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The relationship between dietary NaCl intake and high blood pressure is well-established, and occurs primarily through activation of the sympathetic nervous system. Nax, a Na+-sensitive Na+ channel, plays a pivotal role in driving sympathetic excitability, which is thought to originate from central regions controlling neural outflow. We investigated whether post-ganglionic sympathetic neurons from different ganglia innervating cardiac and vasculature tissue can also directly sense extracellular Na+. Using whole-cell patch clamp recordings we demonstrate that sympathetic neurons from three sympathetic ganglia (superior cervical, stellate and superior mesenteric/coeliac) respond to elevated extracellular NaCl concentration. In sympathetic stellate ganglia neurons, we established that the effect of NaCl was dose-dependent and independent of osmolarity, Cl− and membrane Ca2+ flux, and critically dependent on extracellular Na+ concentration. We show that Nax is expressed in sympathetic stellate ganglia neurons at a transcript and protein level using single-cell RNA-sequencing and immunohistochemistry respectively. Additionally, the response to NaCl was prevented by siRNA-mediated knockdown of Nax, but not by inhibition of other membrane Na+ pathways. Together, these results demonstrate that post-ganglionic sympathetic neurons are direct sensors of extracellular Na+via Nax, which could contribute to sympathetic driven hypertension.

Supplemental Digital Content is available in the text., Single or combinatorial administration of β-blockers is a mainstay treatment strategy for conditions caused by sympathetic overactivity. Conventional wisdom suggests that the main beneficial effect of β-blockers includes resensitization and restoration of β1-adrenergic signaling pathways in the myocardium, improvements in cardiomyocyte contractility, and reversal of ventricular sensitization. However, emerging evidence indicates that another beneficial effect of β-blockers in disease may reside in sympathetic neurons. We investigated whether β-adrenoceptors are present on postganglionic sympathetic neurons and facilitate neurotransmission in a feed-forward manner. Using a combination of immunocytochemistry, RNA sequencing, Förster resonance energy transfer, and intracellular Ca2+ imaging, we demonstrate the presence of β-adrenoceptors on presynaptic sympathetic neurons in both human and rat stellate ganglia. In diseased neurons from the prehypertensive rat, there was enhanced β-adrenoceptor–mediated signaling predominantly via β2-adrenoceptor activation. Moreover, in human and rat neurons, we identified the presence of the epinephrine-synthesizing enzyme PNMT (phenylethanolamine-N-methyltransferase). Using high-pressure liquid chromatography with electrochemical detection, we measured greater epinephrine content and evoked release from the prehypertensive rat cardiac-stellate ganglia. We conclude that neurotransmitter switching resulting in enhanced epinephrine release, may provide presynaptic positive feedback on β-adrenoceptors to promote further release, that leads to greater postsynaptic excitability in disease, before increases in arterial blood pressure. Targeting neuronal β-adrenoceptor downstream signaling could provide therapeutic opportunity to minimize end-organ damage caused by sympathetic overactivity.

Mineral carbonation in ultramafic mine tailings is generally accepted to be a safe and long term means of trapping and storing CO2 within the structures of minerals, but it poses the risk of releasing potentially hazardous metal contaminants from mineral wastes into the environment. Stockpiles of reactive, finely pulverised ultramafic mine tailings are ideal natural laboratories for the observation and promotion of the carbonation of Mg-silicate and Mg-hydroxide waste minerals via reaction with atmospheric or industrial CO2. However, ultramafic mine tailings commonly contain first-row transition metals (e.g., Cr, Co, Cu, Ni) in potentially toxic concentrations within the crystal structures of Mg-silicates, sulphides, and oxides. These transition metals are likely to be mobilised by mineral carbonation reactions, which require mineral dissolution to supply cations for reaction with carbon. At Woodsreef Chrysotile Mine, New South Wales, Australia, transition metals (i.e., Fe, Cr, Ni, Mn, Co, Cu) are most concentrated within minor oxides (magnetite and chromite) and trace alloys (awaruite, Ni2-3Fe and wairauite, CoFe) in serpentine tailings, however, mobilisation of transition metals appears to occur predominantly during dissolution of serpentine and brucite, which are more abundant and reactive phases, respectively. Here, we present new synchrotron X-ray fluorescence mapping data that provide insights into the mobility of first-row transition metals (Fe, Cr, Ni, Mn, Co, Cu) during weathering and carbonation of ultramafic mine tailings collected from the Woodsreef Chrysotile Mine. These data indicate that the recently precipitated carbonate minerals, hydromagnesite [Mg5(CO3)4(OH)2·4H2O] and pyroaurite [Mg6Fe2(CO3)(OH)16·4H2O] sequester trace metals from the tailings at concentrations of 10 s–100 s of ppm, most likely via substitution for Mg or Fe within their crystal structures, or by the physical trapping of small (μm-scale) transition-metal-rich grains (i.e., magnetite, chromite, awaruite), which are stabilised within alkaline carbonate cements. Trace transition metals are present at relatively high concentrations in the bulk tailings (i.e., ∼0.3 wt.% NiO and Cr2O3) and they are largely retained within the unaltered mineral assemblage. The weathering products that occur at the surface of the tailings and form a cement between grains of partially dissolved gangue minerals immobilise transition metals on spatial scales of micrometres and at comparable concentrations to those observed in the unaltered tailings. The end result is that trace metals are not present at detectable levels within mine pit waters. Our observations of metal mobility during passive carbonation suggest that mineral products of accelerated carbonation treatments are likely to sequester trace metals. Thus, accelerated carbonation is unlikely to pose an environmental risk in the form of metalliferous drainage so long as the neutralisation potential of the tailings is not exceeded. Understanding both trace transition metal geochemistry and mineralogy within materials targeted for mineral carbonation could allow optimisation of treatment processes and design for recovery of valuable metals. In ex situ reactors employing acid pre-treatments, trace metals mobilised from reactive phases such as serpentine and brucite could potentially be recovered using pH-swing methods, while recalcitrant metal-rich accessory minerals, including magnetite, awaruite and chromite, could be recovered from treated residue material by conventional mineral separation processes. Recovery of valuable metals (i.e., Ni, Cr and Co) as by-products of accelerated mineral carbonation technologies could also provide an important economic incentive to support broader adoption of this technology.

