The demographics of ageing are changing dramatically such that there will be many more older adults in the near future. This setting likely will produce a new “boomer-driven” epidemic of physiological dysfunction, disability and risk of chronic degenerative disorders, including cardiovascular diseases (CVD). Standing out against this dreary biomedical forecast are Masters athletes, a group of middle-aged and older adults who engage in regular vigorous physical training and competitive sport. Compared with their sedentary/less active (untrained) peers, Masters athletes who perform endurance training-based activities demonstrate a more favorable arterial function-structure phenotype, including lower large elastic artery stiffness, enhanced vascular endothelial function and less arterial wall hypertrophy. As such, they may represent an exemplary model of healthy or “successful” vascular ageing. In contrast, Masters athletes engaged primarily/exclusively in intensive resistance training exhibit less favorable arterial function-structure than their endurance-trained peers and, in some instances, untrained adults. These different arterial properties likely are explained in large part by the different intravascular mechanical forces generated during endurance vs. resistance exercise-related training activities. The more favorable arterial function-structure profile of Masters endurance athletes may contribute to their low risk of clinical CVD.

Background

This study investigated the effect of changes in inspiratory intrathoracic pressure (ITP) on stroke volume (SV) at rest and during moderate exercise in patients with heart failure and reduced ejection fraction (HFREF) as well as healthy individuals.

Methods and Results

SV was obtained by echocardiography during 2 minutes of spontaneous breathing (S), 2 progressive levels of inspiratory unloading (UL1 and UL2) using a ventilator, and 2 progressive levels of inspiratory loading using resistors in 11 patients with HFREF (61±9 years, EF: 32±4 %, NYHA class I-II) and 11 age-matched healthy individuals at rest and during exercise at 60% of maximal aerobic capacity on a semi-recumbent cycle ergometer. At rest, inspiratory unloading progressively decreased stroke volume index (SVI) (S: 35.2 ± 5.4, UL1: 33.3 ± 5.1, UL2: 32.2 ± 4.4 ml/m2) in healthy individuals while it increased SVI (S: 31.4 ± 4.6, UL1: 32.0 ± 5.9, UL2: 34.0 ± 7.2 ml/m2) in patients with HFREF (p=0.04). During moderate exercise, inspiratory unloading similarly decreased SVI (S: 43.9 ± 7.1, UL1: 40.7 ± 4.7, UL2: 39.9 ± 3.7 ml/m2) in healthy individuals while it increased SVI (S: 40.8 ± 6.5, UL1: 42.8 ± 6.9, UL2: 44.1 ± 4.8 ml/m2) in patients with HFREF (p=0.02). Inspiratory loading did not significantly change SVI at rest or during moderate exercise in both groups.

Conclusions

Inspiratory unloading improved SVI at rest and during moderate exercise in patients with HFREF, possibly due to a reduction in LV afterload.

Interstitial cells of Cajal (ICC) generate electrical pacemaker activity in gastrointestinal (GI) smooth muscles. We investigated whether Tmem16a, which encodes Anoctamin 1 (ANO1), a Ca2+-activated Cl− channel might be involved in pacemaker activity in ICC. Tmem16a transcripts and ANO1 were expressed robustly in GI muscles, specifically in ICC in murine, non-human primate (Macaca fascicularis) and human GI tracts. Splice variants of Tmem16a are expressed in GI muscles, as well as other paralogues of the Tmem16 family. Ca2+-activated Cl− (CaCC) channel blocking drugs, niflumic acid and 4,4′-diisothiocyano-2,2′-stillbene-disulfonic acid (DIDS), blocked slow waves in intact muscles of mouse, primate, and human small intestine and stomach. Slow waves failed to develop in Tmem16a knock-out mice (Tmem16atm1Bdh/tm1Bdh). The pacemaker mechanism was investigated in isolated ICC from transgenic mice with constitutive expression of copGFP. Depolarization of ICC activated inward currents due to a Cl− selective conductance. Removal of extracellular Ca2+, replacement of Ca2+ with Ba2+, or extracellular Ni2+ (30 μm) blocked the inward current. Single Ca2+-activated Cl− channels with a unitary conductance of 7.8 pS were resolved in excised patches from ICC. The inward current was blocked in a concentration-dependent manner by niflumic acid (IC50 = 4.8 μm). The role of ANO1 in cholinergic responses in ICC was also investigated. CCh activated Ca2+-activated Cl− currents in ICC, and responses to cholinergic nerve stimulation were blocked by niflumic acid in intact muscles. ANO1 is a prominent conductance in ICC and these channels appear to be involved in pacemaker activity and in responses to enteric excitatory neurotransmitters.

