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1.  Resolution of hypo-osmotic stress in isolated mouse ventricular myocytes causes sealing of t-tubules 
Experimental physiology  2013;98(7):1164-1177.
It has been recently shown that various stress-inducing manipulations in isolated ventricular myocytes may lead to significant remodeling of t-tubules. Osmotic stress is one of the most common complications in various experimental and clinical settings. Therefore, this study was designed to determine the effects of a physiologically relevant type of osmotic stress, hypo-osmotic challenge, to the integrity of t-tubular system in mouse ventricular myocytes using two approaches: (1) electrophysiological measurements of membrane capacitance and inward rectifier (IK1) tail currents originating from K+ accumulation in t-tubules, and (2) confocal microscopy of fluorescent dextrans trapped in sealed t-tubules. Importantly, we found that removal of 0.6 Na (60% NaCl) hypo-osmotic solution, but not its application to myocytes, led to ~27% reduction in membrane capacitance, ~2.5-fold reduction in the amplitude of IK1 tail current and ~2-fold reduction in so-called IK1 ‘inactivation’ (due to depletion of t-tubular K+) at negative membrane potentials – all data being consistent with significant detubulation. Confocal imaging experiments also demonstrated that extracellularly applied dextrans become trapped in sealed t-tubules only upon removal of hypo-osmotic solutions, i.e. during shrinking phase, but not during initial swelling period. In light of these data, relevant previous studies, including those on EC coupling phenomena during hypo-osmotic stress, may need to be reinterpreted and the experimental design of future experiments should take into account the novel findings.
doi:10.1113/expphysiol.2013.072470
PMCID: PMC3746342  PMID: 23585327
cardiac myocytes; t-tubules; inward rectifier potassium channels
2.  Memory consolidation in humans: new evidence and opportunities 
Experimental Physiology  2014;99(3):471-486.
We are endlessly fascinated by memory; we desire to improve it and fear its loss. While it has long been recognized that brain regions such as the hippocampus are vital for supporting memories of our past experiences (autobiographical memories), we still lack fundamental knowledge about the mechanisms involved. This is because the study of specific neural signatures of autobiographical memories in vivo in humans presents a significant challenge. However, recent developments in high-resolution structural and functional magnetic resonance imaging coupled with advanced analytical methods now permit access to the neural substrates of memory representations that has hitherto been precluded in humans. Here, I describe how the application of ‘decoding’ techniques to brain-imaging data is beginning to disclose how individual autobiographical memory representations evolve over time, deepening our understanding of systems-level consolidation. In particular, this prompts new questions about the roles of the hippocampus and ventromedial prefrontal cortex and offers new opportunities to interrogate the elusive memory trace that has for so long confounded neuroscientists.
doi:10.1113/expphysiol.2013.072157
PMCID: PMC4015368  PMID: 24414174
3.  Sources of Intravascular ATP During Exercise in Humans: Critical Role for Skeletal Muscle Perfusion 
Experimental physiology  2013;98(5):988-998.
Exercise hyperemia is regulated by several factors and one factor known to increase with exercise that evokes powerful vasomotor action is extracellular ATP. The origination of ATP detectable in plasma from exercising muscle of humans is, however, a matter of debate and ATP has been suggested to arise from sympathetic nerves, blood sources (e.g. erythrocytes), endothelial cells, and skeletal myocytes, among others. Therefore, we tested the hypothesis that acute augmentation of sympathetic nervous system activity (SNA) results in elevated plasma ATP draining skeletal muscle, and that SNA superimposition during exercise further increases ATP vs exercise alone. We show that increased SNA via −40mmHg lower body negative pressure (LBNP) at rest does not increase plasma ATP (51±8 vs 58±7 nmol/L with LBNP), nor does it increase [ATP] above levels observed during rhythmic handgrip exercise (79±11 exercise alone vs 71±8 nmol/L with LBNP). Secondly, we tested the hypothesis that active perfusion of skeletal muscle is essential to observe increased plasma ATP during exercise. We identify that complete obstruction of blood flow to contracting muscle abolishes exercise-mediated increases in plasma ATP (90±19 to 49±12 nmol/L), and further, that cessation of blood flow prior to exercise completely inhibits the typical rise in ATP (3 vs 61%; obstructed vs intact perfusion). The lack of ATP change during occlusion occurred in the face of continued muscle work and elevated SNA, indicating the rise of intravascular ATP is not resultant from these extravascular sources. Our collective observations indicate that the elevation in extracellular ATP observed in blood during exercise is unlikely to originate from sympathetic nerves or the contacting muscle itself, but rather is dependent on intact skeletal muscle perfusion. We conclude that an intravascular source for ATP is essential and points toward an important role for blood sources (e.g. red blood cells) in augmenting and maintaining elevated plasma ATP during exercise.
doi:10.1113/expphysiol.2012.071555
PMCID: PMC3633621  PMID: 23315195
adenosine triphosphate; muscle blood flow; contractions
4.  Signaling processes in endothelial aging in relation to chronic oxidative stress and their potential therapeutic implications 
Experimental physiology  2008;94(3):305-310.
Aging is an important risk factor for the development of cardiovascular diseases. Vascular aging is mainly characterized by endothelial dysfunction, an alteration of endothelium-dependent signalling processes and vascular remodelling. The underlying mechanisms comprise increased production of reactive oxygen species (ROS), inactivation of nitric oxide (•NO), and subsequent formation of peroxynitrite (ONOO–). Elevated ONOO– may exhibit new messenger functions by posttranslational oxidative modification of intracellular regulatory proteins.