Supplemental Digital Content is available in the text., Reduced cardiomyocyte excitation–contraction coupling and downregulation of the SERCA2a (sarcoendoplasmic reticulum calcium ATPase 2a) is associated with heart failure. This has led to viral transgene upregulation of SERCA2a in cardiomyocytes as a treatment. We hypothesized that SERCA2a gene therapy expressed under a similar promiscuous cytomegalovirus promoter could also affect the cardiac sympathetic neural axis and promote sympathoexcitation. Stellate neurons were isolated from 90 to 120 g male, Sprague–Dawley, Wistar Kyoto, and spontaneously hypertensive rats. Neurons were infected with Ad-mCherry or Ad-mCherry-hATP2Aa (SERCA2a). Intracellular Ca2+ changes were measured using fura-2AM in response to KCl, caffeine, thapsigargin, and carbonylcyanide-p-trifluoromethoxyphenylhydrazine to mobilize intracellular Ca2+ stores. The effect of SERCA2a on neurotransmitter release was measured using [3H]-norepinephrine overflow from 340 to 360 g Sprague–Dawley rat atria in response to right stellate ganglia stimulation. Upregulation of SERCA2a resulted in greater neurotransmitter release in response to stellate stimulation compared with control (empty: 98.7±20.5 cpm, n=7; SERCA: 186.5±28.41 cpm, n=8; P

Lena Succar,1 Julia Lai-Kwon,1 David J Nikolic-Paterson,2 Gopala K Rangan1 1Centre for Transplant and Renal Research, Westmead Millennium Institute, University of Sydney, Westmead Hospital, Sydney, NSW, Australia; 2Department of Nephrology and Monash University Department of Medicine, Monash Medical Centre, Clayton, VIC, Australia Background: The study aimed to test the hypothesis that therapeutic treatment with a mammalian target of rapamycin complex 1 inhibitor reduces renal cell proliferation and attenuates glomerular and tubulointerstitial injury in the early phase of nephrotoxic serum nephritis (NSN) in rats. Methods: Male Wistar-Kyoto rats received a single tail-vein injection of sheep anti-rat glomerular basement membrane serum (day 0) and were treated with vehicle or sirolimus (0.25 mg/kg/day by subcutaneous injection) from day 1 until day 14. Results: Treatment with sirolimus attenuated kidney enlargement by 41% (P

Regular physical exercise reduces the risk of cardiovascular disease and improves outcome in patients with cardiovascular diseases. The dynamic changes in blood pressure and heart rate with acute exercise are independently predictive of prognosis. Quantification of the haemodynamic response to exercise training in genetically modified mouse models may provide insight into the molecular mechanisms underlying the beneficial effects of exercise. We describe, for the first time, the use of radiotelemetry to provide continuous blood pressure monitoring in C57BL/6J mice during a programme of voluntary wheel exercise with continuous simultaneous recording and analysis of wheel rotations and beat-by-beat haemodynamic parameters. We define distinct haemodynamic profiles at rest, during normal cage activity and during episodes of voluntary wheel running. We show that whilst cage activity is associated with significant rises both in blood pressure and in heart rate, voluntary wheel running leads to a further substantial rise in heart rate with only a small increment in blood pressure. With 5 weeks of chronic exercise training, resting heart rate progressively falls, but heart rate during episodes of wheel running initially increases. In contrast, there are minimal changes in blood pressure in response to chronic exercise training. Finally, we have quantified the acute changes in heart rate at the onset of and recovery from individual episodes of wheel running, revealing that changes in heart rate are extremely rapid and that the peak rate of change of heart rate increases with chronic exercise training. The results of this study have important implications for the use of genetically modified mouse models to investigate the beneficial haemodynamic effects of chronic exercise on blood pressure and cardiovascular diseases. © 2011 The Physiological Society.

