neuronal reuptake of NE, and impaired NE synthesis. Tyrosine hydroxylase, a rate-limiting enzyme in NE biosynthesis (37), is reduced in heart failure (16, 53), which may account, at least in part, for the reduced production of NE. Recently, we observed in several animal models of experimental CHF (23, 30, 39, 41) that the sympathetic nerve profiles as measured by NE histofluorescence and tyrosine hydroxylase immunocytochemistry are reduced in CHF, but there is no significant reduction of protein gene product 9.5 (PGP9.5) (41), a panneuronal marker (20, 29, 57). Thus the anatomic integrity of the cardiac sympathetic nerves probably is intact in CHF, and the changes of sympathetic neurotransmitters within the nerve endings are caused by functional abnormalities that are potentially reversible with either effective therapy or removal of primary insult that causes heart failure as discontinuation of cardiac pacing in tachycardia-induced cardiomyopathy (30).
Our laboratories have further shown that the reductions of cardiac sympathetic transmitters, like those in CHF, can also be induced by exogenous administration of NE (40, 41) and inhibited by desipramine, a NE uptake inhibitor (39, 41). The findings suggest that the cardiac sympathetic nerve terminal dysfunction is probably caused by increased interstitial NE in CHF, and the neuronal damaging effect of NE involves the uptake of NE or its oxidative metabolites into the sympathetic nerve endings. However, direct in vivo demonstration of myocardial NE uptake inhibition by desipramine is lacking. In the present study, we employed rapid ventricular pacing to produce left ventricular dilation and failure in rabbits and measured the myocardial interstitial NE concentrations directly using a microdialysis technique (31, 35, 61). We measured the sympathetic neuronal markers NE and tyrosine hydroxylase. Furthermore, desipramine has been shown recently to reduce mitochondrial reactive oxygen species and cell death by inhibiting membrane sphingomyelinase and ceramide production (11, 25, 42). Ceramide is the second messenger in the sphingomyelin-signaling pathway (21). It is highly expressed in the heart and has been shown to play an important role in mediating the cell apoptosis and cardiac dysfunction produced by TNF-α and oxygen free radicals in cardiomyocytes (59). Desipramine has also been shown to reduce ceramide increase, infarct size, and myocyte apoptosis during prolonged ischemia in both isolated perfused hearts (14) and intact rabbits (3). Because reduction of oxidative stress by antioxidants has also been shown to attenuate the neuronal damaging effects of NE (40), we performed additional experiments to determine whether the effects of desipramine on myocardial NE and tyrosine hydroxylase were associated with reductions of interstitial oxygen free radicals and tissue oxidative stress. Cardiac microdialysis of salicylic acid was used to measure myocardial interstitial 2,3-hydroxybenzoic acid (2,3-DHBA), a sensitive and specific marker for hydroxyl radical (45, 50, 54). In addition, myocardial contents of GSH-to-GSSG ratio and mitochondrial DNA (mtDNA) 8-oxo-7,8-dihydro-2 deoxyguanosine (8-oxo-dG) were measured. Finally, to determine whether desipramine exerts a direct effect on sympathetic cells, we administered desipramine to the cultured rat pheochromocytoma PC12 cells treated with various doses of NE. We also measured ceramide by immunocytochemistry in PC12 cells treated with NE and desipramine. Our results indicate that desipramine attenuates the reduction of tyrosine hydroxylase in both the failing myocardium and the NE-treated PC12 cells and that the neuroprotective effects are related to the inhibitory action of desipramine on NE uptake without any effect on oxidant stress or ceramide production.
Animal model.
This study was approved by the University of Rochester Committee on Animal Resources and conformed to the guiding principles approved by the Council of the American Physiological Society and the National Institutes of Health's Guide for the Care and Use of Laboratory Animals (NIH Publications No. 85-23, Revised 1996).
Adult healthy New Zealand White rabbits (2.8 to 3.6 kg, 4–6 mo of age) were chosen for production of experimental heart failure with the use of a modified rapid cardiac pacing technique as described previously (30, 58). Briefly, under general isoflurane anesthesia, subxyphoid thoractomy and pericardiotomy were performed for placement of two shielded pacing leads (model TPW50; Ethicon, Somerville, NJ) onto the left ventricular apex and the left pectoral muscle, respectively. One week after surgery, rabbits were randomly assigned to receive either pacing (CHF animals, 340 beats/min, model 8086 Prevail implantable programmable pacemaker modified for rapid pacing, Medotronic, Minneapolis, MN) or no cardiac pacing (sham-operated animals). The animals developed progressive heart failure beginning 1–2 wk after the start of rapid pacing.
Experimental design.
CHF and sham-operated animals were randomized to receive desipramine or placebo. The drug was delivered at a rate of 10 mg/day for 8 wk with the use of subcutaneous pellets (Innovative Research of America, Sarasota, FL). Animals in a lightly anesthetized state with the use of ketamine (20 mg/kg) and midazolam (0.6 mg/kg) were examined weekly by echocardiograms. At week 8, cardiac pacing was discontinued, and the animal was anesthetized for the measurement of resting hemodynamics and cardiac interstitial NE and 2,3-DHBA with the use of the microdialysis technique. Afterwards, the animal was euthanized with intravenous pentobarbital sodium (>100 mg/kg), and the heart was removed, weighed, and rinsed in ice-cold oxygenated normal saline. The left and right ventricles were separated from the septum and weighed. Transmural muscle blocks from left ventricles were processed immediately or stored in liquid nitrogen for later analysis for cardiac sympathetic neurotransmitters and for total cellular and mitochondrial oxidative stress indexes.
Echocardiographic and hemodynamic measurements.
Two-dimensional and M-mode echocardiograms were obtained by using a 5-MHz transducer on a Toshiba model SSH-60A sonographic system (Toshiba America Medical System, Tustin, CA). Maximal left ventricular end-diastolic dimension (EDD) and end-systolic dimension (ESD) were measured and used to calculate left ventricular fractional shortening (FS) with the following equation: FS = [(EDD − ESD)/EDD] × 100.
For hemodynamic studies, animals were anesthetized with ketamine (35 mg/kg) and midazolam (0.8 mg/kg). A 20-gauge, fluid-filled catheter (Insyte; Desert Medical; Becton, Dickson and Company; Sandy, UT) was inserted into the left carotid artery and connected to a Statham P23 XL pressure transducer (Spectramed, Oxnard) with an amplifier for measuring aortic pressure, while a 2.2 Fr SPR-249 micromanometer-tipped catheter (Millar Instruments, Houston, TX) was advanced into the left ventricle via the right carotid artery for measuring the left ventricular pressure. Electrocardiograms, aortic pressure, and the first derivative of left ventricular pressure (dP/dt) were recorded on a multichannel recorder (Brush model 480, Gould, Instrument Systems Division, Cleveland, OH) and an IOX data acquisition and analysis system (EMKA Technologies, Falls Church, VA). At least 1 h was allowed to elapse after catheterization before the resting hemodynamic data were taken in triplicate over a 15- to 20-min steady state. The measurements taken from five consecutive beats during the expiratory cycles were averaged for statistical analysis.
Myocardial microdialysis for 2,3-DHBA and NE determination.
After completion of resting hemodynamic measurements, rabbits were artificially ventilated with a constant-volume respirator using room air mixed with oxygen. Heart rate, arterial pressure, and ECG were monitored and recorded continuously. The chest was opened through the fifth or sixth rib on the left side. A small incision was made in the pericardium with a fine guiding needle, and a CMA/20 microdialysis probe (10-mm membrane length; CMA Microdialysis, North Chelmsford, MA) was implanted at the midlevel of left ventricular free wall. The dialysis probe was perfused with Ringer solution containing (in mM) 147 NaCl, 2.3 CaCl2, and 4 KCl (pH 7.4) with the use of a CMA/102 microdialysis pump. The outlet side of the probe was connected to a microcentrifuge tube on ice. A 120-min equilibration period was allowed to elapse after probe implantation before the dialysate of 5 μl/min was collected every 4 min into a 5 μl ice-cold solution containing 0.1% Na2EDTA in 0.1 M HClO4 to prevent amine oxidation. Sodium salicylate (1 mM) was added to the microdialysis perfusion to detect the generation of ·OH as reflected by the formation of 2,3-DHBA in the myocardium (46, 47, 50). After baseline measurements were completed, NE was infused intravenously at 2 mmol·kg−1·min−1 for 20 min, and dialysate collections were continued for another 25 min after the discontinuation of NE for immediate assays of NE and 2,3-DHBA with the use of a Bioanalytical System 480 HPLC system (BAS, West Lafayette, IN).
