Characterizing the metabolic capacity of the anoxic hagfish heart.

Pacific hagfish, Eptatretus stoutii, can recover from 36 h of anoxia at 10°C. Such anoxia tolerance demands the mobilization of anaerobic fuels and the removal of metabolic wastes--processes that require a functional heart. The purpose of this study was to measure the metabolic response of the excised, cannulated hagfish heart to anoxia using direct calorimetry. These experiments were coupled with measurements of cardiac pH and metabolite concentrations, at multiple time points, to monitor acid-base balance and anaerobic ATP production. We also exposed hagfish to anoxia to compare the in vitro responses of the excised hearts with the in vivo responses. The calorimetry results revealed a significant reduction in the rate of metabolic heat production over the first hour of anoxia exposure, and a recovery over the subsequent 6 h. This response is likely attributable to a rapid anoxia-induced depression of aerobic ATP-production pathways followed by an upregulation of anaerobic ATP-production pathways such that the ATP production rate was restored to that measured in normoxia. Glycogen-depletion measurements suggest that metabolic processes were initially supported by glycolysis but that an alternative fuel source was used to support the sustained rates of ATP production. The maintenance of intracellular pH during anoxia indicates a remarkable ability of the myocytes to buffer/regulate protons and thus protect cardiac function. Altogether, these results illustrate that the low metabolic demand of the hagfish heart allows for near-routine levels of cardiac metabolism to be supported anaerobically. This is probably a significant contributor to the hagfish's exceptional anoxia tolerance.

Increased brain serotonin turnover in panic disorder patients in the absence of a panic attack: reduction by a selective serotonin reuptake inhibitor.

Since the brain neurotransmitter changes characterising panic disorder remain uncertain, we quantified brain noradrenaline and serotonin turnover in patients with panic disorder, in the absence of a panic attack. Thirty-four untreated patients with panic disorder and 24 matched healthy volunteers were studied. A novel method utilising internal jugular venous sampling, with thermodilution measurement of jugular blood flow, was used to directly quantify brain monoamine turnover, by measuring the overflow of noradrenaline and serotonin metabolites from the brain. Radiographic depiction of brain venous sinuses allowed differential venous sampling from cortical and subcortical regions. The relation of brain serotonin turnover to serotonin transporter genotype and panic disorder severity were evaluated, and the influence of an SSRI drug, citalopram, on serotonin turnover investigated. Brain noradrenaline turnover in panic disorder patients was similar to that in healthy subjects. In contrast, brain serotonin turnover, estimated from jugular venous overflow of the metabolite, 5-hydroxyindole acetic acid, was increased approximately 4-fold in subcortical brain regions and in the cerebral cortex (P < 0.01). Serotonin turnover was highest in patients with the most severe disease, was unrelated to serotonin transporter genotype, and was reduced by citalopram (P < 0.01). Normal brain noradrenaline turnover in panic disorder patients argues against primary importance of the locus coeruleus in this condition. The marked increase in serotonin turnover, in the absence of a panic attack, possibly represents an important underlying neurotransmitter substrate for the disorder, although this point remains uncertain. Support for this interpretation comes from the direct relationship which existed between serotonin turnover and illness severity, and the finding that SSRI administration reduced serotonin turnover. Serotonin transporter genotyping suggested that increased whole brain serotonin turnover most likely derived not from impaired serotonin reuptake, but from increased firing in serotonergic midbrain raphe neurons projecting to both subcortical brain regions and the cerebral cortex.

Tinnitus Stages of Change Questionnaire: psychometric development and validation.

This study describes the development of the Tinnitus Stages of Change Questionnaire (TSOCQ), which aims to assess the readiness of tinnitus patients to change their behaviors and attitudes in relation to tinnitus. Its ability to predict treatment outcome was also investigated. One hundred and fifty-one patients with tinnitus completed the questionnaire. Through repeated principal component analyses, together with judgments of the factors' theoretical relevance, empirically and clinically meaningful scales were derived. A factor solution in accordance with the transtheoretical model (TTM) and with acceptable psychometric properties was found with the five scales of the TSOCQ labeled Precontemplation (Medical Solution), Precontemplation (Helplessness), Contemplation, Preparation and Action/Maintenance. Participants scoring high on Precontemplation (Helplessness) and low on Action/Maintenance had better treatment outcomes. In conclusion, the TSOCQ had some ability to predict treatment success, but further research is required into the utility of the TTM for understanding the rehabilitation process in tinnitus patients.

The neuronal noradrenaline transporter, anxiety and cardiovascular disease.