Background: NO donors exert a positive chronotropic effect by stimulating the pacemaker current If in sino-atrial node cells via a mechanism requiring guanylyl cyclase (GC) and cGMP, and involving mobilization of sarcoplasmic (SR) Ca2+. Cyclic GMP can directly stimulate If but it is ∼10-fold less potent than the 'physiological' If-gating nucleotide, cAMP. In ventricular myocytes, NO can increase cAMP levels through a cGMP-dependent inhibition of PDE3. Aim: To elucidate the role of PDE3, PKA and PKG in mediating the NO-If-dependent increase in HR. Methods and Results: In isolated guinea-pig atria with basal HR (B/L) of 174±3 bpm (n=69), we evaluated the HR response to increasing concentrations (from 0.1 to 100μmol/L) of the NO donors DEA-NO (n=8) or SIN-1 (+SOD 100 U/mL, n=6) or to 10 μmol/L SNP (n=9), (1) alone, and (2) after pretreatment (ca. 40min) with inhibitors of (i) PDE3 (milrinone, trequinsin or Ro-13-6482, n=22) or (ii) PKA (KT5720 or H-89, n=15) or (iii) PKG (KT5823 or Rp-8-pCPT-cGMPs, n=16). Whereas PKG inhibition had no effect on the magnitude of the increase in HR with DEA-NO (see Fig; bars show the peak increase in HR, mean± SEM; =p

Hypertension is associated with impaired nitric oxide (NO) - cyclic nucleotide (CN) - coupled intracellular calcium (Ca2+) homeostasis that enhances cardiac sympathetic neurotransmission. Because neuronal membrane CA2+ currents are reduced by NO-activated S-nitrosylation, we tested whether CNs affect membrane channel conductance directly in neurons isolated from the stellate ganglia of spontaneously hypertensive rats (SHRs) and their normotensive controls. Using voltage-clamp and cAMP-protein kinase A (PKA) FRET sensors, we hypothesized that impaired CN regulation provides a direct link to abnormal signaling of neuronal calcium channels in the SHR and that targeting cGMP can restore the channel phenotype. We found significantly larger whole-cell Ca2+ currents from diseased neurons that were largely mediated by the N-type Ca2+ channel (Cav2.2). Elevating cGMP restored the SHR Ca2+ current to levels seen in normal neurons that were not affected by cGMP. cGMP also decreased cAMP levels and PKA activity in diseased neurons. In contrast, cAMP-PKA activity was increased in normal neurons, suggesting differential swtching in phosphodiesterase (PDE) activity. PDE2A inhibition enhanced the Ca2+ current in normal neurons to a conductance similar to that seen in SHR neurons whereas the inhibitor slightly decreased the current in diseased neurons. Pharmacological evidence supported a switching a cGMP acting via PDE3 in control neurons to PDE2A in SHR neurons in the modulation of the Ca2+ current. Our data suggest that a disturbance in the regulation of PDE-coupled CNs linked to N-type Ca2+ channels is an early hallmark of the prohypertensive phenotype associated with intracellular Ca2+ impairment underpinning sympathetic dysautonomia.

Implantable cardiac vagal nerve stimulators are a promising treatment for ventricular arrhythmia in patients with heart failure. Animal studies suggest the antifibrillatory effect may be nitric oxide (NO) dependent, although the exact site of action is controversial. We investigated whether a stable analogue of acetylcholine could raise ventricular fibrillation threshold (VFT), and whether this was dependent on NO generation and/or muscarinic/nicotinic receptor stimulation.VFT was determined in Langendorff perfused rat hearts by burst pacing until sustained VF was induced. Carbamylcholine (CCh, 200 nmol/L, n = 9) significantly (P