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.

Hypertension caused by chronic infusion of angiotensin II (AngII) in experimental animals is dependent, in part, on increased activity of the sympathetic nervous system. This chronic sympathoexcitatory response is amplified by a high salt diet suggesting an interaction of circulating AngII and dietary salt on sympathetic regulatory pathways in the brain. The present study tested the hypothesis that the subfornical organ (SFO), a forebrain circumventricular organ known to be activated by circulating AngII, is crucial to the pathogenesis of hypertension induced by chronic AngII administration in rats on a high salt diet (AngII-salt model). Rats were randomly selected to undergo either subfornical organ lesion (SFOx) or sham surgery (SHAM) and then placed on a high salt (2% NaCl) diet. One week later rats were instrumented for radiotelemetric measurement of mean arterial pressure (MAP) and heart rate (HR) and placed in metabolic cages to measure sodium and water balance. Baseline MAP was slightly (but not statistically) lower in SFOx compared to SHAM rats during the 5 day control period. During the subsequent 10 days of AngII administration, MAPwas statistically lower in SFOx rats. However, when MAP responses to AngII were analyzed by comparing the change from the 5 day baseline period, only on the 5th day of AngII was MAP significantly different between groups. There were no differences between groups for water or sodium balance throughout the protocol. We conclude that, although the SFO is required for the complete expression of AngII-salt hypertension in the rat, other brains sites are also involved.

The Cystic Fibrosis Transmembrane Conductance Regulator, CFTR, is both an anion channel and a regulator of other transport proteins. CFTR gene mutations underlie the human disease, Cystic Fibrosis (CF). The most common CFTR mutation, ΔF508, produces a misfolded protein which traffics improperly. The availability of transgenic CFTRΔF508/ΔF508 pigs allows measurement of the impact of ΔF508 in native tissue. Thyroid epithelia respond to cAMP-elevating agents by increasing anion transport, a process hinging on functional CFTR. To assess whether endogenous levels of ΔF508 CFTR mediate thyroid transport, primary thyroid epithelial cultures (pThECs) were grown from newborn CFTR+/+ (WT) and CFTRΔF508/ΔF508 (ΔF) pig thyroids and the stimulated, secretory components of short-circuit current (Isc) compared. Surface biotinylation studies assessed the surface presentation of ΔF508 CFTR. Baseline Isc levels of both wt and ΔF pThECs consisted of an amiloride-sensitive component. In ΔF pThECs, this mirrored previous measurements in CFTR−/− (KO) pThECs. Surprisingly, elevation of cAMP transiently increased Isc to peak levels ~ 65% those achieved by wt. In contrast, KO pThECs were indifferent to cAMP activation. In ΔF pThECs, total ΔF508 CFTR expression was ~ 9% that of WT, consistent with misfolding and enhanced degradation. Surface biotinylation studies indicated that ~ 4% of the total ΔF508 resided at the surface and did not increase with cAMP elevation. The present findings show that low endogenous levels of pig ΔF508 CFTR can mediate substantial anion transport by thyroid epithelia. These data suggest that both wt and ΔF508-CFTR regulate additional, thyroid transporters, and together coordinate the overall Isc response.

The Murphy Roths Large (MRL) mouse, a strain capable of regenerating right ventricular myocardium, has a high post-myocardial infarction (MI) survival rate compared with C57BL/6J (C57) mice. The biological processes responsible for this survival advantage are unknown. To assess the effect of genetic background, the LG/J strain, which harbors 75% of the MRL composite genome, was included in the study. The MRL survival advantage versus C57 mice (92% vs. 68%, P < 0.05) occurred primarily in the first 5 days; LG/J survival was intermediate (P = NS). Microarray data analysis revealed an attenuation of apoptotic (P < 0.05) and stress response transcripts in MRL hearts compared with C57 hearts after MI. Supporting the microarray results, there were fewer TUNEL-positive cells 1 day post-MI in MRL infarcts compared with C57 infarcts (P = 0.001) and fewer CD45-positive cells in the MRL infarct border zone 2 days post-MI (P < 0.01). LG/J results were intermediate (P = NS). MRL hearts had smaller infarct scars and attenuated ventricular dilation 30 days post-MI compared with C57 hearts (P < 0.05). We conclude that the early post-MI survival advantage of MRL mice over the C57 strain is mediated at least in part by reductions in apoptosis and inflammatory infiltration, and that these reductions may influence chronic remodeling. The intermediate survival, apoptosis and inflammation profile of LG/J mice suggests this high tolerance for MI in the MRL could be derived from its shared genetic background with the LG/J.