Panic disorder can serve as a clinical model for testing whether mental stress can cause heart disease. Potential neural mechanisms of cardiac risk are the sympathetic activation during panic attacks, continuing release of adrenaline as a co-transmitter in the cardiac sympathetic nerves, and impairment of noradrenaline neuronal reuptake, augmenting sympathetic neural respnses. The phenotype of impaired neuronal reuptake of noradrenaline: an epigenetic mechanism? We suspect that this phenotype, in sensitizing people to heart symptom development, is a cause of panic disorder, and by magnifying the sympathetic neural signal in the heart, underlies increased cardiac risk. No loss of function mutations of the coding region of the norepinephrine transporter (NET) are evident, but we do detect hypermethylation of CpG islands in the NET gene promoter region. Chromatin immunoprecipitation methodology demonstrates binding of the inhibitory transcription factor, MeCP2, to promoter region DNA in panic disorder patients. Cardiovascular illnesses co-morbid with panic disorder. Panic disorder commonly coexists with essential hypertension and the postural tachycardia syndrome. In both of these cardiovascular disorders the impaired neuronal noradrenaline reuptake phenotype is also present and, as with panic disorder, is associated with NET gene promoter region DNA hypermethylation. An epigenetic 'co-morbidity' perhaps underlies the clinical concordance. Brain neurotransmitters. Using internal jugular venous sampling, in the absence of a panic attack we find normal norepinephrine turnover, but based on measurements of the overflow of the serotonin metabolite, 5HIAA, a marked increase (six to sevenfold) in brain serotonin turnover in patients with panic disorder. This appears to represent the underlying neurotransmitter substrate for the disorder. Whether this brain serotonergic activation is a prime mover, or consequential on other primary causes of panic disorder, including cardiac sensitization by faulty neuronal noradrenaline reuptake leading to cardiac symptoms and the enhanced vigilance which accompanies them, is unclear at present.

Arsenic exposure alters hepatic arsenic species composition and stress-mediated gene expression in the common killifish (Fundulus heteroclitus).

In the present paper, we examine how arsenic species accumulate in fish liver and explore the hypothesis that sublethal arsenic concentrations in fish hepatic tissue interfere with stress-mediated gene expression. We exposed killifish (Fundulus heteroclitus) to 787 or 0 microg/L arsenic in tank water for 2 weeks. Arsenic exposure elevated total liver arsenic from 3.4 microg/g wet weight (control fish) to 9.6 microg/g wet weight, and resulted in a higher relative proportion of toxic (e.g. monomethylarsenous acid, dimethylarsenous acid, arsenic V) versus benign (arsenobetaine) arsenic species in this tissue. Following the exposure period, arsenic-treated and control fish were then subjected to a stress protocol: confinement and mechanical chasing for 15 min every 3 h. Liver tissue and blood were sampled from fish not exposed to the stressor at time 0, and at 8, 12, 24 and 40 h following the first stressor. Concentrations of the stress hormone cortisol increased significantly, and glucocorticoid receptor mRNA levels increased and then decreased in both groups, but patterns were nearly identical between arsenic pre-treated and arsenic untreated fish. Prior arsenic exposure prevented the stress-induced increases in stress-responsive LDH-B mRNA levels and enzyme activity observed in fish that had not been exposed to arsenic. However, in another stress-responsive gene, PEPCK, arsenic did not interfere with the stress-induced increase in gene expression, suggesting that the effects of arsenic on stress-mediated gene expression are complex and may involve regulatory pathways that differ between these two genes.

Intraspecific divergence of ionoregulatory physiology in the euryhaline teleost Fundulus heteroclitus: possible mechanisms of freshwater adaptation.

We examined intraspecific variation in ionoregulatory physiology within euryhaline killifish, Fundulus heteroclitus, to understand possible mechanisms of freshwater adaptation in fish. Pronounced differences in freshwater tolerance existed between northern (2% mortality) and southern (19% mortality) killifish populations after transfer from brackish water (10 g l(-1)) to freshwater. Differences in Na(+) regulation between each population might partially account for this difference in tolerance, because plasma Na(+) was decreased for a longer period in southern survivors than in northerns. Furthermore, northern fish increased Na(+)/K(+)-ATPase mRNA expression and activity in their gills to a greater extent 1-14 days after transfer than did southerns, which preceded higher whole-body net flux and unidirectional influx of Na(+) at 14 days. All observed differences in Na(+) regulation were small, however, and probably cannot account for the large differences in mortality. Differences in Cl(-) regulation also existed between populations. Plasma Cl(-) was maintained in northern fish, but in southerns, plasma Cl(-) decreased rapidly and remained low for the duration of the experiment. Correspondingly, net Cl(-) loss from southern fish remained high after transfer, while northerns eliminated Cl(-) loss altogether. Elevated Cl(-) loss from southern fish in freshwater was possibly due to a persistence of seawater gill morphology, as paracellular permeability (indicated by extrarenal clearance rate of PEG-4000) and apical crypt density in the gills (detected using scanning electron microscopy) were both higher than in northern fish. These large differences in the regulation of Cl(-) balance probably contributed to the marked differences in mortality after freshwater transfer. Glomerular filtration rate and urination frequency were also lower in southerns. Taken together, these data suggest that northern killifish are better adapted to freshwater environments and that minimizing Cl(-) imbalance appears to be the key physiological difference accounting for their greater freshwater tolerance.