Mitochondria are a major source of age-associated superoxide (•O2–) formation, as electrons are misdirected from the respiratory chain. Manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, is an integral part of the nucleoids and may protect mitochondrial DNA (mtDNA) from ROS. A model linking •NO, mitochondria, MnSOD and its acetylation/deacetylation by sirtuins (NAD+-dependent class III histone deacetylases) may be the basis for a potentially new powerful therapeutic intervention in the aging process.
doi:10.1113/expphysiol.2008.043315
PMCID: PMC3987961  PMID: 18996949
5.  INTRACEREBROVENTRICULAR LOSARTAN INFUSION MODULATES ANGIOTENSIN TYPE 1 RECEPTOR EXPRESSION IN THE SUBFORNICAL ORGAN AND DRINKING BEHAVIOUR IN BILE DUCT LIGATED RATS 
Experimental physiology  2012;98(4):922-933.
Bile duct ligation (BDL) causes congestive liver failure that initiates hemodynamic changes including peripheral vasodilation and generalized edema. Peripheral vasodilation is hypothesized to then activate compensatory mechanisms including increased drinking behavior and neurohumoral activation. This study tested the hypothesis that changes in the expression of AT1R mRNA and protein in the lamina terminalis is associated with BDL induced hypoosmolality in the rat. All rats received either BDL or sham ligation surgery. The rats were housed in metabolic chambers for measurement of fluid and food intake and urine output. Angiotensin type 1 receptor (AT1R) expression in the lamina terminalis was assessed by western blot and quantitative real-time PCR (RT-qPCR). Average baseline water intake significantly increased in BDL rats compared to sham and upregulation of AT1R protein and AT1aR mRNA were observed in the subfornical organ (SFO) of BDL rats. Separate groups of BDL and sham ligated rats were instrumented with minipumps filled with either losartan (2.0 µg/µl) or 0.9% saline for chronic intracerebroventricular (ICV) or subcutaneous (SC) chronic infusion. Chronic ICV losartan infusion attenuated the increased drinking behavior and prevented the increased abundance of AT1R protein in the SFO in BDL rats. Chronic SC did not affect water intake or AT1R abundance in the SFO. The data presented here indicate a possible role of increased central AT1R expression in the regulation of drinking behavior during congestive cirrhosis.
doi:10.1113/expphysiol.2012.068593
PMCID: PMC3606682  PMID: 23243146
vasopressin; AT1R; drinking behaviour; bile duct ligation
6.  A Real-Time Magnetic Resonance Imaging Technique for Determining Left Ventricle Pressure-Volume Loops 
Experimental physiology  2012;98(3):614-621.
Background
Rapid determination of the left ventricular (LV) pressure-volume (PV) relationship as loading conditions are varied is the gold standard for assessment of LV function. Cine magnetic resonance imaging (MRI) does not have sufficient spatiotemporal resolution to assess beat-to-beat changes of the LV PV relationship required to measure the LV end-systolic elastance (EES) or preload-recruitable stroke work (PRSW). Our aim was to investigate real-time MRI and semi-automated LV measurement of LV volume to measure PV relations in large animals under normal and inotropically stressed physiologic conditions.
Methods and Results
We determined that PV relationships could be accurately measured using an image exposure time Tex < 100 ms and frame rate Tfr < 50 ms at elevated heart rates (~140 bpm) using a golden angle radial MRI k-space trajectory and active contour segmentation. With an optimized exposure time (Tex = 95 ms and frame rate Tfr = 2.8 ms), we found that there was no significant difference between cine and real-time MRI at rest in end-diastolic volume (EDV), end-systolic volume (ESV), ejection fraction (EF), stroke volume (SV) or cardiac output (CO) (n=5, P<0.05) at either normal or elevated heart rates. We found EES increased from 1.9 ± 0.7 to 3.1 ± 0.3 mmHg/mL and PRSW increased from 6.2 ± 1.2 to 9.1 ± 0.9 mmHg during continuous IV dobutamine infusion (n=5, P<0.05).
Conclusions
Real-time MRI can assess LV volumes, EES and PRSW at baseline and elevated inotropic states.
doi:10.1113/expphysiol.2012.064733
PMCID: PMC3939798  PMID: 23064506
7.  Bradycardic effects mediated by activation of G protein-coupled estrogen receptor (GPER) in rat nucleus ambiguus 
Experimental physiology  2012;98(3):679-691.
G protein-coupled estrogen receptor (GPER) has been identified in several brain regions including cholinergic neurons of nucleus ambiguus, which are critical for the parasympathetic cardiac regulation. Using calcium imaging and electrophysiological techniques, microinjection into nucleus ambiguus and blood pressure measurement we examined the in vitro and in vivo effects of GPER activation in nucleus ambiguus neurons. G-1, a GPER selective agonist, produced a sustained increase in cytosolic Ca2+ concentration in a concentration-dependent manner in retrogradely-labeled cardiac vagal neurons of nucleus ambiguus. The increase in cytosolic Ca2+ produced by G-1 was abolished by pretreatment with G36, a GPER antagonist. G-1 depolarized cultured cardiac vagal neurons of nucleus ambiguus. The excitatory effect of G-1 was also identified by whole-cell visual patch-clamp recordings in nucleus ambiguus neurons, in medullary slices. To validate the physiological relevance of our in vitro studies, we carried out in vivo experiments. Microinjection of G-1 into the nucleus ambiguus elicited a decrease in heart rate; the effect was blocked by prior microinjection of G36. Systemic injection of G-1, in addition to a previously reported decrease in blood pressure, also reduced the heart rate. The G-1-induced bradycardia was prevented by systemic injection of atropine, a muscarinic antagonist, or by bilateral microinjection of G36 into the nucleus ambiguus. Our results indicate that GPER-mediated bradycardia occurs via activation of cardiac parasympathetic neurons of the nucleus ambiguus and support the involvement of GPER in the modulation of cardiac vagal tone.
doi:10.1113/expphysiol.2012.069377
PMCID: PMC3578005  PMID: 23104934
calcium; electrophysiology; vagus
8.  THYROID HORMONE IS REQUIRED FOR GROWTH ADAPTATION TO PRESSURE LOAD IN THE OVINE FETAL HEART 
Experimental physiology  2012;98(3):722-733.