The HPLC was equipped with a BAS model 480 pump, a Unijet microbore column (5 μm, 100 × 1 mm), and a LC/4C electrochemical detector with a glassy carbon electrode. Detector potential was +0.70 V with an Ag/AgCl reference electrode. The mobile phase consisted of 0.15% 1-heptansulfonic acid, 0.01% Na2EDTA, 0.3% triethylamine, and 4% acetonitrile (pH 2.8 with phosphoric acid). An aliquot of dialysate was injected into a 10-μl sample loop. A standard solution of 2,3-DHBA and NE was also injected for calibration.
Cardiac sympathetic nerve terminal neurotransmitters.
Left ventricular muscle samples were taken to measure NE with the use of both HPLC and sucrose-potassium phosphate-glyoxylic acid-induced (SPG) histofluorescence (15, 23). For the HPLC assay, muscle samples were minced and suspended in 0.4 N perchloric acid with 5 mM reduced glutathione (pH 7.4), homogenized with a Brinkman Polytron PCU-2 homogenizer, and centrifuged at 500 g. The supernatant was then injected into the BAS HPLC with a LC/4C electrochemical detector. A MP-1 catecholamine mobile phase (Bioanalytical Systems) was used. To measure SPG histofluorescence, tissue blocks were sectioned at a thickness of 16 μm and dipped in SPG solution (23, 39). Tissue sections were viewed under epifluorescent illumination by using a Nikon fluorescence microscope (Nikon Instruments, Melville, NY) and photographed onto 35-mm slides with ×30 magnification. The number of catecholaminergic profiles were counted morphometrically in a 0.221 mm2 (0.003536 mm3) field. Six fields were counted for each animal, and the results were averaged.
To measure tissue tyrosine hydroxylase, left ventricular muscle blocks were homogenized in 1× lysis buffer (Cell Signaling Technology, Beverly, MA) containing 20 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, and 1 mM PMSF) for 30 min on ice. After centrifugation at 12,000 g for 15 min was completed, the supernatant was used for Western blot analysis. Protein content was determined by using a bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). Equal amounts of protein (20 μg) were loaded onto SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membrane (PerkinElmer, Boston, MA) electrically. Blots were then probed with either anti-tyrosine hydroxylase antibody (1:1,000; Cell Signaling Technology) or anti-PGP9.5 antibody (1:1,000; Biogenesis, Kingston, NH). After exposure to horseradish peroxidase-conjugated secondary antibody for 1 h, blots were visualized by using a chemiluminescence detection kit (Cell Signaling Technology). The autoradiograms were scanned on a Microtek model 6800 scanner (Microtek, Carson, CA), and the optical density of bands was determined by using a NIH 1.6 gel image program. The optical density readings of samples were normalized to a control sample in an arbitrary densitometry unit.
Myocardial tyrosine hydroxylase activity assay.
Tyrosine hydroxylase activity was measured by the conversion of [3H]tyrosine to dihydroxyphenylalanine and 3H2O by using a modification (52) of the procedure of Reinhard et al. (56). Briefly, heart tissue was homogenized in five volumes of 1× lysis buffer (Cell Signaling Technology), containing protease inhibitors and phosphatase inhibitors (see Cardiac synaptic nerve terminal neurotransmitters) at 4°C, and centrifuged at 14,000 g. The supernatant was then added to an equal amount of 2× assay buffer to reach the final concentration of 150 mM Tris maleate, 50 μM l-[3,5-3H]tyrosine (51.5 Ci/mM, PerkinElmer), 5 mM ascorbate, 0.45 mg/ml catalase, and 0.5 mM 6-MPH4 at pH 6.8. After 10 min at 37°C, the reaction was stopped on ice. With the use of this reaction, 1 mol of 3H2O is generated for each mole of 3H-tyrosine that is converted to dihydroxyphenylalanine by tyrosine hydroxylase. The tyrosine hydroxylase assay is linear over the reaction time for at least 30 min. Released 3H2O was separated from unreacted 3H-tyrosine by mixing the reaction samples with 7.5% charcoal in 1.0 M HCl and assayed from the supernatant by liquid scintillation counting (TriCarb 2400 TR liquid scintillation counter, Packard Instrument, Meriden, CT). Tyrosine hydroxylase activity was expressed as picomoles of 3H2O formed per milligram of protein in 10 min.
Myocardial glutathione measurement.
A Bioxytech GSH/GSSG-412 test kit (OXIS International, Portland, OR) was used to measure GSH and GSSG. Fresh left ventricular myocardium was homogenized in three volumes of 1% picric acid, and the supernatant was collected for measuring with or without 33 mM 1-methyl-2-vinyl-pyridinium trifluoromethanesulfonate (M2VP), a scavenger of reduced GSH. For GSH estimation, 5 μl of supernatant was mixed with 355 μl of assay buffer containing 100 mM NaPO4 and 5 mM EDTA, pH 7.5. For GSSG estimation, 10 μl of homogenate with M2VP was mixed with GSSG buffer containing 100 mM NaPO4 and 5 mM EDTA, pH 10.05. The samples were mixed with 1.262 mM 5,5-dithiobis-(2-nitrobenzoic acid) and 15 units/ml GSH reductase. The mixture was incubated at room temperature for 5 min, and the absorbance was recorded at 412 nm for 3 min after the addition of 3.8 μmol NADPH by using a SmartSpec 3000 spectrophotometer (Bio-Rad, Hercules, CA).
Myocardial mtDNA 8-oxo-dG measurement.
Left ventricular muscle samples were prepared for isolation of mitochondria (4, 58). The mtDNA was extracted by using QIAamp blood kit (Qiagen, Valencia, CA) per the manufacturer's instructions and digested into deoxynucleosides by nuclease P1 and alkaline phosphatase. The deoxynucleoside mixture was filtrated through a 0.22-μm nylon filter and injected into a YMC Basic S, 3 μ 4.6 × 150 nm column (Waters, Milford, MA) in the BAS 480 HPLC with a mobile phase of 5% methanol in 100 mM lithium acetate buffer (pH 5.2). Detection of 8-oxo-dG and 2′-deoxyguanosine (dG) was performed on a model 5200A Coulochem II electrochemical detector equipped with a model 5011 analytical cell and model 5021 guard cell (Environmental Science Associate, Chelmsford, MA), and dG (Sigma-Aldrich, St. Louis, MO) was used for calibration.
PC12 cell studies.
Liang's laboratory (44) has shown in previous studies that NE reduced NE uptake and NE uptake-1 carrier protein in a dose-dependent manner in PC12 cells without causing significant cell death. Cell death occurred only when the NE concentration exceeded 100 μM (43, 44). In this study, we investigated the effects of NE, as well as the influence of desipramine and antioxidant enzymes on the effects of NE, on the tyrosine hydroxylase content in PC12 cells. PC12 cells were plated in a 60-mm dish for Western blot analysis, or, at a density of 105 cells per well, in a 12-well plate for NE uptake assay and grown for 24 h at 37°C. The medium was then switched to 2% serum RPMI 1640 overnight before exposing the cells to 1, 10, and 100 μM of NE in the presence or absence of 1 μM desipramine for 24 h. PC12 cells were also exposed to NE in the presence and absence of superoxide dismutase (20 μg/ml) and catalase (20 μg/ml) for 24 h. All cell samples were then solubilized in 1× lysis buffer and prepared for Western blot analysis as described above for the heart muscle samples. Equal loading of the protein was confirmed by reblotting the polyvinylidene difluoride membrane with anti-β-actin (1:1,000; Abcam, Cambridge, MA). Finally, to determine whether desipramine reduced the lethal effect of NE on PC12 cells, we measured cell viability of the PC12 cells by 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (44).