Cardiac sympathetic nerve biology and brain monoamine turnover in panic disorder.

Panic disorder serves as a clinical model for testing whether mental stress can cause heart disease. Our own cardiologic management of panic disorder provides case material of recurrent emergency room attendances with angina and electrocardiogram ischemia, triggered arrhythmias (atrial fibrillation, ventricular fibrillation), and documented coronary artery spasm, in some cases with coronary spasm being complicated by coronary thrombosis. Application of radiotracer catecholamine kinetics and clinical microneurography methodology suggests there is a genetic predisposition to panic disorder that involves faulty neuronal norepinephrine uptake, possibly sensitizing the heart to symptom generation. During panic attacks there are large sympathetic bursts, recorded by clinical microneurography in the muscle sympathetic nerve neurogram, and large increases in cardiac norepinephrine spillover, accompanied by surges of adrenal medullary epinephrine secretion. In other conditions such as heart failure and presumably here also, a high level of sympathetic nervous activation can mediate increased cardiac risk. The sympathetic nerve cotransmitter, neuropeptide Y (NPY), is released from the cardiac sympathetics during panic attacks, an intriguing finding given that NPY can cause coronary artery spasm. There is ongoing, continuous release of epinephrine from the heart in panic sufferers, perhaps attributable to epinephrine loading of cardiac sympathetic nerves by uptake from plasma during panic attacks, or possibly to in situ synthesis of epinephrine through the action of intracardiac phenylethanolamine-N-methytransferase (PNMT) activated by repeated cortisol responses. We have used internal jugular venous sampling and measurement of overflowing lipophilic brain monoamine metabolites to quantify brain norepinephrine and serotonin turnover in untreated patients with panic disorder. We find normal norepinephrine turnover but a marked increase in brain serotonin turnover in patients with panic disorder, in the absence of a panic attack, which presumably represents an underlying neurotransmitter substrate for the condition.

Changes in gene expression in gills of the euryhaline killifish Fundulus heteroclitus after abrupt salinity transfer.

Maintenance of ion balance requires that ionoregulatory epithelia modulate ion flux in response to internal or environmental osmotic challenges. We have explored the basis of this functional plasticity in the gills of the euryhaline killifish Fundulus heteroclitus. The expression patterns of several genes encoding ion transport proteins were quantified after transfer from near-isosmotic brackish water [10 parts/thousand (ppt)] to either freshwater (FW) or seawater (SW). Many changes in response to SW transfer were transient. Increased mRNA expression occurred 1 day after transfer for Na(+)-K(+)-ATPase-alpha(1a) (3-fold), Na(+)-K(+)-2Cl(-)-cotransporter 1 (NKCC1) (3-fold), and glucocorticoid receptor (1.3-fold) and was paralleled by elevated Na(+)-K(+)-ATPase activity (2-fold). The transient increase in NKCC1 mRNA expression was followed by a later 2-fold rise in NKCC protein abundance. In contrast to the other genes studied in the present work, mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel generally remained elevated (2-fold) in SW. No change in protein abundance was detected, however, suggesting posttranscriptional regulation. The responses to FW transfer were quite different from those to SW transfer. In particular, FW transfer increased Na(+)-K(+)-ATPase-alpha(1a) mRNA expression and Na(+)-K(+)-ATPase activity to a greater extent than did SW transfer but had no effect on V-type H(+)-ATPase expression, supporting the current suggestion that killifish gills transport Na(+) via Na(+)/H(+) exchange. These findings demonstrate unique patterns of ion transporter expression in killifish gills after salinity transfer and illustrate important mechanisms of functional plasticity in ion-transporting epithelia.

Palmitate movement across red and white muscle membranes of rainbow trout.