One hundred years ago in this journal, Krogh and Lindhard published a seminal paper highlighting the importance of the brain in the control of breathing during exercise. This symposium report reviews the historical developments that have taken place since 1913, and attempts to place the detailed neurocircuitry thought to underpin exercise hyperpnoea into context by focusing on key structures that might form the command network. With the advent of enhanced neuroimaging and functional neurosurgical techniques, a unique window of opportunity has recently arisen to target potential circuits in humans. Animal studies have identified a priori sites of interest in mid-brain structures, in particular the subthalamic locomotor region (subthalamic nucleus, STN) and the periaqueductal grey (PAG), which have now been recorded from in humans during exercise. When all data are viewed in an integrative manner, the PAG, in particular the lateral PAG, and aspects of the dorsal lateral PAG, appear to be key communicating circuitry for 'central command'. Moreover, the PAG also fulfils many requirements of a command centre. It has functional connectivity to higher centres (dorsal lateral prefrontal cortex) and the basal ganglia (in particular, the STN), and receives a sensory input from contracting muscle, but, importantly, it sends efferent information to brainstem nuclei involved in cardiorespiratory control.

The traditional model of efferent cardiac noradrenaline and acetylcholine release being driven solely via brainstem integration of circulatory reflex afferent input needs to be modified in the light of the discovery of numerous local cardiac factors that impact on peripheral neuronal neurotransmitter release. These neuromodulators can be intrinsic to sympathetic ganglia or vagal neurons (such as neuronal nitric oxide synthase), act as cotransmitters between these neuronal populations (such as neuropeptide Y) or are released from the myocardium itself to act on neurons in a paracrine manner (such as natriuretic peptides). Both myocardial infarction and congestive heart failure are characterized by enhanced regulation of these neuromodulators. This review will focus on recent evidence that nitric oxide, natriuretic peptides and neuropeptide Y act by converging on neuronal cyclic nucleotide-dependent pathways to alter the autonomic phenotype in both health and disease.

Steep action potential duration (APD) restitution has been shown to facilitate wavebreak and ventricular fibrillation. The global APD restitution properties in cardiac patients are unknown. We report a combined clinical electrophysiology and computer modelling study to: (1) determine global APD restitution properties in cardiac patients; and (2) examine the interaction of the observed APD restitution with known arrhythmia mechanisms. In 14 patients aged 52-85 years undergoing routine cardiac surgery, 256 electrode epicardial mapping was performed. Activation-recovery intervals (ARI; a surrogate for APD) were recorded over the entire ventricular surface. Mono-exponential restitution curves were constructed for each electrode site using a standard S1-S2 pacing protocol. The median maximum restitution slope was 0.91, with 27% of all electrode sites with slopes1 over a range of diastolic intervals of at least 30 ms; and 0.3% for at least 50 ms. Activation-recovery interval restitution was spatially heterogeneous, showing regional organization with multiple discrete areas of steep and shallow slope. We used a simplified computer model of 2-D cardiac tissue to investigate how heterogeneous APD restitution can influence vulnerability to, and stability of re-entry. Our model showed that heterogeneity of restitution can act as a potent arrhythmogenic substrate, as well as influencing the stability of re-entrant arrhythmias. Global epicardial mapping in humans showed that APD restitution slopes were organized into regions of shallow and steep slopes. This heterogeneous organization of restitution may provide a substrate for arrhythmia.

Inhibition of major histocompatibility complex (MHC) class II expression by macrophages is the primary mechanism by which interleukin-10 (IL-10) exerts immune suppression. Little, however, is known of the effects of IL-10 on other types of cells which can be induced to express MHC class II during an inflammatory response. We therefore studied the effects of IL-10 treatment on the expression of MHC class II molecules in a rat model of immunologically induced glomerulonephritis. MHC class II mRNA levels in whole kidney were increased in saline-treated (control) animals with glomerulonephritis (2.6-fold increase versus normal, P = 0.028) and this was partially inhibited by treatment with IL-10 (P = NS). Double immunostaining of tissue sections was used to compare MHC class II expression by infiltrating macrophages and resident glomerular cells. IL-10 treatment reduced the proportion of glomerular macrophages which expressed detectable MHC class II (70% reduction, P = 0.03). In contrast, IL-10 treatment was associated with an increase in the number of resident glomerular cells expressing MHC class II, particularly within mesangial areas. Therefore, the effects of IL-10 on macrophages and mesangial cells were compared in vitro. IL-10 reduced constitutive MHC class II mRNA and cell surface expression by peritoneal macrophages. In contrast, IFN-gamma-stimulated mesangial cells (1097 cell line) cultured with IL-10 for 24 hr showed increased MHC class II mRNA (26% increase) and surface expression (72% increase in percentage MHC II+ by flow cytometry, P = 0.04) as compared with cells stimulated with IFN-gamma alone. IL-10 also directly up-regulated expression of ICAM-1 by 1997 cells. In conclusion, IL-10 was found to have contrasting effects on the production and cell surface expression of MHC class II molecules by mesengial cells and by macrophages, both in vitro and in vivo. The implications of these findings for IL-10-mediated immunosuppression are discussed.