Thyroid hormone exerts broad effects on the adult heart, however little is known regarding the role of thyroid hormone on regulating cardiac growth early in development and in response to pathophysiological conditions. To address this issue, we determined the effects of fetal thyroidectomy on cardiac growth and growth related gene expression in control and pulmonary artery banded fetal sheep. Fetal thyroidectomy (THX) and placement of a restrictive pulmonary artery band (PAB) was performed at 126 ± 1 d gestation (term 145 d). Four groups of animals (n = 5–6 in each group): 1) control; 2) fetal THX; 3) fetal PAB; and 4) fetal PAB + THX; were monitored for 1 week prior to being euthanized. Fetal heart rate was significantly lower in the two THX groups compared with the non-THX groups while mean arterial blood pressure was similar among groups. Combined left and right ventricle free wall + septum weight, expressed per kg fetal weight, was significantly increased in PAB (6.27 ± 0.85 g/kg) compared to control animals (4.72 ± 0.12 g/kg). THX significantly attenuated the increase in cardiac mass associated with PAB (4.94 ± 0.13 g/kg) while THX alone had no detectable effect on heart mass (4.95 ± 0.27 g/kg). The percentage of binucleated cardiomyocytes was significantly decreased in THX and PAB +THX (~16%) compared to the non-THX groups (~27%). No differences in levels of activated Akt, ERK or JNK were detected among the groups. Markers of cellular proliferation but not apoptosis or expression of growth related genes were lower in the THX and THX+ PAB groups relative to thyroid intact animals. These findings suggest that in the late gestation fetal heart, thyroid hormone has important cellular growth functions in both physiologic and pathophysiologic states. Specifically, thyroid hormone is required for adaptive fetal cardiac growth in response to pressure overload.
doi:10.1113/expphysiol.2012.069435
PMCID: PMC3578172  PMID: 23104936
cardiac; development; hypothyroidism
9.  PULMONARY SYSTEM LIMITATIONS TO ENDURANCE EXERCISE PERFORMANCE IN HUMANS 
Experimental physiology  2011;97(3):311-318.
Accumulating evidence over the past 25 years depicts the healthy pulmonary system as a limiting factor of whole body endurance exercise performance. This brief overview emphasizes three respiratory system-related mechanisms which impair O2 transport to the locomotor musculature [arterial O2 content (CaO2) × leg blood flow (QL)], i.e. the key determinant of an individual’s aerobic capacity and ability to resist fatigue. First, the respiratory system often fails to prevent arterial desaturation substantially below resting values and thus compromises CaO2. Especially susceptible to this threat to convective O2 transport are well-trained endurance athletes characterized by high metabolic and ventilatory demands and, likely due to anatomical and morphologic gender differences, active females. Second, fatiguing respiratory muscle work (Wresp) associated with strenuous exercise elicits sympathetically-mediated vasoconstriction in limb-muscle vasculature which compromises QL. This impact on limb O2 transport is independent of fitness level and affects all individuals, however, only during sustained, high-intensity endurance exercise performed above ~85% VO2max. And third, excessive fluctuations in intrathoracic pressures accompanying Wresp can limit cardiac output and therefore QL. Exposure to altitude exacerbates the respiratory system limitations observed at sea level and further reduces CaO2 and substantially increases exercise-induced Wresp. Taken together, the intact pulmonary system of healthy endurance athletes impairs locomotor muscle O2 transport during strenuous exercise by failing to ensure optimal arterial oxygenation and compromising QL. This respiratory system-related impact exacerbates the exercise-induced development of fatigue and compromises endurance performance.
doi:10.1113/expphysiol.2011.058800
PMCID: PMC3934509  PMID: 22125308
pulmonary ventilation; blood flow distribution; gas exchange; exercise-induced arterial hypoxemia; arterial oxygen saturation; work of breathing
10.  Beta-Adrenoceptor Gene Variation and Intermediate Physiologic Traits: Prediction of Distant Phenotype 
Experimental physiology  2010;95(7):757-764.
Intermediate physiological phenotype is the genetic and environmental influence on functional physiological characteristics with direct prognostic relevance to distant, more complex phenotypes such as cardiovascular and metabolic disease. Increasingly available and affordable genotyping techniques have created an explosion of information on candidate gene variation and its relationship to intermediate physiological traits. Variation in beta-adrenoceptor genes is an intense focus of investigation because beta-adrenoceptors are 1) ubiquitous in organ system distribution; 2) integral to a multitude of physiological processes; 3) well-described in cardiovascular and metabolic disease; and 4) major pharmacologic treatment targets. Furthermore, knowledge of functional gene variants in these receptors predates the description of the human genome. This review highlights the influence of common gene variation in the three beta-adrenoceptor subtypes on intermediate physiologic phenotype predictive of cardiovascular disease and obesity. Although further information is needed to replicate this information across populations, this review condenses and summarizes growing trends in specific pleiotropic effects of beta-adrenoceptor polymorphisms, and suggests which variants may be predictive of distant phenotype.
doi:10.1113/expphysiol.2009.048330
PMCID: PMC3919627  PMID: 20382665
11.  Atomic Force Microscopy to Characterize Binding Properties of α7-Containing Nicotinic Acetylcholine Receptors on NK1-Expressing Medullary Respiratory Neurons 
Experimental physiology  2012;98(2):415-424.