We examined the movement of [3H]palmitate across giant sarcolemmal vesicles prepared from red and white muscle of rainbow trout (Oncorhynchus mykiss). Red and white muscle fatty acid carriers have similar affinities for palmitate (apparent Km = 26 +/- 6 and 33 +/- 8 nM, respectively); however, red muscle has a higher maximal uptake compared with white muscle (Vmax = 476 +/- 41 vs. 229 +/- 23 pmol.mg protein-1.s-1, respectively). Phloretin (250 microM) inhibited palmitate influx in red and white muscle vesicles by approximately 40%, HgCl2 (2.5 mM) inhibited palmitate uptake by 20-30%, and the anion-exchange inhibitor DIDS (250 microM) inhibited palmitate influx in red and white muscle vesicles by approximately 15 and 30%, respectively. Western blot analysis of red and white muscle vesicles did not detect a mammalian-type fatty acid transporter (FAT); however, preincubation of vesicles with sulfo-N-succinimidyloleate, a specific inhibitor of FAT in rats, reduced palmitate uptake in red and white muscle vesicles by approximately 15 and 25%, respectively. A mammalian-type plasma membrane fatty acid-binding protein was identified in trout muscle using Western blotting, but the protein differed in size between red and white muscle. At low concentrations of free palmitate (2.5 nM), addition of high concentrations (111 microM total) of oleate (18:0) caused approximately 50% reduction in palmitate uptake by red and white muscle vesicles, but high concentrations (100 microM) of octanoate (8:0) caused no inhibition of uptake. Five days of aerobic swimming at approximately 2 body lengths/s and 9 days of chronic cortisol elevation in vivo, both of which stimulate lipid metabolism, had no effect on the rate of palmitate movement in red or white muscle vesicles.

Na+/K+-ATPase alpha-isoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer.

We identified five Na+/K+-ATPase alpha-isoforms in rainbow trout and characterized their expression pattern in gills following seawater transfer. Three of these isoforms were closely related to other vertebrate alpha1 isoforms (designated alpha1a, alpha1b and alpha1c), one isoform was closely related to alpha2 isoforms (designated alpha2) and the fifth was closely related to alpha3 isoforms (designated alpha3). Na+/K+-ATPase alpha1c- and alpha3-isoforms were present in all tissues examined, while all others had tissue specific distributions. Four Na+/K+-ATPase alpha-isoforms were expressed in trout gills (alpha1a, alpha1b, alpha1c and alpha3). Na+/K+-ATPase alpha1c- and alpha3-isoforms were expressed at low levels in freshwater trout gills and their expression pattern did not change following transfer to 40% or 80% seawater. Na+/K+-ATPase alpha1a and alpha1b were differentially expressed following seawater transfer. Transfer from freshwater to 40% and 80% seawater decreased gill Na+/K+-ATPase alpha1a mRNA, while transfer from freshwater to 80% seawater caused a transient increase in Na+/K+-ATPase alpha1b mRNA. These changes in isoform distribution were accompanied by an increase in gill Na+/K+-ATPase enzyme activity by 10 days after transfer to 80% seawater, though no significant change occurred following transfer to 40% seawater. Isoform switching in trout gills following salinity transfer suggests that the Na+/K+-ATPase alpha1a- and alpha1b-isoforms play different roles in freshwater and seawater acclimation, and that assays of Na+/K+-ATPase enzyme activity may not provide a complete picture of the role of this protein in seawater transfer.

Treatment of PTSD: stress inoculation training with prolonged exposure compared to EMDR.

The effectiveness of Stress Inoculation Training with Prolonged Exposure (SITPE) was compared to Eye Movement Desensitization and Reprocessing (EMDR). Twenty-four participants who had a diagnosis of Post Traumatic Stress Disorder (PTSD) were randomly assigned to one of the treatment conditions. Participants were also their own wait-list control. Outcome measures included self-report and observer-rated measures of PTSD, and self-report measures of depression. On global PTSD measures, there were no significant differences between the treatments at the end of therapy. However on the subscale measures of the degree of intrusion symptoms, EMDR did significantly better than SITPE. At follow-up EMDR was found to lead to greater gains on all measures.

Substrate utilization during graded aerobic exercise in rainbow trout.