After considerable debate and key experimental evidence, the importance of the arterial baroreflex in contributing to and maintaining the appropriate neural cardiovascular adjustments to exercise is now well accepted. Indeed, the arterial baroreflex resets during exercise in an intensity-dependent manner to continue to regulate blood pressure as effectively as at rest. Studies have indicated that the exercise resetting of the arterial baroreflex is mediated by both the feed-forward mechanism of central command and the feed-back mechanism associated with skeletal muscle afferents (the exercise pressor reflex). Another perhaps less appreciated neural mechanism involved in evoking and maintaining neural cardiovascular responses to exercise is the cardiopulmonary baroreflex. The limited information available regarding the cardiopulmonary baroreflex during exercise provides evidence for a role in mediating sympathetic nerve activity and blood pressure responses. In addition, recent investigations have demonstrated an interaction between cardiopulmonary baroreceptors and the arterial baroreflex during dynamic exercise, which contributes to the magnitude of exercise-induced increases in blood pressure as well as the resetting of the arterial baroreflex. Furthermore, neural inputs from the cardiopulmonary baroreceptors appear to play an important role in establishing the operating point of the arterial baroreflex. This symposium review will highlight recent studies in these important areas indicating that the interactions of four neural mechanisms (central command, the exercise pressor reflex, the arterial baroreflex and cardiopulmonary baroreflex) are integral in mediating the neural cardiovascular adjustments to exercise.

Power spectral analysis of heart rate variability (HRV) has been used frequently to assess cardiac autonomic function; however, the relationship of low frequency (LF) power of HRV to cardiac sympathetic tone has been unclear. With or without adjustment for high frequency (HF) power, total power, or respiration, LF power seems to provide an index not of cardiac sympathetic tone but of baroreflex function. Manipulations and drugs that change LF power or LF:HF may do so not by affecting cardiac autonomic outflows directly but by affecting modulation of those outflows by baroreflexes.

An in vitro motility assay approach was used to investigate the mechanisms of the functional differences between myosin isoforms, by studying the effect of MgATP and MgADP on actin sliding velocity (Vf) of pure slow and fast rat skeletal myosin at different temperatures. The value of Vf depended on [MgATP] according to Michaelis–Menten kinetics, with an apparent constant (Km) of 54.2, 64.4 and 200 μm for the fast isoform and 18.6, 36.5 and 45.5 μm for the slow isoform at 20, 25 and 35°C, respectively. The presence of 2 mm MgADP decreased Vf and yielded an inhibition constant (Ki) of 377, 463 and 533 μm for the fast isoform at 20, 25 and 35°C, respectively, and 120 and 355 μm for the slow isoform at 25 and 35°C, respectively. The analysis of Km and Ki suggested that slow and fast isoforms differ in the kinetics limiting Vf. Moreover, the higher sensitivity of the fast myosin isoform to a drop in [MgATP] is consistent with the higher fatigability of fast fibres than slow fibres. From the Michaelis–Menten relation in the absence of MgADP, we calculated the rate of actomyosin dissociation by MgATP (k+ATP) and the rate of MgADP release (k–ADP). We found values of k+ATP of 4.8 × 106, 6.5 × 106 and 6.6 × 106 M−1 s−1 for the fast isoform and 3.3 × 106, 2.9 × 106 and 6.7 × 106 M−1 s−1 for the slow isoform and values of k–ADP of 263, 420 and 1320 s−1 for the fast isoform and 62, 107 and 306 s−1 for the slow isoform at 20, 25 and 35°C, respectively. The results suggest that k–ADP could be the major determinant of functional differences between the fast and slow myosin isoforms at physiological temperatures.

In rats with salt-induced hypertension or post myocardial infarction (MI), AT1 receptor (AT1R) densities and oxidative stress increase and neuronal nitric oxide synthase (nNOS) levels decrease in the paraventricular nucleus (PVN). The present study was designed to determine whether these changes may depend on activation of the aldosterone – “ouabain” neuromodulatory pathway. After intracerebroventricular (icv) infusion of aldosterone (20ng/h) for 14 days, blood pressure (BP) and heart rate (HR) were recorded in conscious Wistar rats, and mRNA and protein for nNOS, endothelial NOS (eNOS), AT1R and NADPH oxidase subunits were assessed in brain tissue. BP and HR were significantly increased by aldosterone. Aldosterone significantly increased mRNA and protein of AT1R, P22phox, P47phox, P67phox and Nox2, and decreased nNOS but not eNOS mRNA and protein in the PVN, and increased angiotensin converting enzyme (ACE) and AT1R binding densities in the PVN and SON. The increases in BP and HR as well as changes in mRNA, proteins and ACE and AT1R binding densities were all largely prevented by concomitant icv infusion of Digibind (to bind “ouabain”) or benzamil (to block presumably epithelial sodium channels). These data indicate that aldosterone via “ouabain” increases in the PVN ACE, AT1R and oxidative stress but decreases nNOS, and suggest that endogenous aldosterone may cause this similar pattern of changes observed in salt sensitive hypertension and heart failure post MI.