NE uptake activity in PC12 cells after NE and desipramine treatment was measured by specific uptake of [3H]NE in an incubation medium containing 35.7 nM l-[7-3H(N)]NE (13.8 Ci/mmol; New England Nuclear, Boston, MA) at 37°C for 15 min (44). After the wash was completed, cells were solubilized and counted for radioactivity in the TriCarb 2400 TR liquid scintillation counter. Nonspecific accumulation of radioactivity was determined by parallel incubation of PC12 cells in the presence of 10 μM nisoxetine in the incubation medium.
Immunocytochemistry of ceramide in PC12 cells.
Ceramide was measured in PC12 cells with an anti-ceramide antibody (12), by using an immunocytochemistry protocol for avidin-biotin complex (ABC) detection by Cell Signaling Technology. To study the effects of NE and desipramine on ceramide generation, PC12 cells were treated with 100 μM NE, 1 μM desipramine, or 100 μM NE plus 1 μM desipramine for 24 h and compared with those cells without NE or desipramine treatment. After treatment was completed, PC12 cells were washed by 1× TBS and immersed in 4% paraformaldehyde for 10 min at 4°C. The cells were then permeabilized on coverslips with 0.2% Triton X-100, blocked with a 5.5% goat serum in TBS-Triton buffer, and incubated with a primary anti-ceramide mouse IgM antibody (1:100) in 3% bovine serum albumin in TBS overnight at 4°C. The cells were then treated with a biotinylated anti-mouse IgM secondary antibody and prepared for ABC detection using hydrogen peroxide and Vectastain ABC kits (Vector, Burlingame, CA).
Statistical analysis.
Results are presented as means ± SE. Experimental data were managed and analyzed by the RS/I Research System (Bolt, Beraneck and Newman Software Products, Cambridge, MA). The statistical significance of differences among the different experimental groups was analyzed by ANOVA. Student's t-test with Bonferroni correction was used to determine the statistical significance of difference between two individual groups. For serial measurements, two-grouping (CHF and drug treatment), repeated-measures ANOVA (SYSTAT version 11, SYSTAT Software, Point Richmond, CA) was used to determine the significance of influence of CHF and desipramine treatment. The significance of changes over a period of time in the same animals was determined by pairwise Bonferroni t-tests. Differences were considered statistically significant if P < 0.05.
Cardiac function and resting hemodynamics.
Rapid ventricular pacing caused a progressive increase in left ventricular EDD and decline in left ventricular FS over 8 wk of pacing (Fig. 1). Desipramine treatment alone had no effects in the cardiac function in sham-operated animals. However, in animals receiving rapid cardiac pacing, desipramine appeared to hasten the dilation of the left ventricle, but the net increase of left ventricular EDD at the end of 8 wk did not differ significantly between the CHF animals with and without desipramine (2.3 ± 0.3 vs. 2.7 ± 0.3 mm). Desipramine also produced no statistically significant differences in left ventricular FS in CHF animals.
Table 1 shows body weight, heart weight, resting heart rate, mean aortic blood pressure, and left ventricular end-diastolic pressure and dP/dt in the sham-operated and CHF animals. Cardiac dysfunction was evidenced by the increased left ventricular end-diastolic pressure and reduced left ventricular dP/dt. Body weight, left ventricular weight, heart rate, and aortic blood pressure did not differ between the CHF and sham-operated groups. Desipramine produced no changes in any of the hemodynamic parameters in either the sham-operated or the CHF animals.
Microdialysis measurements of myocardial interstitial NE and responses to NE infusion.
Figure 2 illustrates the changes of interstitial NE concentration during 20 min of NE infusion and 25 min of recovery. The baseline interstitial NE (in ng/ml) taken within 5 min immediately before NE infusion differed significantly among the four groups of animals (F = 23.79, degree of freedom = 3, 20, P < 0.001). When compared with baseline value in the sham-operated placebo animals (0.26 ± 0.03 ng/ml), interstitial NE was increased in animals treated with desipramine (0.74 ± 0.09 ng/ml), CHF (0.89 ± 0.11 ng/ml), and CHF plus desipramine (1.28 ± 0.12 ng/ml). NE infusion produced significant increases in interstitial NE in all experimental animals, but compared with the other groups, the increase of interstitial NE during NE infusion was relatively small in the sham-operated placebo group. The peak interstitial NE (obtained between 12 and 20 min of NE infusion) was greatest in the desipramine-treated CHF animals (4.21 ± 0.76 ng/ml), compared with the sham-operated placebo (0.76 ± 0.09 ng/ml), sham-operated desipramine (2.80 ± 0.27 ng/ml), and CHF placebo (3.13 ± 0.63 ng/ml) groups. Interstitial NE returned to baseline quickly in the sham-operated placebo animals (0.29 ± 0.04 ng/ml), but the values in the other three groups remained elevated above baseline 12 min after the end of NE infusion and were higher than that in the sham-operated placebo group at 20–25 min after end of NE infusion. NE infusion also increased mean aortic blood pressure in all animals. The net increase of mean aortic blood pressure during NE infusion, however, was smaller in CHF animals (23 ± 5 mmHg) compared with sham-operated animals (43 ± 5 mmHg). In addition, consistent with inhibition of NE uptake, desipramine treatment enhanced the pressor response to NE in the CHF animals (37 ± 6 mmHg).
Effect of desipramine on myocardial interstitial 2,3-DHBA.
Figure 3 shows the changes of interstitial 2,3-DHBA at rest and during NE infusion in sham-operated and CHF animals. Interstitial 2,3-DHBA was increased sixfold at baseline in CHF animals compared with the sham-operated animals. NE infusion increased 2,3-DHBA in both groups of animals. These changes in sham-operated animals were further exaggerated by desipramine administration.
Myocardial tissue glutathione and mtDNA 8-oxo-dG.
Rapid cardiac pacing reduced myocardial GSH and increased GSSG. As a result, the GSH-to-GSSG ratio decreased markedly in the failing myocardium (Fig. 4). It also increased mtDNA 8-oxo-dG. Because there was no change in mtDNA dG, the mtDNA 8-oxo-dG-to-dG ratio increased more than twofold in animals with CHF. The figure also showed desipramine treatment caused a slight increase of 8-oxo-dG-to-2′-dG ratio in sham-operated animals but had no effect on 8-oxo-dG-to-dG ratio in CHF animals.
Effects of desipramine on tissue NE and tyrosine hydroxylase.
Table 2 shows the changes in tissue NE, SPG histofluorescence, and tyrosine hydroxylase in sham-operated and CHF animals. As shown before, myocardial NE and tyrosine hydroxylase protein were reduced in CHF animals. Similar to the Western blot analysis, tyrosine hydroxylase activity was reduced by 50–60% in CHF animals. Furthermore, because PGP9.5 did not differ between the groups, the ratio of tyrosine hydroxylase to PGP9.5 was reduced to a similar extent as tyrosine hydroxylase in CHF. The administration of desipramine reduced tissue NE and SPG histofluorescence in sham-operated animals but had no significant effect in tyrosine hydroxylase protein or activity. In contrast to the sham-operated animals, desipramine increased SPG histofluorescence profiles and tyrosine hydroxylase protein and activity in CHF animals.
Effects of desipramine in PC12 cells.
Figure 5 shows that NE reduced tyrosine hydroxylase in a dose-dependent fashion in PC12 cells. A 34% reduction of tyrosine hydroxylase was seen with 100 μM NE. We also found that the reduction of tyrosine hydroxylase was abolished by 1 μM desipramine and superoxide dismutase plus catalase (Fig. 5). This dose of desipramine was chosen from our preliminary experiments showing an 88% reduction of NE uptake activity in PC12 cells (39 ± 4 vs. 313 ± 9 fmol·mg−1·min−1, t = 27.4, degree of freedom = 10, P < 0.001). This dose of desipramine was also sufficient to reduce cell death, as measured by MTT, produced by 500 μM NE in PC12 cells from 51 ± 6% to 29 ± 3% (P < 0.01).