In the present study we used atomic force microscopy (AFM) to examine the ligand binding properties of α7-containing nicotinic acetylcholine receptors (nAChRs) expressed on neurons from the ventral respiratory group. We also determined the effect of acute and prolonged exposure to nicotine on the binding probability of nAChRs. Neurons from neonatal (P5–P10) and juvenile (3–4 wk) rats were cultured. Internalization of Alexa Fluor 488–conjugated substance P was used to identify respiratory neurons that expressed neurokinin-1 receptors (NK1-R); a recognized marker of ventral respiratory group neurons. To assess functional changes in nAChRs, AFM probes conjugated with anti-α7 subunit nAChR antibody were used to cyclically interact the soma surface of NK1-R positive neurons. Measurements were made of the frequency of antibody adhesion to the α7-receptor subunit and of the detachment forces between the membrane attached receptor and the AFM probe tip. Addition of α-bungarotoxin (a specific antagonist of α-7 subunit-containing nAChRs) to the cell bath produced a 69% reduction in binding to the α-7 subunit (P<0.05, n=10), supporting specificity of binding. Acute exposure to nicotine (1 μM added to culture media) produced an 80% reduction in nAChR antibody binding to the α-7 subunit (P<0.05, n=9). Prolonged incubation (72 hours) of cell culture in nicotine significantly reduced α-7 binding in a concentration-dependent manner. Collectively, these findings demonstrate that AFM is a sensitive tool for assessment of functional changes in nAChRs expressed on the surface of living NK1-R expressing medullary neurons. Moreover, these data demonstrate that nicotine exposure decreases the binding probability of α-7 subunit containing nAChRs.
doi:10.1113/expphysiol.2012.067660
PMCID: PMC3548956  PMID: 22962286
Atomic force microscopy; respiration; nicotine
12.  ATM impacts IGF-1 signaling in skeletal muscle 
Experimental physiology  2012;98(2):526-535.
Reports that ataxia telangiectasia mutated (ATM) is required for full activation of Akt raise the hypothesis that ATM plays a role in IGF-1 signaling through the Akt/mTOR pathway. Differentiated C2C12 cells harboring either ATM-targeting shRNA or non-targeting shRNA and myotubes from a C2C12 lineage previously exposed to empty vector lentivirus were incubated in the presence or absence of 10 nM IGF-1 followed by western blot analysis. Parallel experiments were performed in isolated soleus muscles from mice expressing only one functional ATM allele (ATM+/−) compared to muscles from wild-type (ATM+/+) mice. IGF-1 increased phosphorylation of Akt S473, Akt T308, and p70 S6 kinase (S6K) in myotubes expressing non-targeting shRNA and in empty vector controls, but the IGF-1 effects were significantly reduced in myotubes with shRNA-mediated ATM knockdown. Likewise, IGF-1-stimulated phosphorylation of Akt S473, Akt T308, mTOR, and S6K was lower in isolated soleus muscles from ATM+/− mice compared to muscles from ATM+/+ mice. The ATM inhibitor KU55933 prevented stimulation of S6K phosphorylation in C2C12 myotubes exposed to IGF-1, suggesting that decreased IGF-1 action is not limited to chronic conditions of decreased ATM function. Stimulation of IRS-1 tyrosine 612 phosphorylation by IGF-1 was unaffected by ATM deficiency, though IGF-1 phosphatidylinositol 3-kinase activity tended to be lower in muscle from ATM haploinsufficient mice compared to wild type muscle. The data suggest that ATM is a modulator of IGF-1 signaling downstream of IRS-1 in skeletal muscle.
doi:10.1113/expphysiol.2012.066357
PMCID: PMC3553258  PMID: 22941977
Akt; IGF-1; mTOR
13.  Physiology of invertebrate oxytocin and vasopressin neuropeptides 
Experimental physiology  2013;99(1):10.1113/expphysiol.2013.072561.
Neuropeptides and regulatory peptide hormones control many developmental, physiological and behavioural processes in animals, including humans. The nonapeptides oxytocin and arginine vasopressin are produced and released by the pituitary gland and have actions on many organs and tissues. Receptive cells possess particular receptors to which the peptides bind as ligands, leading to activation of G-protein-coupled receptors, hence cellular responses. In humans and other mammalian species, oxytocin and vasopressin mediate a range of peripheral and central physiological functions that are important for osmoregulation, reproduction, complex social behaviours, memory and learning. The origin of the oxytocin/vasopressin signalling system is thought to date back more than 600 million years. All vertebrate oxytocin- and vasopressin-like peptides have presumably evolved from the ancestral nonapeptide vasotocin by gene duplication and today are present in vertebrates, including mammals, birds, reptiles, amphibians and fish. Oxytocin- and vasopressin-like peptides have been identified in several invertebrate species, including molluscs, annelids, nematodes and arthropods. Members of this peptide family share high sequence similarity, and it is possible that they are functionally related across the entire animal kingdom. However, it is evident that not all animals express oxytocin/vasopressin neuropeptides and that there is little information available about the biology and physiology of this signalling system of invertebrates and, in particular, of insects, which represent more than half of all known living organisms. This report describes the discovery of novel oxytocin- and vasopressin-like peptides in arthropods and summarizes the status quo of the functional relevance of this neuropeptide signalling system in invertebrates, which will have beneficial implications for the design of selective and potent ligands to human oxytocin and vasopressin receptors.
doi:10.1113/expphysiol.2013.072561
PMCID: PMC3883647  PMID: 23955310
14.  Effect of exercise on mouse liver and brain bioenergetic infrastructures 
Experimental physiology  2012;98(1):207-219.