Ghrelin is a gut peptide that has been studied extensively for its role in food intake and energy balance. More recent studies show that ghrelin reduces water intake in rats and some non-mammalian species. Despite the importance of the regulation of NaCl intake in body fluid homeostasis, the effects of ghrelin on saline intake have not been investigated. Accordingly, we tested the effect of ghrelin on water and 1.8% NaCl intake in two-bottle test conditions under five stimuli that increase hypertonic saline intake: central angiotensin II administration, 24 h fluid deprivation, water deprivation followed by partial rehydration, dietary sodium deficiency, and polyethylene glycol administration combined with dietary sodium deficiency. We found that ghrelin attenuated saline intake stimulated by angiotensin II, by water deprivation followed by partial rehydration, and by dietary sodium deficiency. We did not detect an effect of ghrelin on saline intake after 24 h fluid deprivation without partial rehydration or after the combination of polyethylene glycol and dietary sodium deficiency. The finding that ghrelin reduced hypertonic saline intake under some, but not all, natriorexigenic conditions mirrors the previously published findings that in one-bottle tests of drinking, ghrelin reduces water intake under only some conditions. The results provide evidence for a new role for ghrelin in the regulation of body fluid homeostasis.

We recently observed a marked increase in brachial artery (BA) diameter during prolonged leg cycling exercise. The purpose of the present study was to test the hypothesis that this increase in BA diameter during lower limb exercise is shear stress mediated. Accordingly, we determined whether recapitulation of cycling-induced BA shear rate with forearm heating, a known stimulus evoking shear-induced conduit artery dilatation, would elicit comparable profiles and magnitudes of BA vasodilatation to those observed during cycling. In 12 healthy men, BA diameter and blood velocity were measured simultaneously using Doppler ultrasonography at baseline and every 5 min during 60 min of either steady-state semi-recumbent leg cycling (120 W) or forearm heating. At the onset of cycling, the BA diameter was reduced (−3.9 ± 1.2% at 5 min; P < 0.05), but it subsequently increased throughout the remainder of the exercise bout (+15.1 ± 1.6% at 60 min; P < 0.05). The increase in BA diameter during exercise was accompanied by an approximately 2.5-fold rise in BA mean shear rate (P < 0.05). Similar increases in BA mean shear with forearm heating elicited an equivalent magnitude of BA vasodilatation to that observed during cycling (P > 0.05). Herein, we found that in the absence of exercise the extent of the BA vasodilator response was reproduced when the BA was exposed to comparable magnitudes of shear rate via forearm heating. These results are consistent with the hypothesis that shear stress plays a key role in signalling brachial artery vasodilatation during dynamic leg exercise.

New ideas about the relative importance of the autonomic nervous system (and especially its sympathetic arm) in long-term blood pressure regulation are emerging. It is well known that mean arterial blood pressure is normally regulated in a fairly narrow range at rest and that blood pressure is also able to rise and fall ‘appropriately’ to meet the demands of various forms of mental, emotional and physical stress. By contrast, blood pressure varies widely when the autonomic nervous system is absent or when key mechanisms that govern it are destroyed. However, 24 h mean arterial pressure is still surprisingly normal under these conditions. Thus, the dominant idea has been that the kidney is the main long-term regulator of blood pressure and the autonomic nervous system is important in short-term regulation. However, this ‘renocentric’ scheme can be challenged by observations in humans showing that there is a high degree of individual variability in elements of the autonomic nervous system. Along these lines, the level of sympathetic outflow, the adrenergic responsiveness of blood vessels and individual haemodynamic patterns appear to exist in a complex, but appropriate, balance in normotension. Furthermore, evidence from animals and humans has now clearly shown that the sympathetic nervous system can play an important role in longer term blood pressure regulation in both normotension and hypertension. Finally, humans with high baseline sympathetic traffic might be at increased risk for hypertension if the ‘balance’ among factors deteriorates or is lost. In this context, the goal of this review is to encourage a comprehensive rethinking of the complexities related to long-term blood pressure regulation in humans and promote finer appreciation of physiological relationships among the autonomic nervous system, vascular function, ageing, metabolism and blood pressure.