The effects of desipramine and NE on ceramide content in PC12 cells are shown in Fig. 6, which shows that desipramine (1 μM) treatment resulted in a marked reduction of ceramide. In contrast, NE, at a dose that effectively reduced tyrosine hydroxylase by 34% (Fig. 5), produced no significant effect on ceramide in either control or desipramine-treated PC12 cells.
Our present experiments extend our prior studies on the reduction of cardiac sympathetic neurotransmitters in the failing heart. Our present study is the first to provide direct evidence that the reduction of cardiac sympathetic neurotransmitters in CHF is associated with increases in interstitial NE and concomitant production of ·OH with the use of the cardiac microdialysis technique. The findings suggest that the changes in neuronal transmitters are probably causally related to the increases in interstitial NE or its oxidative products, which is consistent with our prior findings that exogenous NE infusion reduced cardiac sympathetic neuronal profiles (40, 41). Direct demonstration of the effect of NE on neuronal tyrosine hydroxylase was also provided in our present study using PC12 cells.
Our present study also shows that the effect of NE on the cardiac sympathetic nerve endings requires an intact NE uptake mechanism. The administration of desipramine attenuated the reduction of tyrosine hydroxylase protein and enzyme activity in CHF. However, desipramine had a comparatively smaller effect on the restoration of tissue NE, probably because of the increased cardiac release of NE in CHF and the inhibition of NE uptake by desipramine. Inhibition of NE uptake in PC12 cells by desipramine also abolished the reduction of tyrosine hydroxylase produced by NE. Desipramine also reduced the lethal effects of high-dose NE in PC12 cells. Similar effects were produced by superoxide dismutase and catalase in PC12 cells (43). However, desipramine did not affect the changes of either cardiac GSH-to-GSSG or mtDNA 8-oxo-dG-to-dG ratios in the CHF animals. In fact, desipramine actually increased myocardial ·OH as measured by interstitial 2,3-DHBA in the sham-operated animals. Thus the effects of desipramine on the preservation of sympathetic neuronal transmitters are independent of tissue oxidative stress. In addition, the neuroprotective effects of desipramine and superoxide dismutase are additive on sympathetic nerves (1). Thus the loss of noradrenergic transmitters in the failing myocardium is most likely caused by oxidation metabolites of NE that exert effects on the sympathetic nerve endings via the desipramine-sensitive NE uptake site.
Desipramine is a classical NE uptake inhibitor. It has been shown to reduce the NE uptake activity in the ventricular muscle preparation ex vivo (39, 40). At the dose administered, desipramine was well tolerated and produced no untoward clinical or hemodynamic effects in our present study. The dose was sufficient to potentiate the pressor response to exogenous administration of NE and to exaggerate the increase in myocardial interstitial NE during NE infusion. The prolonged elevation of myocardial interstitial NE after the discontinuation of NE infusion in the desipramine-treated animals was also consistent with the pharmacological inhibition of NE uptake by desipramine in the heart. In a prior study of open-chest anesthetized dogs, Levy and Blattberg (38) reported that desipramine potentated and prolonged the chronotropic, pressor, and inotropic responses of the animals to exogenous NE. Desipramine also inhibited the extraction of exogenous NE in the heart, which is consistent with the greater and more prolonged elevations of interstitial NE after desipramine treatment in our study. Our microdialysis study also showed that NE infusion produced a much greater increase in myocardial interstitial NE in CHF placebo animals than in the sham-operated placebo group. These findings are consistent with the reduced myocardial NE uptake activity known to exist in the failing heart. However, unlike the exaggeration of interstitial NE, the magnitude of pressor response to NE infusion was smaller in CHF animals compared with the sham-operated control. The latter phenomenon, which has been described previously (39, 41), probably was related to the reduced responsiveness of vascular postjunctional adrenoceptors to NE that is known to exist in heart failure (19).
The microdialysis method has been used to demonstrate the cardiac release of NE after coronary ischemia-repurfusion (36, 47, 50), NE administration (48), and cardiac sympathetic nerve stimulation (46). The earlier studies have also shown that cardiac sympathetic stimulation could raise interstitial NE to a level high enough to generate oxygen free radicals. In these studies, salicylate was administered to the dialysis perfusate and reacts with the interstitial hydroxyl free radical to form ·OH adducts, such as 2,3-DHBA, which was detected in the dialysate and used as an index of interstitial ·OH radicals (45). A positive correlation has been shown between the release of NE and the formation of ·OH in the heart (49). Likewise, our present study demonstrates a temporal correlation between the amounts of interstitial NE and 2,3-DHBA in the heart during NE infusion.
Our present study demonstrates that CHF is associated with increased myocardial interstitial ·OH. An increase in cardiac ·OH has been described by Ide et al. (26) in dogs with pacing-induced heart failure with the use of electron resonance spectroscopy. In the latter study, the authors also found that the increase of ·OH in freeze-clamp failing heart myocardium was abolished by desferrioxamine (an iron chelater), Tiron (a nonenzymatic O2−· scavenger), and catalase. The findings suggest that O2−· is produced and converted into H2O2 and further to ·OH via the iron-catalyzed Harber-Weiss reaction and Fenton reaction in the failing heart. Ide et al. (27) have also shown that O2−· is probably produced in the failing myocardium by a functional block of electron transport at mitochondrial electron transport complex I. In addition, O2−· may react with nitric oxide, which is increased in the failing myocardium as a result of increased expression and activity of the inducible nitric oxide synthase (60), to form another strong oxidant mediator peroxynitrite (24); the latter also has been shown to cause cardiac and vascular endothelial dysfunction (8, 22, 51, 55). The functional importance of the reactive oxygen and nitrogen species in heart failure is a topic of intense current research and beyond the scope of our present investigation.
Desipramine at the dose employed produced no significant hemodynamic effects in our studies but increased myocardial interstitial NE in sham-operated and CHF animals. Desipramine treatment has also been shown to impair NE clearance and increased plasma NE in patients with CHF (10, 38). The hemodynamic measurements in our studies, however, should be interpreted with caution because of the use of anesthesia that could have modified animals' responses. In addition, desipramine has been shown to have a sympathoinhibitory effect via stimulation of presynaptic α2-receptors (18). Levy and Blattberg (38) reported that acute administration of desipramine diminished the release of NE from the cardiac nerve endings after stimulation of the left ansa subclavia. On the other hand, after prolonged exposure, desipramine desensitized the central and peripheral α2-adrenoceptors, causing a decrease of sympathoinhibition and an increase of NE release (13). Thus the effects of desipramine on hemodynamics and plasma NE may vary depending on the experimental condition and the duration of its administration. The higher interstitial NE in the desipramine-treated CHF animals could have exerted a greater inotropic effect and accounted for the greater average left ventricular FS compared with the CHF placebo animals. However, the differences between the two groups were not statistically significant. Furthermore, because desipramine promoted earlier dilation of the left ventricle (Fig. 1), NE uptake inhibitors are ultimately detrimental in the failing heart despite the potential beneficial effects on the sympathetic nerve endings and FS.
Immunocytochemistry demonstrated that desipramine reduced endogenous ceramide in PC12 cells, indicating that, at the dose employed, desipramine inhibited sphingomyelinase activity and ceramide production. On the other hand, the administration of 100 μM NE did not increase ceramide. The findings suggest that at doses ≤100 μM, the increases in reactive oxygen species and cellular oxidative stress produced by NE (44) are not caused by the formation of ceramide. This is consistent with our results in intact animals that desipramine did not reduce cardiac interstitial ·OH radicals or myocardial oxidative stress in CHF animals. However, a possibility that the apoptotic effects of higher doses of NE are mediated via an action of ceramide exists and warrants further evaluation.