To assess the effects of exercise on liver and brain bioenergetic infrastructures, we exposed C57BL/6 mice to 6 weeks of moderate-intensity treadmill exercise. During the training period, fasting blood glucose was lower in exercised mice than in sedentary mice, but serum insulin levels were not reduced. At week 6, trained mice showed a paradoxical decrease in plasma lactate during exercise, which was accompanied by an increase in the liver monocarboxylate transporter 2 protein level (~30%, P < 0.05). Exercise increased liver peroxisomal proliferator-activated receptor-γ coactivator 1α expression (approximately twofold, P < 0.001), NAD-dependent deacetylase sirtuin-1 protein (~30%, P < 0.05), p38 protein (~15%, P < 0.05), cytochrome c oxidase subunit 4 isoform 1 protein (~50%, P < 0.05) and AMP-activated protein kinase phosphorylation (~40%, P < 0.05). Despite this, liver mitochondrial DNA copy number (~30%, P = 0.05), mitochondrial transcription factor A expression (~15%, P < 0.05), cytochrome c oxidase subunit 2 expression (~10%, P < 0.05), cAMP-response element binding protein phosphorylation (~60%, P < 0.05) and brain-derived neurotrophic factor expression (~40%, P < 0.05) were all reduced, while cytochrome oxidase and citrate synthase activities were unchanged. The only altered brain parameter observed was a reduction in tumour necrosis factor α expression (~35%, P < 0.05); tumour necrosis factor α expression was unchanged in liver. Our data suggest that lactate produced by exercising muscle modifies the liver bioenergetic infrastructure, and enhanced liver uptake may in turn limit the ability of exercise-generated lactate to modify brain bioenergetics. Also, it appears that, at least in the liver, a dissociated mitochondrial biogenesis, in which some components are strategically enhanced while others are minimized, can occur.
doi:10.1113/expphysiol.2012.066688
PMCID: PMC3540163  PMID: 22613742
15.  Angiotensin-(1-12) in the rostral ventrolateral medullary pressor area of the rat elicits sympathoexcitatory responses 
Experimental physiology  2012;98(1):94-108.
The rostral ventrolateral medullary pressor area (RVLM) is known to be critical in the regulation of cardiovascular function. In this study, it was hypothesized that the RVLM may be one of the sites of cardiovascular actions of a new angiotensin, angiotensin-(1-12) [ANG-(1-12)]. Experiments were carried out in urethane-anaesthetized, artificially ventilated, adult male Wistar rats. RVLM was identified by microinjections of L-glutamate (5 mM). The volume of all microinjections into the RVLM was 100 nl. Microinjections of ANG-(1-12) (0.1–1.0 mM) into the RVLM elicited increases in mean arterial pressure (MAP) and heart rate (HR). Maximum cardiovascular responses were elicited by 0.5 mM ANG-(1-12); this concentration was used in other experiments described below. Microinjections of ANG-(1-12) increased greater splanchnic nerve activity (GSNA). The tachycardic responses to ANG-(1-12) were not altered by bilateral vagotomy. The cardiovascular responses elicited by ANG-(1-12) were attenuated by microinjections of an angiotensin II type 1 receptor (AT1R) antagonist (losartan), but not AT2R antagonist (PD123319), into the RVLM. Combined inhibition of angiotensin converting enzyme (ACE) and chymase in the RVLM abolished ANG-(1-12)-induced responses. ANG-(1-12)-immunoreactive cells were present in the RVLM. AT1Rs and phenylethanolamine-N-methyl-transferase (PNMT) were present in the RVLM neurons retrogradely labeled by microinjections of Fluoro-Gold into the intermediolateral cell column of the thoracic spinal cord. ANG-(1-12)-containing neurons in the hypothalamic paraventricular nucleus did not project to the RVLM. These results indicated that: 1) microinjections of ANG-(1-12) into the RVLM elicited increases in MAP, HR, and GSNA, 2) both ACE and chymase were needed to convert ANG-(1-12) into angiotensin II, and 3) AT1Rs, but not AT2Rs, in the RVLM mediated ANG-(1-12)-induced responses.
doi:10.1113/expphysiol.2012.067116
PMCID: PMC3470822  PMID: 22707504
angiotensin II; blood pressure; captopril; chymostatin; heart rate; losartan; sympathetic nerve activity
16.  Differential changes in vascular mRNA levels between rat iliac and renal arteries produced by cessation of voluntary running 
Experimental physiology  2012;98(1):337-347.
Early vascular changes at the molecular level caused by adoption of a sedentary lifestyle are incompletely characterized. Herein, we employed the rodent wheel lock model to identify mRNAs in the arterial wall that are responsive to the acute transition from higher to lower levels of daily physical activity. Specifically, we evaluated whether short-term cessation of voluntary wheel running alters vascular mRNA levels in rat conduit arteries previously reported to have marked increases (i.e. iliac artery) versus marked decreases (i.e. renal artery) in blood flow during running. We used young female Wistar rats with free access to voluntary running wheels. Following 23-days of voluntary running (average distance of ~15-km/night; ~4.4-hrs/night), rats in one group were rapidly transitioned to a sedentary state by locking the wheels for seven days (n=9) or remained active (n=9) in a second group for an additional seven days. Real-time PCR was conducted on total RNA isolated from iliac and renal arteries to evaluate expression of 25 pro-atherogenic and anti-atherogenic genes. Compared to iliac arteries of wheel lock 0-day rats, iliac arteries of wheel lock 7-day rats exhibited increased expression of TNFR1 (+19%), ET1 (+59%), and LOX-1 (+31%) (p<0.05). Moreover, compared to renal arteries of wheel lock 0-day rats, renal arteries of wheel lock 7-day rats exhibited decreased expression of ETb (−23%), p47phox (−32%), and p67phox (−19%) (p<0.05). These data demonstrate that cessation of voluntary wheel running for seven days produces modest, but differential changes in mRNA levels between the iliac and renal arteries of healthy rats. This heterogeneous influence of short-term physical inactivity could be attributed to the distinct alteration in hemodynamic forces between arteries.
doi:10.1113/expphysiol.2012.066076
PMCID: PMC3474882  PMID: 22709650
17.  NF-kappaB signaling and transcriptional regulation in skeletal muscle atrophy 
Experimental physiology  2012;98(1):19-24.
