Role of proline residues in the expression and function of the human noradrenaline transporter

The aim was to investigate the roles of proline residues in extracellular loop 2 (P172, P183, P188 and P209) and transmembrane domains 2, 5, 11 and 12 (P108, P270, P526, P551, P552 and P570) in determining noradrenaline transporter (NET) expression and function. Mutants of human NET with these residues mutated to alanine were pharmacologically characterized. Mutation of P108, P270 and P526 disrupted cell surface expression, from [H-3]nisoxetine binding and confocal microscopy data. Mutations of P526, P551 and P570 reduced transporter turnover (V-max of [H-3]noradrenaline uptake/B-max of [H-3]nisoxetine binding) by 1.5-1.7-fold compared with wild-type NET, so these residues might be involved in conformational changes associated with substrate translocation. Conversely, mutations of P172, P183, P188 and P209 increased V-max/B-max by 2-3-fold compared with wild-type, indicating that the presence of these proline residues limits turnover of the NET. The mutations had few effects on apparent affinities of substrates or affinities of inhibitors, except decreases in inhibitor affinities after mutations of the P270 and P570 residues, and increases after mutation of the P526 residue. Hence, proline residues in extracellular loop 2 and in transmembrane domains have a range of roles in determining expression and function of the NET.

Molecular recognition of sub-micromolar inhibitors by the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase

The crystal structures of human phenylethanolamine N-methyltransferase in complex with S-adenosyl-L-homocysteine (7, AdoHcy) and either 7-iodo-1,2,3,4-tetrahydroisoquinoline (2) or 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine (3, LY134046) were determined and compared with the structure of the enzyme complex with 7 and 7-aminosulfonyl-1,2,3,4-tetrahydroisoquinoline (1, SK&F 29661). The enzyme is able to accommodate a variety of chemically disparate functional groups on the aromatic ring of the inhibitors through adaptation of the binding pocket for this substituent and by subtle adjustments of the orientation of the inhibitors within the relatively planar binding site. In addition, the interactions formed by the amine nitrogen of all three inhibitors reinforce the hypothesis that this functional group mimics the beta-hydroxyl of norepinephrine rather than the amine. These studies provide further clues for the development of improved inhibitors for use as pharmacological probes.

Conotoxins as selective inhibitors of neuronal ion channels, receptors and transporters

Cone snails have evolved a vast array of peptide toxins for prey capture and defence. These peptides are directed against a wide variety of pharmacological targets, making them an invaluable source of ligands for studying the properties of these targets in normal and diseased states. A number of these peptides have shown efficacy in vivo, including inhibitors of calcium channels, the norepinephrine transporter, nicotinic acetylcholine receptors, NMDA receptors and neurotensin receptors, with several having undergone pre-clinical or clinical development for the treatment of pain.

L-arginine attenuates cardiovascular impairment in DOCA-salt hypertensive rats

Nitric oxide (NO) is essential for normal function of the cardiovascular system. This study has determined whether chronic administration of L-arginine, the biological precursor of NO, attenuates the development of structural and functional changes in hearts and blood vessels of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Uninephrectomized rats treated with DOCA (25 mg every 4th day sc) and 1% NaCl in the drinking water for 4 wk were treated with L-arginine (5% in food, 3.4 +/- 0.3 g.kg body wt(-1).day(-1)). Changes in cardiovascular structure and function were determined by echocardiography, microelectrode studies, histology, and studies in isolated hearts and thoracic aortic rings. DOCA-salt hypertensive rats developed hypertension, left ventricular hypertrophy with increased left ventricular wall thickness and decreased ventricular internal diameter, increased inflammatory cell infiltration, increased ventricular interstitial and perivascular collagen deposition, increased passive diastolic stiffness, prolonged action potential duration, increased oxidative stress, and inability to increase purine efflux in response to an increased workload. L-Arginine markedly attenuated or prevented these changes and also normalized the reduced efficacy of norepinephrine and acetylcholine in isolated thoracic aortic rings of DOCA-salt hypertensive rats. This study suggests that a functional NO deficit in blood vessels and heart due to decreased NO synthase activity or increased release of reactive oxygen species such as superoxide may be a key change initiating many aspects of the cardiovascular impairment observed in DOCA-salt hypertensive rats. These changes can be prevented or attenuated by administration of L-arginine.

Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis

The aim was to investigate the roles of transmembrane domain 2 and the adjacent region of the first intracellular loop in determining human noradrenaline transporter (hNET) function by pharmacological and substituted-cysteine accessibility method (SCAM) analyses. It was first necessary to establish a suitable background NET for SCAM. Alanine mutants of endogenous hNET cysteines, hC86A, hC131A and hC339A, were examined and showed no marked effects on expression or function. hNET and the mutants were also resistant to methanethiosulfonate (MTS), ethylammonium (MTSEA) and MTStrimethylammonium (MTSET). Hence, wild-type hNET is an appropriate background for production of cysteine mutants for SCAM. Pharmacological investigation showed that all mutants except hT99C and hL109C showed reduced cell-surface expression, while all except hM107C showed a reduction in functional activity. The mutations did not markedly affect the apparent affinities of substrates, but apparent affinities of cocaine were decreased 7-fold for hP97C and 10-fold for hF101C and increased 12-fold for hY98C. [H-3]Nisoxetine binding affinities were decreased 13-fold for hP97C and 5-fold for hF101C. SCAM analysis revealed that only hL102C was sensitive to 1.25 mM MTSEA, and this sensitivity was protected by noradrenaline, nisoxetine and cocaine. The results suggest that this region of hNET is important for interactions with antidepressants and cocaine, but it is probably not involved in substrate translocation mechanisms.

The role of BNP testing in heart failure

Brain natriuretic peptide (BNP) levels are simple and objective measures of cardiac function. These measurements can be used to diagnose heart failure, including diastolic dysfunction, and using them has been shown to save money in the emergency department setting. The high negative predictive value of BNP tests is particularly helpful for ruling out heart failure. Treatment with angiotensin-converting enzyme inhibitors, angiotensin-II receptor blockers, spironolactone, and diuretics reduces BNP levels, suggesting that BNP testing may have a role in monitoring patients with heart failure. However, patients with treated chronic stable heart failure may have levels in the normal range (i.e., BNP less than 100 pg per mL and N-terminal proBNP less than 125 pg per mL in patients younger than 75 years). Increases in BNP levels may be caused by intrinsic cardiac dysfunction or may be secondary to other causes such as pulmonary or renal diseases (e.g., chronic hypoxia). BNP tests are correlated with other measures of cardiac status such as New York Heart Association classification. BNP level is a strong predictor of risk of death and cardiovascular events in patients previously diagnosed with heart failure or cardiac dysfunction.

Noradrenergic modulatoins of odor learning and odor representation in the rat

How experience alters neuronal ensemble dynamics and how locus coeruleus-mediated norepinephrine release facilitates memory formation in the brain are the topics of this thesis. Here we employed a visualization technique, cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH), to assess activation patterns of neuronal ensembles in the olfactory bulb (OB) and anterior piriform cortex (aPC) to repeated odor inputs. Two associative learning models were used, early odor preference learning in rat pups and adult rat go-no-go odor discrimination learning. With catFISH of an immediate early gene, Arc, we showed that odor representation in the OB and aPC was sparse (~5-10%) and widely distributed. Odor associative learning enhanced the stability of the rewarded odor representation in the OB and aPC. The stable component, indexed by the overlap between the two ensembles activated by the rewarded odor at two time points, increased from ~25% to ~50% (p = 0.004-1.43E⁻4; Chapter 3 and 4). Adult odor discrimination learning promoted pattern separation between rewarded and unrewarded odor representations in the aPC. The overlap between rewarded and unrewarded odor representations reduced from ~25% to ~14% (p = 2.28E⁻⁵). However, learning an odor mixture as a rewarded odor increased the overlap of the component odor representations in the aPC from ~23% to ~44% (p = 0.010; Chapter 4). Blocking both α- and β-adrenoreceptors in the aPC prevented highly similar odor discrimination learning in adult rats, and reduced OB mitral and granule ensemble stability to the rewarded odor. Similar treatment in the OB only slowed odor discrimination learning. However, OB adrenoceptor blockade disrupted pattern separation and ensemble stability in the aPC when the rats demonstrated deficiency in discrimination (Chapter 5). In another project, the role of α₂-adrenoreceptors in the OB during early odor preference learning was studied. OB α2-adrenoceptor activation was necessary for odor learning in rat pups. α₂-adrenoceptor activation was additive with β-adrenoceptor mediated signalling to promote learning (Chapter 2). Together, these experiments suggest that odor representations are highly adaptive at the early stages of odor processing. The OB and aPC work in concert to support odor learning and top-down adrenergic input exerts a powerful modulation on both learning and odor representation.