Our present study provides no molecular mechanism to explain the reduction of tyrosine hydroxylase produced by the oxidative metabolites of NE, but several plausible mechanisms have been postulated. Kuhn et al. (34) reported that tyrosine hydroxylase activity was reduced by various catechol-quinones by converting tyrosine hydroxylase to a redox-cycling quinoprotein. This mechanism of action is probably not important in the reduction of tyrosine hydroxylase produced by NE in our study, because unlike our present study, the catechol-quinone-mediated inactivation of tyrosine hydroxylase, which is sensitive to reducing agents, is not inhibited by antioxidant enzymes such as superoxide dismutase or catalase (34). Tyrosine hydroxylase has also been shown to be inhibited by a mechanism involving peroxynitrite-induced tyrosine nitration (2, 28) or sulfhydryl oxidation of the enzyme (6, 33). Borges et al. (6) showed that peroxynitrite-induced inactivation of tyrosine hydroxylase is associated with dithiothreitol-reversible S-glutathionylation of the enzyme. Studies (32) have also shown that the peroxynitrite-induced inhibition of tyrosine hydroxylase can be reversed by reduced forms of nicotinamide nucleotides. These studies suggest that the reduction of tyrosine hydroxylase during oxidative stress is caused primarily by S-glutathionylation, whereas tyrosine nitration of the enzyme occurs relatively late when extensive depletion of endogenous catecholamines have occurred (32). In a recent study, we observed that NE reduced NE uptake transporter receptor in PC12 cells by endoplasmic reticulum stress (43). Because endoplasmic reticulum stress may also affect biosynthesis of tyrosine hydroxylase, studies are needed to explore the relationships between the increased endoplasmic reticulum stress produced by NE and tyrosine hydroxylase biosynthesis.
In summary, our present study showed that the reductions of cardiac sympathetic neurotransmitters in CHF are associated with elevated interstitial NE and ·OH. Desipramine treatment blocked NE uptake and increased interstitial NE but attenuated the reduction of myocardial tyrosine hydroxylase in CHF. These results suggest the neurotoxic effect of NE or its oxidative metabolites depend on the normal NE reuptake mechanism of the sympathetic nerve endings. However, despite the potential neuroprotective of desipramine, the medication probably has limited therapeutic value because it increased myocardial interstitial NE and could potentially exaggerate the progressive cardiac remodeling in CHF.
The study was supported in part by National Heart, Lung, and Blood Institute Grant HL-68151.
The authors gratefully acknowledge the excellent technical support provided by Janice Gerloff, Megan Simeone, and Robin Stuart-Buttles.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2006 by the American Physiological Society


text:
February 4, 2004
Is Colin Farrell mellowing his bad boy ways? Though he's been in Bangkok since the beginning of January filming the new Oliver Stone drama Alexander (in which he plays the title role), so far he hasn't taken in any of the raunchy nightlife in the city's infamous red-light district. "We have been working 19-hour days, so I haven't had time to see much," he told us during last weekend's Bangkok Film Festival, adding: "I can't wait to get back to Los Angeles to see my son." What happened to everyone's favorite party boy?
Actress Michelle Rodriguez may have found fame starring as a boxer in 2000's Girlfight, but lately she has been giving her girly side its due. While in Thailand for the Bangkok Film Festival, she had a purple spaghetti-strap dress custom-made for herself. Why the change? "I am kind of primming it up right now so that I don't only attract muscle-heads and complete geeks," she told PEOPLE.com. "I do not want to retire with 20 or 30 cats. I think eventually after 27 or 30 – the sexual peak – I would like to have a husband and have kids."
Ah, the life of a hip-hop star. Producer-performer Pharrell was in London recently promoting the new N.E.R.D. album (due in March) when he decided to hop over to Paris to watch his friend, model Karolina Kurkova, walk the runway in the Versace Couture show. Then he high-tailed it back to New York for a Paper magazine/Svedka Vodka party at nightclub APT in his honor. By the time he got to the party, all he wanted was some peace … and a cheeseburger. Unfortunately, the kitchen was closed – even for him – so he had to survive on vodka alone. Also at the party: Gina Gershon, Sopranos star Drea De Matteo and the now-grown brothers of the teen rock group Hanson: Taylor, Zach and Isaac.
New York's Chelsea Piers sports facility has been a welcome reprieve for celebrities with kids ever since below-freezing temperatures hammered the city. Hugh Jackman, in town to star in the Broadway musical The Boy from Oz, took his young son, Oscar, for a romp in the indoor toddler gym there. Director Spike Lee was also spotted leaving Chelsea Piers with his son, Jackson. Father and son, dressed in similar fur-collared parkas, braved the cold for about five minutes in the taxi queue before a cab finally arrived.
Reported by: SEAN DALY, MARK DAGOSTINO and JOANNE FOWLER

subset: commoncrawl


text:
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Really interesting medieval architecture. I looked it up online as I had never heard of it be fore and it has an interesting history as well. Thanks for sharing the picture with us.


text:
Photographer's Note
Here we are going up one of the needles,(peaks) of cerro Catedral in Bariloche. As I already had described, we were camped next to I take refuge it that I showed in the photo of yesterday, that can be seen in the superior part of the Toncek lake. It was the beginning of the summer, but still it had much snow, it blew very and he was cold, the ascent, however it is not difficult, the rocks has many points where to support the hands.
Aqui estamos subindo uma das agulhas, (picos) do cerro Catedral em Bariloche. Como eu já havia descrito, estávamos acampados próximo ao refugio que eu exibi na foto de ontem, que pode ser visto na parte superior do lago Toncek.
Era o inicio do verão, mas ainda havia muita neve, ventava muito e fazia bastante frio, a subida, porém não é difícil, as pedras tem muitos pontos onde apoiar as mãos.
Thanks for visits.
Critiques | Translate
TeresaT (10806) 2006-04-24 14:56
Olá antonio,
WOW vertiginosa esta imagem.
Muito bem captada, num POV perfeito.
Gosto principalmente da sensação de altura que conseguuiste transmitir através do angulo escolhido.
Os restos de neve na montanha tambem dão um "toque" interessante à imagem.
fica bem.
um abraço
Wahclellaspirit (4125) 2006-04-24 15:17
Hello Antonio,
Rough terrain to climb, but what a place to do it at. Good DoF and exposure. Looking at the brown rock, is it Volcanic?
Olhando a rocha marrom, e vulcanico?
Excellent view down to the valley floor and across...
tcht (7565) 2006-04-24 15:27
Olá Antonio,
Tiny person, massive landscape. Incredible sky and perfect depth. Texture of stones & colours r good too. Also great sharpness. You've got everything what a landscape pic should have! Well done!!
peter1892 (1681) 2006-04-24 15:53
Hello Antonio - a stunning location, great colurs, sharpness & DOF. The inclusion of the person in this shot is a good idea - it helps to give a bit of scale to the shot, especially to the large rocks in the foreground.
robiuk (10807) 2006-04-24 16:46
Hey Antonio,
what a huge amount of rocks you managed to squeeze in this frame!
Wonderful wild environment and good POV down to the lake and up to the blue sky carrying fluffy clouds.
huahoa (3698) 2006-04-24 16:51
Hi Antonio,
Wonderful shot again, with so many depth, great sharpness too.
Beautiful sky with white clouds, i always like the presence of somebody in a landscape, this gives us the good idea of the nature size.
Thanks a lot
Gerrit (52469) 2006-04-24 17:28
Hi Antonio,
a majestic landscape and good grahics with the diagonal stripes of snow crossing the main diagonal.
Thanks and regards, Gerrit
izmirli (3989) 2006-04-24 17:48
merhaba dear antonio
wanderful nature.wanderful mountains and sky.well done. regards.
gürkan akçakır.
lasscor (7640) 2006-04-24 17:52
Hi Antonio,
Wonderful gigantic landscape.
Great colours, light, sharpness and dof.