The NF-kappaB signaling pathway is a necessary component of adult skeletal muscle atrophy due to systemic illnesses or disuse. Studies showing a role for the NF-kappaB pathway in muscle disuse focus on unloading, denervation, or immobilization, and studies showing a role for NF-kappaB in systemic illnesses include cancer, chronic heart failure, and acute septic lung injury. Muscle atrophy due to most of these triggers is associated with activation of NF-kappaB transcriptional activity. With the exception of muscle unloading however, there is a paucity of data on the NF-kappaB transcription factors that regulate muscle atrophy and there little known about which genes are targeted by NF-kappaB transcription factors during atrophy. Interestingly, in some cases it appears that the amelioration of muscle atrophy by genetic inhibition of NF-kappaB signaling proteins is due to effects that are independent of the downstream NF-kappaB transcription factors. These questions are prime areas for investigation if we are to understand a key component of muscle wasting in adult skeletal muscle.
doi:10.1113/expphysiol.2011.063321
PMCID: PMC3505235  PMID: 22848079
18.  Exercise partially reverses the effect of maternal separation on hippocampal proteins in 6-hydroxydopamine lesioned rat brain 
Experimental physiology  2012;98(1):233-244.
Animals subjected to maternal separation stress during the early stages of development display behavioural, endocrine and growth factor abnormalities that mirror the clinical findings in anxiety/depression. In addition, maternal separation has been shown to exacerbate the behavioural deficits induced by 6-hydroxydopamine (6-OHDA) in a rat model of Parkinson's disease. In contrast, voluntary exercise reduced the detrimental effects of 6-OHDA in the rat model. The beneficial effects of exercise appeared to be largely due to compensation in the non-lesioned hemisphere. The aim of the present study was to investigate whether voluntary exercise for 3 weeks could reverse the effects of maternal separation in rats challenged with the neurotoxin 6-OHDA infused into the medial forebrain bundle after 1 week of exercise, at postnatal day 60 (P60). The rats were killed 2 weeks later, at P74. Their brains were dissected and the hippocampus rapidly removed for proteomic analysis - isobaric tagging (iTRAQ) and quantification of peptides by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). Maternal separation up-regulated hippocampal proteins functionally involved in energy metabolism (nucleoside diphosphate kinase B, enolase, triosephosphate isomerase) and synaptic plasticity (alpha-synuclein, tenascin-R, Ba1-667, brevican and neurocan core protein) in the non-lesioned hemisphere. Exercise reversed many of these changes by down-regulating the levels of hippocampal proteins functionally associated with energy metabolism (nucleoside diphosphate kinase B, enolase, triosephosphate isomerase) and synaptic plasticity (alpha-synuclein, tenascin-R, Ba1-667, brevican and neurocan core protein) in the non-lesioned hemisphere of rats subjected to maternal separation. Exercise and maternal separation therefore appeared to have opposing effects on the hippocampus in the non-lesioned hemisphere of the rat brain. Exercise seemed to partially reverse the effects of maternal separation stress on these proteins in the non-lesioned hemisphere. The partial reversal of maternal separation-induced proteins by exercise in the non-lesioned side sheds some insight into the mechanism by which exercise alters the molecular role players involved in determining the consequences of early life stress.
doi:10.1113/expphysiol.2012.066720
PMCID: PMC3509240  PMID: 22636255
Maternal separation; Exercise; Proteomics
19.  A ROLE FOR NITRIC OXIDE WITHIN THE NUCLEUS TRACTUS SOLITARIUS IN THE DEVELOPMENT OF MUSCLE MECHANOREFLEX DYSFUNCTION IN HYPERTENSION 
Experimental physiology  2012;97(12):1292-1304.
Evidence suggests the muscle mechanoreflex, a circulatory reflex that raises blood pressure and heart rate (HR) upon activation of mechanically sensitive afferent fibers in skeletal muscle, is overactive in hypertension. However, the mechanisms underlying this abnormal reflex function have yet to be identified. Sensory input from the mechanoreflex is processed within the nucleus tractus solitarius (NTS) in the medulla oblongata. Within the NTS, the enzymatic activity of nitric oxide synthase (NOS) produces nitric oxide (NO). This centrally-derived NO has been shown to modulate muscle reflex activity and serves as a viable candidate for mediating the mechanoreflex dysfunction that develops in hypertension. We hypothesized that mechanoreflex dysfunction in hypertension is mediated by abnormal alterations in NO production in the NTS. Mechanically sensitive afferent fibers were stimulated by passively stretching hindlimb muscle before and after blocking the endogenous production of NO within the NTS via microdialysis of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 1 and 5 mM) in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Changes in HR and mean arterial pressure (MAP) in response to stretch were significantly larger in SHR compared to WKY prior to L-NAME dialysis. Attenuating NO production via L-NAME in normotensive rats recapitulated the exaggerated cardiovascular response to stretch observed in SHR. Dialyzing L-NAME in SHR further accentuated the increases in HR and MAP elicited by stretch. These findings support the contention that reductions in NO production within the NTS contribute to the generation of abnormal cardiovascular control by the skeletal muscle mechanoreflex in hypertension.
doi:10.1113/expphysiol.2012.065433
PMCID: PMC3480555  PMID: 22581746
blood pressure; heart rate; exercise
20.  Mitogen-activated protein kinase is required for the behavioral desensitization that occurs after repeated injections of angiotensin II 
Experimental physiology  2012;97(12):1305-1314.