A biochemical approach was employed to examine the oxidative utilization of carbohydrate and lipid in red muscle of rainbow trout (Oncorhynchus mykiss) during sustained swimming at 30 and 60% of their critical swimming speed (U(crit); for 2, 15 and 240 min) and during non-sustainable swimming at 90% U(crit) (for 2, 15 and 45 min). Measurements included pyruvate dehydrogenase (PDH) activity, creatine phosphate, ATP, glycogen, glycolytic intermediates, acetyl-CoA, acetyl-, total-, free-, short-chain fatty acyl- and long-chain fatty acyl- carnitine, intramuscular triacylglycerol and malonyl-CoA concentrations, and whole body oxygen consumption ((O)(2)). During the first 2 min at 30 and 60% U(crit), oxidation of endogenous glycogen by PDH activation increased 4- and 8-fold, respectively, yielding 1.5- to 2.5-fold increases in acetyl-CoA and 2- to 6-fold increases in acetyl-carnitine concentrations. Within 15 min, PDH activity returned to control values (153.9+/-30.1 nmol g(-1) wet tissue min(-1)); after 240 min there were small 1.7- to 2.6-fold increases in long-chain fatty acyl-carnitine and approx. 50% decreases in malonyl-CoA concentrations, indicating an overall enhancement of lipid oxidation. Sustainable swimming at 30 and 60% U(crit) was further characterized by 1.5- and 2.2-fold increases in M(O(2)), respectively. Non-sustainable swimming at 90% U(crit) was characterized by a sustained tenfold (approx.) elevation of red muscle PDH activity (approx. 1600 nmol g(-1) wet tissue min(-1)). Significant 67% decreases in white muscle creatine phosphate and 73% decreases in glycogen levels, without matching increases in lactate levels, point to significant recruitment of white muscle during high-speed swimming for power production, and the potential export of white muscle lactate to red muscle for oxidation. Overall, sustainable exercise at 30 and 60% U(crit) is supported by approximately equal contributions of carbohydrate (approx. 45%) and lipid (approx. 35%) oxidation, whereas non-sustainable swimming is supported primarily by carbohydrate oxidation with only moderate contributions from lipid oxidation.

Glycogen phosphorylase and pyruvate dehydrogenase transformation in white muscle of trout during high-intensity exercise.

We examined the regulation of glycogen phosphorylase (Phos) and pyruvate dehydrogenase (PDH) in white muscle of rainbow trout during a continuous bout of high-intensity exercise that led to exhaustion in 52 s. The first 10 s of exercise were supported by creatine phosphate hydrolysis and glycolytic flux from an elevated glycogenolytic flux and yielded a total ATP turnover of 3.7 micromol x g wet tissue(-1) x s(-1). The high glycolytic flux was achieved by a large transformation of Phos into its active form. Exercise performed from 10 s to exhaustion was at a lower ATP turnover rate (0.5 to 1.2 micromol x g wet tissue(-1) x s(-1)) and therefore at a lower power output. The lower ATP turnover was supported primarily by glycolysis and was reduced because of posttransformational inhibition of Phos by glucose 6-phosphate accumulation. During exercise, there was a gradual activation of PDH, which was fully transformed into its active form by 30 s of exercise. Oxidative phosphorylation, from PDH activation, only contributed 2% to the total ATP turnover, and there was no significant activation of lipid oxidation. The time course of PDH activation was closely associated with an increase in estimated mitochondrial redox (NAD(+)-to-NADH concentration ratio), suggesting that O2 was not limiting during high-intensity exercise. Thus anaerobiosis may not be responsible for lactate production in trout white muscle during high-intensity exercise.

Lipid oxidation fuels recovery from exhaustive exercise in white muscle of rainbow trout.

The oxidative utilization of lipid and carbohydrate was examined in white muscle of rainbow trout (Oncorhynchus mykiss) at rest, immediately after exhaustive exercise, and for 32-h recovery. In addition to creatine phosphate and glycolysis fueling exhaustive exercise, near maximal activation of pyruvate dehydrogenase (PDH) at the end of exercise points to oxidative phosphorylation of carbohydrate as an additional source of ATP during exercise. Within 15 min postexercise, PDH activation returned to resting values, thus sparing accumulated lactate from oxidation. Glycogen synthase activity matched the rate of glycogen resynthesis and represented near maximal activation. Decreases in white muscle free carnitine, increases in long-chain fatty acyl carnitine, and sustained elevations of acetyl-CoA and acetyl carnitine indicate a rapid utilization of lipid to supply ATP for recovery. Increases in malonyl-CoA during recovery suggest that malonyl-CoA may not regulate carnitine palmitoyltransferase-1 in trout muscle during recovery, but instead it may act to elongate short-chain fatty acids for mitochondrial oxidation. In addition, decreases in intramuscular triacylglycerol and in plasma nonesterified fatty acids indicate that both endogenous and exogenous lipid fuels may be oxidized during recovery.