We have reported that airway nociceptors [C fiber receptors (CFRs) and high threshold A-delta fiber receptors (HTARs)] are activated during oleic acid (OA) induced acute lung injury. In the current studies, we tested the hypothesis that this nociceptor activation is mediated by arachidonic acid products. In anesthetized, open chest, and mechanically ventilated rabbits, we examined the response of the nociceptors to intravenous injection of OA before and after blocking the cyclo-oxygenase pathways by indomethacin. Pre-treatment with indomethacin (20 mg/kg) decreased the background activities of both CFRs (from 0.48±0.12 to 0.25±0.08, n=7, p<0.05) and HTARs (from 0.54±0.14 to 0.23±0.08, n=10, p<0.01). It also blocked the nociceptors’ response to OA. Similarly, pre-treatment with thromboxane synthase inhibitor (ketoconazole) also blocked the nociceptor response to OA. In addition, local microinjection or intravenous injection of a thromboxane mimetic stimulated CFRs and HTARs. The current results clearly indicate that arachidonic acid metabolites mediate airway nociceptor activation during OA-induced acute lung injury and suggest that thromboxane may be a key mediator.

Arterial compliance, the inverse of arterial stiffness, is a prognostic indicator of arterial health. Central and peripheral arterial compliance decrease with acute cold stress and may increase post exercise when exercise-induced elevations in core temperature are likely still present. Increased blood flow through the conduit arteries associated with elevated core temperature increases shear stress which in turn releases nitric oxide and other endothelial derived factors. These changes, in conjunction with supportive in vitro data, suggest that elevated core temperature may indirectly increase central and peripheral arterial compliance (i.e., decrease arterial stiffness). The purpose of this study was to test the hypothesis that increased core temperature decreases central and peripheral arterial stiffness, as measured with pulse wave velocity (PWV). Using Doppler ultrasound, carotid-femoral (central) and carotid-radial (peripheral) arterial PWVs were measured from eight subjects (age 37 ± 11 years; mass 68.8 ± 11.1 kg; height 171 ± 3 cm) before and during passive heat-stress induced increases in core temperature of 0.47 ± 0.05, 1.03 ± 0.12, and 1.52 ± 0.07°C (i.e., baseline, 0.5, 1.0, and 1.5°C, respectively). Changes in PWV were evaluated with a one-way repeated measures ANOVA. When analyzed as group means, neither central (677 ± 161, 617 ± 72, 659 ± 74, and 766 ± 207 cm/s; P=0.12) nor peripheral (855 ± 192, 772 ± 95, 759 ± 49, and 858 ± 247 cm/s; P=0.56) PWV changed as core temperature increased from baseline to 0.5, 1.0, and 1.5°C, respectively. However, individual changes in central (average r = −0.89, P < 0.05) and peripheral (average r = −0.93, P < 0.05) PWV with heat stress were significantly correlated with normothermic baseline PWV. In conclusion, these data suggest that the magnitude by which heat stress reduced PWV was predicated upon normothermic PWV, with the individuals having the highest normothermic PWV being most responsive to the heat stress-induced reductions in PWV.

The electroneutral Na/HCO3 cotransporter NBCn1 (SLC4A7) contributes to intracellular pH maintenance and transepithelial HCO3− movement. In this study, we expressed NBCn1 in Xenopus oocytes and examined the effect of NBCn1 on oocyte NH4+ transport by analyzing changes in membrane potential, current, and intracellular pH mediated by NH4Cl. In the presence of HCO3−/CO2, applying NH4Cl (20 mM) produced intracellular acidification of oocytes. The acidification was faster in oocytes expressing NBCn1 than in control oocytes injected with water. However, NH4Cl-mediated membrane depolarization was smaller in oocytes expressing NBCn1. In HCO3−/CO2-free solution, NH4Cl produced a smaller inward current in NBCn1-expressing oocytes (56% inhibition by 20 mM NH4Cl; measured at −60 mV), while minimally affecting intracellular acidification. The inhibition of the current by NBCn1 was unaffected when BaCl2 replaced KCl. Current-voltage relationships showed a positive and nearly linear relationship between NH4Cl-mediated current and voltage, which was markedly reduced by NBCn1. Large basal currents (before NH4Cl exposure) were produced in NBCn1-expressing oocytes due to the previously characterized channel-like activity of NBCn1. Inhibiting this channel-like activity by Na+ removal abolished NBCn1’s inhibitory effect on NH4Cl-mediated currents. The currents were progressively reduced over 72–120 h after NBCn1 cRNA injection, during which the channel-like activity was high. These results indicate that NBCn1 by its Na/HCO3 cotransport activity stimulates NH4+ transport, while reducing NH4+ conductance by its channel-like activity.