Angiotensin II (AngII) acts on central angiotensin type 1 (AT1) receptors to increase water and saline intake. Prolonged exposure to AngII in cell culture models results in a desensitization of the AT1 receptor that is thought to involve receptor internalization, and a behavioral correlate of this desensitization has been shown in rats after repeated central injections of AngII. Specifically, rats given repeated injections of AngII drink less water than controls after a subsequent test injection of AngII. Under the same conditions, however, repeated injections of AngII have no effect on AngII-induced saline intake. Given earlier studies indicating that separate intracellular signaling pathways mediate AngII-induced water and saline intake, we hypothesized that the desensitization observed in rats may be incomplete, leaving the receptor able to activate mitogen-activated protein (MAP) kinases (ERK1/2), which play a role in AngII-induced saline intake without affecting water intake. In support of this hypothesis, we found no difference in MAP kinase phosphorylation after an AngII test injection in rats given prior treatment with repeated injections of vehicle, AngII, or Sar1,Ile4,Ile8-AngII (SII), an AngII analog that activates MAP kinase without G protein coupling. In addition, we found that pretreatment with the MAP kinase inhibitor U0126 completely blocked the desensitizing effect of repeated AngII injections on water intake. Furthermore, AngII-induced water intake was reduced similarly by repeated injections of AngII or SII. The results suggest that G protein-independent signaling is sufficient to produce behavioral desensitization of the angiotensin system and that the desensitization requires MAP kinase activation.
doi:10.1113/expphysiol.2012.065771
PMCID: PMC3721319  PMID: 22581747
21.  Cardiovascular actions of angiotensin-(1-12) in the hypothalamic paraventricular nucleus of the rat are mediated via angiotensin II 
Experimental physiology  2011;97(9):1001-1017.
The role of hypothalamic paraventricular nucleus (PVN) in cardiovascular regulation is well established. In this study, it was hypothesized that the PVN may be one of the sites of cardiovascular actions of a new angiotensin, angiotensin-(1-12). Experiments were carried out in urethane-anaesthetized, artificially ventilated, adult male Wistar rats. PVN was identified by microinjections of N-methyl-D-aspartic acid (NMDA, 10 mM). Microinjections (50 nl) of angiotensin-(1-12) (1 mM) into the PVN elicited increases in mean arterial pressure (MAP), heart rate (HR) and renal nerve activity (RSNA). The tachycardic responses to angiotensin-(1-12) were attenuated by bilateral vagotomy. The cardiovascular responses elicited by angiotensin-(1-12) were attenuated by microinjections of an angiotensin II type 1 receptor (AT1R) antagonist (losartan), but not AT2R antagonist (PD123319), into the PVN. Combined inhibition of angiotensin converting enzyme (ACE) and chymase in the PVN abolished angiotensin-(1-12)-induced responses. Angiotensin-(1-12)-immunoreactive cells and fibres were more numerous in the middle and caudal regions of the PVN. Angiotensin-(1-12) was present in many, but not all, vasopressinergic PVN cells. This peptide was also present in some non-vasopressinergic PVN cells but not in oxytocin containing PVN cells. These results indicated that: 1) microinjections of angiotensin-(1-12) into the PVN elicited increases in MAP, HR, and RSNA, 2) HR responses were mediated via both sympathetic and vagus nerves, 3) both ACE and chymase were needed to convert angiotensin-(1-12) to angiotensin II in the PVN, and 4) AT1Rs, but not AT2Rs, in the PVN mediated angiotensin-(1-12)-induced responses. It was concluded that the cardiovascular actions of angiotensin-(1-12) in the PVN are mediated via its conversion to angiotensin II.
doi:10.1113/expphysiol.2011.062471
PMCID: PMC3303999  PMID: 22125313
blood pressure; captopril; chymostatin; heart rate; losartan; sympathetic nerve activity
22.  Lentivirus-mediated overexpression of angiotensin-(1–7) attenuated ischaemia-induced cardiac pathophysiology 
Experimental physiology  2011;96(9):863-874.
Myocardial infarction (MI) results in cell death, development of interstitial fibrosis, ventricular wall thinning and ultimately, heart failure. Angiotensin-(1–7) [Ang-(1–7)] has been shown to provide cardioprotective effects. We hypothesize that lentivirus-mediated overexpression of Ang-(1–7) would protect the myocardium from ischaemic injury. A single bolus of 3.5 ×108 transducing units of lenti-Ang-(1–7) was injected into the left ventricle of 5-day-old male Sprague–Dawley rats. At 6 weeks of age, MI was induced by ligation of the left anterior descending coronary artery. Four weeks after the MI, echocardiography and haemodynamic parameters were measured to assess cardiac function. Postmyocardial infarction, rats showed significant decreases in fractional shortening and dP/dt (rate of rise of left ventricular pressure), increases in left ventricular end-diastolic pressure, and ventricular hypertrophy. Also, considerable upregulation of cardiac angiotensin-converting enzyme (ACE) mRNA was observed in these rats. Lentivirus-mediated cardiac overexpression of Ang-(1–7) not only prevented all these MI-induced impairments but also resulted in decreased myocardial wall thinning and an increased cardiac gene expression of ACE2 and bradykinin B2 receptor (BKR2). Furthermore, in vitro experiments using rat neonatal cardiac myocytes demonstrated protective effects of Ang-(1–7) against hypoxia-induced cell death. This beneficial effect was associated with decreased expression of inflammatory cytokines (tumour necrosis factor-α and interleukin-6) and increased gene expression of ACE2, BKR2 and interleukin-10. Our findings indicate that overexpression of Ang-(1–7) improves cardiac function and attenuates left ventricular remodelling post-MI. The protective effects of Ang-(1–7) appear to be mediated, at least in part, through modulation of the cardiac renin–angiotensin system and cytokine production.
doi:10.1113/expphysiol.2011.056994
PMCID: PMC3755594  PMID: 21685447
23.  Changes in the control of skin blood flow with exercise training: where do cutaneous vascular adaptations fit in? 
Experimental physiology  2011;96(9):822-828.