Ghrelin suppresses glucose-stimulated insulin secretion and deteriorates glucose tolerance in healthy humans.

OBJECTIVE: The orexigenic gut hormone ghrelin and its receptor are present in pancreatic islets. Although ghrelin reduces insulin secretion in rodents, its effect on insulin secretion in humans has not been established. The goal of this study was to test the hypothesis that circulating ghrelin suppresses glucose-stimulated insulin secretion in healthy subjects. RESEARCH DESIGN AND METHODS: Ghrelin (0.3, 0.9 and 1.5 nmol/kg/h) or saline was infused for more than 65 min in 12 healthy patients (8 male/4 female) on 4 separate occasions in a counterbalanced fashion. An intravenous glucose tolerance test was performed during steady state plasma ghrelin levels. The acute insulin response to intravenous glucose (AIRg) was calculated from plasma insulin concentrations between 2 and 10 min after the glucose bolus. Intravenous glucose tolerance was measured as the glucose disappearance constant (Kg) from 10 to 30 min. RESULTS: The three ghrelin infusions raised plasma total ghrelin concentrations to 4-, 15-, and 23-fold above the fasting level, respectively. Ghrelin infusion did not alter fasting plasma insulin or glucose, but compared with saline, the 0.3, 0.9, and 1.5 nmol/kg/h doses decreased AIRg (2,152 +/- 448 vs. 1,478 +/- 2,889, 1,419 +/- 275, and 1,120 +/- 174 pmol/l) and Kg (0.3 and 1.5 nmol/kg/h doses only) significantly (P < 0.05 for all). Ghrelin infusion raised plasma growth hormone and serum cortisol concentrations significantly (P < 0.001 for both), but had no effect on glucagon, epinephrine, or norepinephrine levels (P = 0.44, 0.74, and 0.48, respectively). CONCLUSIONS: This is a robust proof-of-concept study showing that exogenous ghrelin reduces glucose-stimulated insulin secretion and glucose disappearance in healthy humans. Our findings raise the possibility that endogenous ghrelin has a role in physiologic insulin secretion, and that ghrelin antagonists could improve beta-cell function.

Characterizing bupropion metabolism, pharmacokinetics and drug-drug interaction liability

Thesis (Ph.D.)--University of Washington, 2016-06

Time-Dependent Effects of Acute Exercise-Induced Arousal on Long-Term Memory for Emotional and Neutral Stimuli

Psychological research has strongly documented the memory-enhancing effects of emotional arousal, while the effects of acute aerobic exercise on memory are not well understood. Manipulation of arousal has been shown to enhance long-term memory for emotional stimuli in a time-dependent fashion. This presents an opportunity to investigate the role of acute exercise in memory modulation. The purpose of this study was to determine the time-dependent relationship between acute exercise-induced arousal and long-term emotional memory. Participants viewed pleasant, neutral, and unpleasant images before or after completing a high-intensity session of cycling exercise. Salivary alpha-amylase, a biomarker of central norepinephrine, was measured as an indicator of arousal. No effects of exercise on recognition memory were revealed, however; a single session of high-intensity cycling increased salivary alpha-amylase. Our results also indicate that the influence of exercise on emotional responsiveness should be considered in further exploration of the memory-enhancing potential of acute exercise.

A systematic review of the psychobiological burden of informal caregiving for patients with dementia: focus on cognitive and biological markers of chronic stress

As the physiological impact of chronic stress is difficult to study in humans, naturalistic stressors are invaluable sources of information in this area. This review systematically evaluates the research literature examining biomarkers of chronic stress, including neurocognition, in informal dementia caregivers. We identified 151 papers for inclusion in the final review, including papers examining differences between caregivers and controls as well as interventions aimed at counteracting the biological burden of chronic caregiving stress. Results indicate that cortisol was increased in caregivers in a majority of studies examining this biomarker. There was mixed evidence for differences in epinephrine, norepinephrine and other cardiovascular markers. There was a high level of heterogeneity in immune system measures. Caregivers performed more poorly on attention and executive functioning tests. There was mixed evidence for memory performance. Interventions to reduce stress improved cognition but had mixed effects on cortisol. Risk of bias was generally low to moderate. Given the rising need for family caregivers worldwide, the implications of these findings can no longer be neglected.