Human limb muscle and skin blood flow increases significantly with elevations in temperature, possibly through physiological processes that involve temperature-sensitive regulatory mechanisms. Here we tested the hypothesis that the release of the vasodilator ATP from human erythrocytes is sensitive to physiological increases in temperature both in vitro and in vivo, and examined potential channel/transporters involved. To investigate the source of ATP release, whole blood, red blood cells (RBCs), plasma and serum were heated in vitro to 33, 36, 39 and 42°C. In vitro heating augmented plasma or ‘bathing solution’ ATP in whole blood and RBC samples, but not in either isolated plasma or serum samples. Heat-induced ATP release was blocked by niflumic acid and glibenclamide, but was not affected by inhibitors of nucleoside transport or anion exchange. Heating blood to 42°C enhanced (P < 0.05) membrane protein abundance of cystic fibrosis transmembrane conductance regulator (CFTR) in RBCs. In a parallel in vivo study in humans exposed to whole-body heating at rest and during exercise, increases in muscle temperature from 35 to 40°C correlated strongly with elevations in arterial plasma ATP (r2 = 0.91; P = 0.0001), but not with femoral venous plasma ATP (r2 = 0.61; P = 0.14). In vitro, however, the increase in ATP release from RBCs was similar in arterial and venous samples heated to 39°C. Our findings demonstrate that erythrocyte ATP release is sensitive to physiological increases in temperature, possibly via activation of CFTR-like channels, and suggest that temperature-dependent release of ATP from erythrocytes might be an important mechanism regulating human limb muscle and skin perfusion in conditions that alter blood and tissue temperature.

The neuropeptides substance P (SP) and calcitonin gene-related peptide are believed to be involved in the axon reflex-mediated component of cutaneous thermal hyperaemia, but no studies have specifically addressed this issue. The purpose of this study was to determine whether neurokinin-1 (NK1) receptors, which preferentially bind SP, contribute to the axon-reflex component of cutaneous thermal hyperaemia. Nine subjects were equipped with four microdialysis fibres and received one of four treatments: 1) lactated Ringer’s (control); 2) 10mM L-NAME to inhibit NO synthase; 3) 10μM SP; and 4) 10μM SP + 10mM L-NAME. Skin blood flow was monitored via laser-Doppler flowmetry (LDF) and local skin temperature was controlled using local heating devices. Sites 3 and 4 were perfused with 10μM SP for 15 minutes at a rate of 4μl min−1 and the ensuing vasodilatation was allowed to return to baseline. Following SP-induced vasodilatation, all skin sites were locally heated from a baseline temperature of 33°C to 42°C at a rate of 0.5°C every 5 seconds. Cutaneous vascular conductance (CVC) was calculated as LDF/MAP and normalized to maximal (%CVCmax) via 28mM nitroprusside and local heating to 43°C. Initial peak did not differ between control (79±3%CVCmax) and SP only sites (79±2%CVCmax). Initial peak at L-NAME (43±3%CVCmax) and SP + L-NAME (53±3%CVCmax) sites were significantly reduced compared to both control and SP only sites (p<0.001 for both) and L-NAME sites were attenuated compared to SP + L-NAME sites (p<0.01). There was no observable nadir response at sites pretreated with SP. Compared to control sites (57±4%CVCmax), nadir at L-NAME (14±2%CVCmax) and SP + L-NAME (31±5%CVCmax) sites were significantly reduced (p<0.01 for all conditions). L-NAME significantly reduced the nadir compared to SP + L-NAME (p<0.01). Plateau CVC values did not differ between control (86±3%CVCmax) and SP sites (91±1%CVCmax). At L-NAME (36±4%CVCmax) and SP + L-NAME (56±6%CVCmax) sites, plateau CVC was significantly reduced compared to control and SP only sites (p<0.01 for all conditions). The plateau at L-NAME sites was significantly reduced compared to SP + L-NAME sites (p<0.01). These data suggest NK1 receptors contribute to both the axon reflex component and secondary plateau phase of cutaneous thermal hyperaemia.