Heat is the most abundant byproduct of cellular metabolism. As such, dynamic exercise in which a significant percentage of muscle mass is engaged generates thermoregulatory demands that are met in part by increases in skin blood flow. Increased skin blood flow during exercise adds to the demands on cardiac output and confers additional circulatory strain beyond that associated with perfusion of active muscle alone. Endurance exercise training results in a number of physiological adaptations which ultimately reduce circulatory strain and shift thermoregulatory control of skin blood flow to higher levels of blood flow for a given core temperature. In addition, exercise training induces peripheral vascular adaptations within the cutaneous microvasculature indicative of enhanced endothelium-dependent vasomotor function. However, it is not currently clear how (or if) these local vascular adaptations contribute to the beneficial changes in thermoregulatory control of skin blood flow following exercise training. The purpose of this Hot Topic review is to synthesize the literature pertaining to exercise training-mediated changes in cutaneous microvascular reactivity and thermoregulatory control of skin blood flow. In addition, we address mechanisms driving changes in cutaneous microvascular reactivity and thermoregulatory control of skin blood flow, and pose the question: what (if any) is the functional role of increased cutaneous microvascular reactivity following exercise training?
doi:10.1113/expphysiol.2010.056176
PMCID: PMC3754812  PMID: 21602295
exercise training; physical activity; vascular adaptations; skin
24.  Increasing brain serotonin corrects CO2 chemosensitivity in methyl-CpG-binding protein 2 (Mecp2)-deficient mice 
Experimental physiology  2012;98(3):842-849.
Mice deficient in the transcription factor methyl-CpG-binding protein 2 (Mecp2), a mouse model of Rett syndrome, display reduced CO2 chemosensitivity, which may contribute to their breathing abnormalities. In addition, patients with Rett syndrome and male mice that are null for Mecp2 show reduced levels of brain serotonin (5-HT). Serotonin is known to play a role in central chemosensitivity, and we hypothesized that increasing the availability of 5-HT in this mouse model would improve their respiratory response to CO2. Here we determined the apnoeic threshold in heterozygous Mecp2-deficient female mice and examined the effects of blocking 5-HT reuptake on the CO2 response in Mecp2-null male mice. Studies were performed in B6.129P2(C)-Mecp2τm1.1Bird null males and heterozygous females. In an in situ preparation, seven of eight Mecp2-deficient heterozygous females showed arrest of phrenic nerve activity when arterial CO2 was lowered to 3%, whereas the wild-types maintained phrenic nerve amplitude at 53 ± 3% of maximal. In vivo plethysmography studies were used to determine CO2 chemosensitivity in null males. These mice were exposed sequentially to 1, 3 and 5% CO2. The percentage increase in minute ventilation in response to increased inspired CO2 was less in Mecp2−/y than in Mecp2+/y mice. Pretreatment with citalopram, a selective 5-HT reuptake inhibitor (2.5 mg kg−1 I.P.), 40 min prior to CO2 exposure, in Mecp2−/y mice resulted in an improvement in CO2 chemosensitivity to wild-type levels. These results suggest that decreased 5-HT in Mecp2-deficient mice reduces CO2 chemosensitivity, and restoring 5-HT levels can reverse this effect.
doi:10.1113/expphysiol.2012.069872
PMCID: PMC3696880  PMID: 23180809
25.  Chronic Activation of Endogenous Angiotensin-Converting Enzyme 2 Protects Diabetic Rats from Cardiovascular Autonomic Dysfunction 
Experimental Physiology  2012;97(6):699-709.
In this study we evaluated whether the activation of endogenous angiotensin-converting enzyme (ACE) 2 would improve the cardiovascular autonomic dysfunction of diabetic rats. Ten days after type 1 diabetes induction (Streptozotocin, STZ, 50mg/kg i.v.), the rats were orally treated with 1-[(2-dimethylamino)ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one (XNT), a newly discovered ACE2 activator (1mg/kg/day), or saline (equivalent volume) during 30 days. Autonomic cardiovascular parameters were evaluated in unanesthetized animals and an isolated heart preparation was used to analyze the cardiac function. Diabetes induced a significant decrease in the baroreflex bradycardia sensibility, as well as in the chemoreflex chronotropic response and parasympathetic tone. The XNT treatment improved these parameters by ~76% (0.82±0.09 vs. 1.44±0.17ΔPI/ΔmmHg), ~85% (−57±9 vs. −105±10 Δbpm) and ~205% (22±2 vs. 66±12 Δbpm), respectively. Also, XNT administration enhanced the bradycardia induced by the chemoreflex activation by ~74% in non-diabetic animals (−98±16 vs. −170±9 Δbpm). No significant changes were observed in the mean arterial pressure, baroreflex tachycardia sensibility, chemoreflex pressor response and sympathetic tone among any of the groups. Furthermore, chronic XNT treatment ameliorated the cardiac function of diabetic animals. However, the coronary vasoconstriction observed in diabetic rats was unchanged by ACE2 activation. These findings indicate that XNT protects against the autonomic and cardiac dysfunction induced by diabetes. Thus, our results evidenced the viability and effectiveness of oral administration of an ACE2 activator for the treatment of the cardiovascular autonomic dysfunction caused by diabetes.
doi:10.1113/expphysiol.2011.063461
PMCID: PMC3350618  PMID: 22286369
Baroreflex; Chemoreflex; ACE2 activation

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