I like the texture of the rocks in the foreground, the colour of the water in the middle, the clouds and the sky in the background.
singuanti (15250) 2006-04-24 22:22
Hi Antonio. What a huge landscape this is. I get this is the sort of place where no matter what wide-angle you have it will never be enough. That depth goes on and on. Very nice one Antonio!
vagabondtravels (6511) 2006-04-25 0:14
Great colours, light, pov, sharpness and dof. Everything else has been said. Very well done.
rbcy1974 (20758) 2006-04-25 7:24
Hola Antonio
Esta magnifica foto me hace desear haber hecho la subida de este lugar,
Una vista magnifica, el personaje agrega una sensacion de dimension.
nwoehnl (122) 2006-04-25 8:19
Beautiful scenic photo, Antonio. The way in which the rocky landscape details are arranged in the vertical frame gives a good idea about the expanse of land, and a nice sense of perspective and depth. The person in the red jacket is well included for interest and scale. Nice work.
repaulson (607) 2006-04-25 9:45
Antonio, Amazing colors! The vegetation really stands out amongst the harsh terrain. The sky is brilliantly colorful. Nice post! Ross
anesugur (18789) 2006-04-25 16:16
Hi Antonio,
Very good work.
Well seen and composed.
Best regards. Uğur
cjmm (4479) 2006-04-26 10:16
Hola Antonio,
Hoje estou com um pouco de preguiça, entáo... aulas de espanhol meu amigo?
ME gusta mucho el FoV y la gran sensación de profundidad que hay en la imagen, con el lago apareciendo muy pequeño al fondo.
También, el incluir a una persona en la composición ha sido ideal, ya que le da una verdadera sensación de dimensión a las rocas.
Un abrazo grande,
Henryk_Bilor (20803) 2006-04-26 11:08
Ola Antonio
As Teresa wrote - zdjêcie przyprawia o zawrót g³owy... As you force me to read your critiques in Portuguese, I will do the same for you ;).
Anyway, I find Portuguese quite similiar to Polish language...
Back to the image - great perspective, and the rocks. I like the colours of the sky and the lake in the valley.
carper (96) 2006-04-26 11:14
You made a very nice tribute for Fenando as so many members did, Yes Fernando was a very nice and good man, we will remember him.
Wel about the shot I like it, the composition is technical very good the pov is good and the dof is very good too, very good job.
gr. jaap
gilou530 (34) 2006-04-26 12:29
re antonio
une grande photo encore d'un endroit grandiose, avec une vue a couper le souffle, bravo pour la grimpette
Albrecht (11283) 2006-04-26 14:12
Hi Antoniop
What an impressive sight;great POV of course and wonderful scene.
>take care, Albrecht<
saguzar (862) 2006-04-26 18:37
Hola Antonio,
otra espectacular foto. magnífico punto de vista, al igual que la nitidez, composición, la luz y los colores. siempre nos maravillas con tus fotos!
Un abrazo, amigo.
riclopes (35577) 2006-04-28 4:34
Espectáculo António!
É pena o frio e o vento porque a paisagem é super atractiva e eu adoraria estar aí. Fabulosa vista, tirada num ângulo fantástico. A vista daquelas nuvens até ao horizonte ainda torna a foto mais impressionante. Parabéns!
Uma abraço amigo
ChristianS (2997) 2006-04-28 11:14
Hi Antonio,
Impressive landscape shot from and equally impressive perspective overlooking the valley and the lake below. the sky is also very nice. Well done!
bend1k (934) 2006-05-13 6:22
Hi Antonio, great photo. very nice landscape. i like the way you included the person at the photo. go on climbing :)
quantabeh (5205) 2006-05-17 1:40
hi Antonio... a superb shot. the scenery is really breathtaking. The colors are exposed superbly. the composition is rich and beautiful. nice shot!!!
aralda (1240) 2006-06-22 11:44
Hi Antonio,
Nice idea to post a photo from this hike. It looks like great exercise. Good to see a person in this rocky shot.
Hellas (7035) 2007-09-17 23:27
Hello Antonio,
this is a wonderful landscape captured from a very good point of view. I like very much the vivid colours as well as the perspective. Thanks for sharing.
My warmest wishes,
Silversnow (2319) 2008-04-06 6:37
Absolutely stunning picture, the Box composition really defines the massive mountain and emptiness of distance. The fellow climber adds a sense of awe-inspiring scale, and the colors are strong yet natural.
Photo Information
- Copyright: Antonio Negrao (aznegrao) (10316)
- Genre: Places
- Medium: Color
- Date Taken: 2005-12-29
- Categories: Nature, Decisive Moment
- Photo Version: Original Version
- Theme(s): My mountains adventures. [view contributor(s)]
- Date Submitted: 2006-04-24 14:24
- Favorites: 1 [view]
- To Wahclellaspirit: Volcanic (1)
by aznegrao, last updated 2006-04-24 03:58


text:
" Author: Robert Rankin / Genre: Fiction "
* Prices may differ from that shown
Imagine we have been lied to and that all the fantasy fiction of the Victorian era was in fact true. That 19th Century England was in fact full of airships and automata, where Oscar Wilde was a notorious ladies' man and Queen Victoria was the Empire Bike.
Airships and automata are familiar enough to any reader of alternative histories. They crop up in anything where Rome didn't fall or the Nazis weren't defeated. To combine this kind of earnest but far-fetched academic speculation with outrageous humour should be a sure-fire hit.
And I suspect it would be if it was written by anyone other than Robert Rankin.
A man who must surely only have got into print by virtue of people getting him confused with the man who created Rebus, Rankin is the slow-thinking man's Terry Pratchett. The author of a string of 'comedy' novels set around the London borough of Brentford, these books lure you in with admittedly brilliant punning titles such as '101 Damnations' and things.
The contents are generally (I've read three or four over the years, hopefully I've learned my lesson now) meandering tales full of one-note characters, interminable and unfunny authorial digressions and euphemisms for the male member. I have never read anything aimed more solidly at the physics undergraduate.
In this particular tale of not-very-much, a young chap called Will Starling goes on a time-travelling voyage of discovery after he discovers a wristwatch on a painting in the Tate. I was just about giving this wandering narrative the benefit of the doubt when 'Barry the Time Sprout' was introduced. Starling wanders around avoiding his destiny, which is apparently to defeat the evil cabal of witches known as the Chiswick Townswoman's Guild. Along the way, he enjoys an orgy with the Elephant Man, steals Mary Poppins's clothes and solves the case of Jack the Ripper, all of which sounds funnier than it is.
Isaac Asimov once observed that comedy is the hardest style of all, because while a love story can be moderately romantic, and a tragedy can be almost upsetting while retaining some dramatic credibility, a joke which is 'nearly funny' is still a failed joke, whichever way you look at it.
Any page of this novel will yield a good half-dozen illustrations of why this is such a good point. Rankin fires gag after gag at the reader in the hope that some will stick. Tastes vary, but I was tickled roughly once every five pages.
Rankin's status as the Vauxhall Conference Pratchett is further confirmed by his use of footnotes to deliver punchlines and further authorial intrusions. These are rarely worth even reading, and certainly never up to the standard of the asides in the Discworld novels.
Having realised that the humour was duff, I then turned my attention to the storyline. Big mistake. At one point one of the characters even makes a joke about writers making things up as they go along. Astonishingly, this seems to be how the book was written. Twists in the plot occur almost at random before being dropped a couple of pages later. Key characters are introduced far too late in the story. Other key characters seem to be killed off and resurrected with cartoon-like implausibility. This is an untidy, rambling story, and deliberately so.
The idea of making up a novel as you go along is an interesting one, but frankly a lot of people do it and it's one of the main reasons there are so many unpublished authors out there. Robert Rankin is not a Beat Poet and he's not James Joyce, his wordsmithery is simply inadequate to construct a comedy fantasy novel out of whatever comes into his head at any given moment.
To be honest, in assuming he was making it up as he went along, I'm giving him the benefit of the doubt.
There are some patches of quality, obviously, or I'd never have lasted through the whole book, even if I was bored on a train. The promiscuous Elephant Man is crude but engaging (right up until we learn he's an alien hybrid from Mars), the fashionably skint Hugo Rune is an endearing old rogue (right up until he wanders into the final scene having been absent for most of the book and yet still does bog all), and the invisible H.G. Wells makes for a briefly amusing take on that novel.