The production of vocalizations is intimately linked to the respiratory system. Despite our understanding of neural circuits that generate normal respiratory patterns, very little is understood regarding how these ponto-medullary circuits become engaged during vocal production. Songbirds offer a potentially powerful model system for addressing this relationship. Songs dramatically alter the respiratory pattern in ways that are often highly predictable and songbirds have a specialized telencephalic vocal motor circuit that provides massive innervation to a brainstem respiratory network that shares many similarities with its mammalian counterpart. In this review, we highlight interactions between the song motor circuit and the respiratory system, describing how both systems likely interact to produce the complex respiratory patterns that are observed during vocalization. We also discuss how the respiratory system, through its bilateral bottom-up projections to thalamus, might play a key role in sending precisely timed signals that synchronize premotor activity in both hemispheres.

Central chemoreception is the mechanism by which CO2/pH-sensitive neurons (i.e., chemoreceptors) regulate breathing presumably in response to changes in tissue pH. A region of the brainstem called the retrotrapezoid nucleus (RTN) is thought to be an important site of chemoreception (Guyenet et al., 2010); select neurons (i.e., chemoreceptors) in this region sense changes in CO2/H+ and send excitatory glutamatergic drive to respiratory centers to modulate the depth and frequency of breathing. Purinergic signaling may also contribute to chemoreception, e.g., it was shown in vivo that CO2/H+ facilitates ATP release within the RTN to stimulate breathing (Gourine et al., 2005), and recent evidence suggests that CO2/H+-sensitive RTN astrocytes are the source of this purinergic drive to breathe (Gourine et al., 2010; Huckstepp et al., 2010b; Wenker et al., 2010). In this review, we will summarize evidence that RTN astrocytes sense changes in CO2/H+, identify mechanisms that likely confer CO2/H+-sensitivity to RTN astrocytes, including inhibition of heteromeric Kir4.1–Kir5.1 channels and activation of a depolarizing inward current generated by the sodium/bicarbonate cotransporter, and discuss the extent to which astrocytes contribute to respiratory drive.

Adrenaline activates transient Cl−-secretion and sustained K+-secretion across isolated distal colonic mucosa of guinea pig. The Ca++-activated Cl− channel inhibitor CaCCinh-A01 [30μM] significantly reduced electrogenic K+-secretion, detected as short-circuit current (Isc). This inhibition supported the cell model for K+-secretion in which basolateral membrane Cl− channels provide an exit pathway for Cl− entering the cell via Na+/K+/2Cl−-cotransporters. CaCCinh-A01 inhibited both Isc and transepithelial conductance in a concentration dependent manner, IC50 = 6.3μM. GlyH-101, another Cl− channel inhibitor, also reduced sustained adrenaline-activated Isc (IC50 = 9.4μM). Adrenaline activated whole-cell Cl− current in isolated intact colonic crypts, confirmed by ion substitution. This adrenaline-activated whole-cell Cl− current also was inhibited by CaCCinh-A01 or GlyH-101. In contrast to K+-secretion, CaCCinh-A01 augmented the electrogenic Cl−-secretion activated by adrenaline as well as that activated by PGE2. Synergistic Cl−-secretion activated by cholinergic/PGE2 stimulation was insensitive to CaCCinh-A01. Colonic expression of the Ca++-activated Cl− channel protein Tmem16A was supported by RT-PCR detection of Tmem16A-mRNA, by immuno-blot with a Tmem16A-antibody, and by immuno-fluorescence detection in lateral membranes of epithelial cells. Alternative splices of Tmem16A were detected for exons that are involved in channel activation. Inhibition of K+-secretion and augmentation of Cl−-secretion by CaCCinh-A01 supports a common colonic cell model for these two ion secretory processes, such that activation of basolateral membrane Cl− channels contributes to the production of electrogenic K+-secretion and limits the rate of Cl−-secretion. Maximal physiological Cl−-secretion occurs only for synergistic activation mechanisms that close these basolateral membrane Cl− channels.

The metabolic syndrome is associated with elevated peripheral vascular disease risk, characterized by mismatched blood flow delivery/distribution and local metabolism. The obese Zucker rat (OZR) model of the metabolic syndrome exhibits myriad vascular impairments, although their integrated impact on functional hyperemia remains unclear. In this study, arterial pressor responses and skeletal muscle perfusion were assessed in lean Zucker rats (LZR) and OZR during adrenergic stimulation (phenylephrine), challenge with thromboxane (U46619) and endothelium-dependent dilation (methacholine). OZR were hypertensive versus LZR, but this was abolished by adrenoreceptor blockade (phentolamine); pressor responses to U46619 were similar between strains and were abolished by blockade with the PGH2/TxA2 receptor antagonist, SQ-29548. Depressor reactivity to methacholine was impaired in OZR, but was improved by antioxidant treatment (TEMPOL). Across levels of metabolic demand, blood flow to in situ gastrocnemius muscle was restrained by adrenergic constriction in OZR, although this diminished with increased demand. O2 extraction, reduced in OZR vs. LZR across levels of metabolic demand, was improved by TEMPOL or SQ-29548; treatment with phentolamine did not impact extraction and neither TEMPOL nor SQ-29548 improved muscle blood flow in OZR. While VO2 and muscle performance were consistently reduced in OZR vs. LZR, treatment with all three agents improved outcomes, while treatment with individual agents was less effective. These results suggest that contributions of vascular dysfunction to perfusion, VO2 and muscle performance are spatially distinct, with adrenergic constriction impacting proximal resistance and endothelial dysfunction impacting distal microvessel-tissue exchange. Further, these data suggest that increasing skeletal muscle blood flow in OZR is not sufficient to improve performance, unless distal perfusion inhomogeneities are rectified.

As part of the insulin signaling pathway, AKT influences growth and metabolism. The AKT1 gene G205T (rs1130214) polymorphism has potential functional effects. Thus, we determined whether the G205T polymorphism influences metabolic variables and their responses to aerobic exercise training. Following dietary stabilization, healthy, sedentary, 50-75 yr old Caucasian men (n = 51) and women (n = 58) underwent 6 months of aerobic exercise training. Before and after completing the intervention, dual-energy x-ray absorptiometry measured percent body fat, computed tomography measured visceral and subcutaneous fat, and oral glucose tolerance testing measured glucose total area under the curve (AUC), insulin AUC, and insulin sensitivity. Taqman assay determined AKT1 G205T genotypes. At baseline, men with the GG genotype (n = 29) had lower VO2max values (p = 0.026), and higher percent body fat (p = 0.046), subcutaneous fat (p = 0.021), and insulin AUC (p = 0.003) values than T allele carriers (n = 22). Despite their rather disadvantageous starting values, men with the GG genotype seemed to respond to exercise training more robustly than men with the T allele, highlighted by significantly greater fold change improvements in insulin AUC (p = 0.012) and glucose AUC (p = 0.035). Although the GG group also significantly improved VO2max with training, the change in VO2max was not as great as that of the T allele carriers (p = 0.037). In contrast, after accounting for hormone replacement therapy use, none of the variables differed in the women at baseline. As a result of exercise training, women with the T allele (n = 20) had greater fold change improvements in fasting glucose (p = 0.011), glucose AUC (p = 0.017), and insulin sensitivity (p = 0.044) than GG genotype women (n = 38). Our results suggest that the AKT1 G205T polymorphism influences metabolic variables and their responses to aerobic exercise training in older previously sedentary individuals.

Our previous studies have indicated that chronic treatment with XNT (1-[(2-dimethylamino) ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one), an angiotensin-converting enzyme2 (ACE2) activator, reverses hypertension-induced cardiac and renal fibrosis in spontaneously hypertensive rats (SHR). Furthermore, XNT prevented pulmonary vascular remodeling and right ventricular hypertrophy and fibrosis in a rat model of monocrotaline-induced pulmonary hypertension. The aim of this study was to determine the mechanisms underlying the protective effects of XNT against cardiac fibrosis. Hydroxyproline assay was used to measure cardiac collagen content in control and XNT-treated (200ng/kg/min for 28 days) SHR. Cardiac ACE2 activity and protein levels were determined using the fluorogenic peptide assay and western blot analysis, respectively. Extracellular signal-regulated kinases (p44 and p42; ERKs) and AT1 receptor levels were quantitated by western blotting. Cardiac ACE2 protein levels were ~15% lower in SHR compared with WKY controls (1.00±0.02 vs. 0.87±0.01 ACE2/GAPDH ratio in SHR). However, treatment of SHR with XNT completely restored the decreased cardiac ACE2 levels. Also, chronic infusion of XNT significantly increased cardiac ACE2 activity in SHR. This increase in ACE2 activity was associated with decreased cardiac collagen content. Furthermore, the anti-fibrotic effect of XNT correlated with increased cardiac Ang-(1–7) immunostaining, though no change in cardiac AT1 protein levels was observed. The beneficial effects of XNT were also accompanied by a reduction in ERK phosphorylation (WKY: 1.00±0.04; Control-SHR: 1.46±0.25; SHR-treated: 0.86±0.02 phospho ERK/total ERK ratio). Our observations demonstrate that XNT activates cardiac ACE2 and inhibits fibrosis. These effects are associated with increases in Ang-(1–7) and inhibition of cardiac ERK signaling.