But the whole thing is just such a MESS that it's impossible to recommend it to anyone with any conscience. I paid 20p for the book at a car boot sale and I'm still feeling cheated. The cover price on my copy is £6.99, and I imagine it's at least a quid more than that these days.
Perhaps if you'd just had surgery and needed something to read while you recovered in hospital but didn't want to have to concentrate too hard, then this might be the ideal book to read.
It's the only way you'll be in stitches while reading it.
Robert Rankin's fondness for demented conspiracy theories is complicated by time travel in The Witches of Chiswick--which demonstrates again that everything you know is wrong, that Brentford is the true centre of the multiverse, and that nobody is quite as weird as Robert Rankin. Will Starling lives in a dystopian 23rd century where Brentford Utility Conurbation is crammed with 303-storey tower blocks and synthetic food has made everyone vastly obese. Except for Will, who's mocked for morbid slimness and eccentric tastes--art, for example. When he notices the digital watch in a well-known Victorian painting, a murderous cover-up begins. The sinister Witches of Chiswick are determined to erase all traces of the other past. Time-travelling Terminator-style automata keep arriving, not from the future but from that lost Victorian age of Babbage supercomputers, flying cabs running on beamed power from Tesla transmitters and the imminent launch of Her Majesty's Moonship Victoria. Thanks to the convenient time machine of a Mr Wells, Will finds himself in that other 19th century, complicating the stories of his own ancestors. There he's tutored by the flamboyant guru or conman Hugo Rune. He stands in for Sherlock Holmes--called away to a Dartmoor case--and investigates the Jack-the-Ripper murders. As tends to happen in the Rankin universe, he acquires a Holy Guardian Sprout called Barry. Will even meets himself, another Will from a very different future. Even aided by his best friend Tim, by the Brentford Snail Boy (raised like Tarzan by wild animals, not apes but snails), and by the deadly martial art Dimac, can Will hope to foil a witchy plan to reprogram time and send high-tech Britain back to gaslight as midnight strikes on December 31, 1899?


text:
Viking Speedway: Hall of famers
By Larry Holverson
Contributing writer
Ron Pease and DuWayne Nelson were inducted into the Viking Speedway Hall of Fame for 2011 on Saturday, August 13.
Both men have played integral parts in the success enjoyed during the 46-year existence of the local oval.
Nelson served as a crewmember for the late Wayne Kluver and was the track maintenance director, earning the reputation as one of the best blade operators in the area if not the state.
Nelson actually got involved in auto racing in 1967 when Kluver approached him, asking if he would be interested in being partners with him in building a race car.
That was the beginning of the hard charging and popular No. 9.
"We were blessed in having several outstanding pit crew members," pointed out Nelson.
They included Jim Novak, Chuck Spaulding, Don Domine, Tom Blade and Gary Vickerman.
Nelson recalled that Kluver was a very easy-going person to work with and enjoyed the success the team had.
During that time span, Kluver and his gang brought home one season championship and recorded three top five finishes.
At that point in time, Nelson dropped out of the partnership to spend more time on work and family obligations, which marked the end of his affiliation with racing.
Kluver was inducted into the Viking Speedway Hall of Fame in 2006.
Nelson returned to racing in 1980 when Jerry Chlian and Les Tschida approached him to blade and maintain the track.
Nelson enjoyed molding the big half-mile, oftentimes with less than desirable material, for the next 20 years.
He spent countless hours working on the track each week.
During that time span, Nelson served two terms on the board of directors.
His wife Barb also served as track secretary during that time and assisted Nelson in maintaining the track.
Nelson is also involved in the Runestone Raceway go-kart oval here in Alexandria.
He takes care of the track and is also a board member, a post he will relinquish following the 2011 campaign.
Pease competed at the Viking Speedway for six seasons and was also on the board of directors for nine years.
He was on the board beginning in the 1973 season and served as president of the local oval the last three years of his time on the board.
"Looking back on my years as president of the speedway, the most important change I tried to make was that of attitude," pointed out Pease.
"I knew we had to treat everyone involved better, starting with the drivers and the car owners.
"I told the board of directors I wanted our racers to be treated like company in our own homes. I put on a smile and did my best to greet everyone I came in contact with and it was amazing, everything changed," Pease recalled.
He also pointed out that the guaranteed purse helped a lot, but mostly everyone got encouraged about racing again and we all had a winning attitude," Pease noted.
"I surrounded myself with the best people I could find. The Viking Speedway's success was obtained by a lot of hard work, by a lot of folks, not just one individual."
His best season behind the wheel of his Chevrolet product was in 1974 when he recorded 31 wins while competing at the Viking Speedway, Sauk Centre and Herman.
During that season he recorded seven clean sweeps.
"We got booed a lot, it was fun," recalled Pease.
"I wouldn't trade this time for anything," pointed out Pease.
"I thank the Viking Speedway for the opportunity to serve and compete."
Pease now resides in a small town of 30,000 just south of Nashville named LaVergne, Tennessee.
He and his wife Anne moved to that community in 2002 and Pease points out they enjoy small town living.
Pease is retired but has a part-time job working three days a week at O'Reilly Auto Parts.
He is no longer involved in racing other than being an avid fan.
A total of 22 individuals are now enshrined in the Viking Speedway Hall of Fame.
2001 - Jerry Geris, Frankie Herzog.
2002 - Dean Peterson, Butch Peterson.
2003 - Jim Johnson, Florian Sprinzel.
2004 - Dave Gilbertson, Walt Anderson.
2005 - Roy Snyder, Dick Block.
2006 - Wayne Kluver, Larry Holverson.
2007 - Oliver Buse, Don Domine.
2008 - Bill Peterson, Jerry Kluver.
2009 - Jim Nieland, Jerry VanKempen.
2010 - Greg Muzik, Stu Olson.
2011 - Duwayne Nelson, Ron Pease


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Barbra Streisand stars as struggling singer becomes a star but loses the man she loves--who will not allow himself to hinder her rise to fame once A Star is Born.
In this film based on the Academy Award-winning 1937 and 1955 films, the career of hopeful, young singer Esther Hoffman (Streisand) is launched by fading rock star John Norman Howard (Kris Kristofferson), who also wins the young singer's heart. Hoffman becomes a leading lady on stage and in Howard's life, but as Howard's career flounders, he sinks into an abyss of alcohol and cocaine. Hoffman chooses to sacrifice her stardom to care for Howard, but he will not allow her to abandon her dreams.
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"We but half express ourselves, and are ashamed of that divine idea which each of us represents" ~Ralph Waldo Emerson
"The will to do springs from the knowledge that we can do. Doubt and fear are the great enemies of knowledge, and he who encourages them, who does not slay them, thwarts himself at every step." ~James Allen
In the last article, I reflected on how Erik Erikson's First Stage of Personality Development, Trust vs. Mistrust, could be looked at from a spiritual perspective in addition to a psycho-social perspective. This issue we will look at stage two: Autonomy vs. Shame and Doubt and its corresponding virtue, Will.
According to Erickson's Theory of personality, the Autonomy vs. Shame and Doubt Stage is developed between 18 months and 3 years of age. This occurs when a child begins to assert their independence by distancing themselves from their mother in order to make choices about which toy they want to play with or what they'd like to eat, etc. If the child is supported and encouraged in their increased independence they build confidence and therefore feel safe to survive on their own in the world. When criticized, controlled or not given the opportunity for independence for whatever reason, they feel inadequate and therefore fearful that they cannot survive on their own in the world. This can result in an overly dependent person who lacks self-esteem and whose life is colored through the lens of shame and doubt. The question at this stage is: "Can I do things myself or must I always rely on others?"
Wanting to explore the world with curiosity and excitement is a natural human need. This stage creates the foundation for how we do so in subsequent stages and therefore, throughout our life. Many factors can impact a parent's ability to lend healthy support during this stage. A fearful, controlling, or criticizing parent may unknowingly pass on extreme feelings of doubt and inadequacy in their child in an attempt to protect them. A neglectful, unavailable or absent parent contributes to feelings of inadequacy by their perceived disinterest.
When I was age three, my mother attempted suicide. This, of course, was a significant and defining trauma in my young life. However, imagine the state of my mother up to that point and the impact that also had on me. This is where the basis of my shame, my life lesson, was formed. I was not a wanted pregnancy, and therefore, my mother treated me as an overwhelming burden more than a welcome surprise. She was also prone to depression due to the abuse she too experienced as a child.
Prior to this defining event in my life, my mother was often nervous, depressed, unhappy, intoxicated and angry. I had to walk on eggshells and felt more "in the way" than I felt safe. I internalized my mother's problems and assumed her inability to handle the world was how the world was and thus my perceived inability to handle the world competently developed during this stage.
Loss of Freedom:
• Freedom to think and evaluate • Freedom to feel and have compassion and passion • Freedom of choice • Freedom to imagine • Freedom to give and receive • Freedom to heal yourself and others • Freedom to ask for help
From a spiritual perspective, toxic shame can be an opportunity to seek spiritual guidance and learn to ask Spirit for help. The fear of being unable to survive on one's own can catapult one on a quest for meaning, identity and spiritual connection as a way to alleviate the feelings of inadequacy. Therefore, a sense of WILL is the virtue born from this state. Willpower or lack thereof defines our sense and strength of self through the eyes of our ego.
Although spirituality often has a goal of leaving our ego behind, our ego is necessary and therefore, having a healthy, strong ego can lead to action-oriented choices in life that can be to our benefit. Ego and Will are not the same. Without a strong will our ego will attempt to "steal the show" making choices that further disconnect us from Spirit but garner superficial and instant gratification. With a healthy ego, we actually have a better chance of connecting with Spirit and remembering the divine being we are.
Although these early experiences created a shame-based foundation I perceived my reality through, I am very clear that this was part of Spirit's Divine plan for me. Being able to reach the virtue of Will through my experiences was my lesson as well as the intense experience of shame. Since I'm happy with my life as it currently is, I can't really express discontent at my early experiences. In fact, I have arrived at forgiveness and gratitude for them.
Further, I realize part of why I experienced them in such a way and got past them was through my own sense of Will combined with Spirit's help and undoubtedly so I could be a living example for those struggling with the same issues. As Gandhi said, " My life is my teaching." and I intend to teach others through my life's experiences so they can overcome any perceived obstacles, exercise their will and let their Divine Light shine in the world as we were all meant to do.
The beauty of Erickson's theory is his belief that whether you experience the negative or positive extremes of each developmental stage we always find our way to the inherent virtue. I believe his spirit channelled through him this universal knowledge, suggesting that in reality, it's impossible to "fall off" your spiritual path, even if you take different roads to get there and have varied experiences. If we arrive at the development of each virtue regardless of the experiences we've had, then we can always trust that Spirit is guiding us and therefore THY WILL, in addition to our own Will through free choice, will be done.
Shame is a spiritual affliction at its core, one where John Bradshaw, author of Healing the Shame that Binds You termed "spiritual bankruptcy". Therefore shame can only be truly healed through spirituality and our connection to source. It may be minimized with a temporary band-aid through psychological tools such as psychotherapy and medication, but a true healing cannot occur without divine intervention.
Toxic shame inhibits us from sharing who we are with others. When we are ashamed of who we are and think we are bad, we also hide our soul's essence, talents and gifts in an attempt to protect ourselves from further shaming. Even those closest to us may not get a glimpse of our fullest potential.
Give yourself back the freedoms you lost.
Find a place where you won't be interrupted. Get comfortable whether lying down or sitting up. Rid yourself of any distractions such as uncomfortable clothing or jewelry, outside noises, hard chairs, tickly blankets, etc.
Get your body ready to meditate by taking five long, deep breaths.
Go to your safe place in your mind's eye. Some place in nature that is just yours and completely safe. Call on any Spirit Helper you wish to be present. I always invite my Higher Self. You may call on deceased loved ones, angels, ascended masters or guides as well. Get acquainted through a greeting of your choice: a bow, a handshake, a hug, etc.
Your Spirit Helper will guide you to an altar with some items present. There will be a fire, a pot, some herbs, and some significant items such as crystals, feathers etc. Don't make them up, wait for your Spirit Helper to show you what they are. These will be clues to the guidance you are getting. Once all the items have been gathered and identified, the two of you (or more) will begin working on creating a potion that restores all of your lost freedoms. Each item gifted to you will represent one of your lost freedoms. The item itself or the energy of each item will be infused into the potion you are brewing. Your Spirit Helper will determine the appropriate method for each. Let them show you and trust the guidance you receive. Each of you will bless and/or say a prayer over the boiling brew. A healing potion will be the final product.
Your spirit helper will pour the brew into a container and present it to you for drinking. You will drink the potion with the knowledge that your lost freedoms, due to shame, will be restored to you by drinking this "magic" potion. Place the container back on the altar and thank your Spirit Helper. As your Spirit Helper walks away, you will slowly come back into consciousness.
• Rehearse the future with these freedoms present through meditation, daydreaming, storytelling or writing.
• Develop Remorse. Forgive yourself and others for making mistakes in life.
• Ask for help from your Higher Self, Guides, Angels or Ascended Masters on a daily basis to lift your shame and continue the restoration of your freedoms.
• Create an altar that includes the items you received in your meditation which represent your lost freedoms. Seeing these items and praying intentionally over them often will help invoke Spirit's help as well as re-train your sub-conscious.
About The Author: Jesseca Camacho is a writer, teacher, spiritual counselor, wife and mother. She is a graduate of the Clearsight Clairvoyant Program, completed a 2-year channeling program with world renowned channeling teacher, Shawn Randall and received her Massage, Reflexology, Energetic Medicine and Reiki I & II Certification through the Institute of Psycho-Structural Balancing. Jesseca has a B.A. in Early Childhood Education and completed the Parenting from the Heart Program. In addition, she incorporates the ancient systems of numerology and enneagram into her spiritual counseling sessions to facilitate awareness and healing on a soul level. She is also the co-author of the Children's Spirit Animal Cards with Dr. Steven Farmer.


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A plan viewed as a source of much-needed flood relief in some areas of Augusta and an unnecessary government fee elsewhere goes before the city commission Tuesday for further approval.
The commission, which approved an initial phase of implementing the stormwater fee last year, will vote on spending $484,453 in Sales Tax 6 funds with consultant AMEC Inc., to advance the fee program, including the development of a rate structure, billing system and public awareness campaign.
The year's record rainfalls have served as a frequent reminder of inadequate stormwater drainage systems in the urban District 1 represented by Commissioner Bill Fennoy, a proponent of the fee.
"We have a lot of flooding that goes on in Augusta," he said. "Any time there's a real big rain we have some communities that have multiple water breaks and they don't have any drinking water, they don't have any bathing water."
While other areas of the city may not experience as severe of flooding, Fennoy said he hoped all would buy into the program and pay the fee, as many pay school taxes even without having children in Richmond County schools.
"I think it's fair for everybody to participate in it," he said.
On another side of the stormwater debate is Commissioner Wayne Guilfoyle, who likened his mostly rural District 8 to rural parts of Columbia County, which lack storm drainage infrastructure and were exempted when the suburban county implemented a stormwater management program in 2000.
"Columbia County focused on the urban area," Guilfoyle said. "It's the only fair and equitable way."
Columbia County exempts no one within the district from the fee, including residential, commercial, nonprofit and even governmental users, said Gary Bennett, stormwater utility department manager. The county even charges the fee against its own buildings.
Commissioner Bill Lockett agreed with the plan and Guilfoyle's desire to opt out areas lacking stormwater systems beyond unmaintained ditches.
"I think Commissioner Guilfoyle has a brilliant idea about us treating parts of his district somewhat differently because they are primarily rural areas," Lockett said.
Pushed as necessary under federal stormwater mandates and a way to help avoid raising taxes, Engineering Director Abie Ladson estimated the fee of a few dollars a month for a typical household will generate up to $10 million annually to maintain the city's stormwater infrastructure.