Experimental Physiology

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Intrinsic chemosensitivity of rostral ventrolateral medullary sympathetic premotor neurons in the in situ arterially perfused preparation of rats

01 November 2014

Brainstem hypoperfusion is a major excitant of sympathetic activity triggering hypertension, but the exact mechanisms involved remain incompletely understood. A major source of excitatory drive to preganglionic sympathetic neurons originates from the ongoing activity of premotor neurons in the rostral ventrolateral medulla (RVLM sympathetic premotor neurons). The chemosensitivity profile of physiologically characterized RVLM sympathetic premotor neurons during hypoxia and hypercapnia remains unclear. We examined whether physiologically characterized RVLM sympathetic premotor neurons can sense brainstem ischaemia intrinsically. We addressed this issue in a unique in situ arterially perfused preparation before and after a complete blockade of fast excitatory and inhibitory synaptic transmission. During hypercapnic hypoxia, respiratory modulation of RVLM sympathetic premotor neurons was lost, but tonic firing of most RVLM sympathetic premotor neurons was elevated. After blockade of fast excitatory and inhibitory synaptic transmission, RVLM sympathetic premotor neurons continued to fire and exhibited an excitatory firing response to hypoxia but not hypercapnia. This study suggests that RVLM sympathetic premotor neurons can sustain high levels of neuronal discharge when oxygen is scarce. The intrinsic ability of RVLM sympathetic premotor neurons to maintain responsivity to brainstem hypoxia is an important mechanism ensuring adequate arterial pressure, essential for maintaining cerebral perfusion in the face of depressed ventilation and/or high cerebral vascular resistance.

Renal sympathetic denervation modulates ventricular electrophysiology and has a protective effect on ischaemia-induced ventricular arrhythmia

01 November 2014

Recently, a beneficial effect of renal sympathetic denervation (RSD) has been seen in patients with ventricular electrical storm. However, the effect of RSD on ventricular electrophysiology remains unclear. Thirty-three mongrel dogs were included in the present study. Renal sympathetic denervation was performed by radiofrequency ablation of the adventitial surface of the renal artery. In group 1 (n = 8), programmed stimulation was performed before and after RSD to determine the ventricular effective refractory period (ERP) and action potential duration (APD) restitution properties. The same parameters were measured in five other animals that underwent sham RSD to serve as controls. In group 2 (n = 10), acute myocardial ischaemia (AMI) was induced by ligating the proximal left anterior descending coronary artery after the performance of RSD, and the incidence of ventricular arrhythmia (VA) was calculated during 1 h of recording. In another 10 dogs (group 3), AMI was induced and VA was measured with sham RSD. In group 1, RSD significantly prolonged ventricular ERP and APD, reduced the maximal slope (Smax) of the restitution curve and suppressed APD alternans at each site. Renal sympathetic denervation also significantly decreased the spatial dispersion of ERP, APD and Smax. In the five control animals, no significant electrophysiological change was detected after sham RSD. The occurrence of spontaneous VA during 1 h of AMI in group 2 was significantly lower than that in group 3. These data suggest that RSD stabilizes ventricular electrophysiological properties in normal hearts and reduces the occurrence of VA in hearts experiencing AMI.

Influence of muscle metaboreceptor stimulation on middle cerebral artery blood velocity in humans

01 November 2014

Regional anaesthesia to attenuate skeletal muscle afferent feedback abolishes the exercise-induced increase in middle cerebral artery mean blood velocity (MCA Vmean). However, such exercise-related increases in cerebral perfusion are not preserved during post exercise muscle ischaemia (PEMI) where the activation of metabolically sensitive muscle afferents is isolated. We tested the hypothesis that a hyperventilation-mediated decrease in the arterial partial pressure of CO2, hence cerebral vasoconstriction, masks the influence of muscle metaboreceptor stimulation on MCA Vmean during PEMI. Ten healthy men (20 ± 1 years old) performed two trials of fatiguing isometric hand-grip exercise followed by PEMI, in control conditions and with end-tidal CO2 (P ET ,CO2) clamped at ~1 mmHg above the resting partial pressure. In the control trial, P ET ,CO2 decreased from rest during hand-grip exercise and PEMI, while MCA Vmean was unchanged from rest. By design, P ET ,CO2 remained unchanged from rest throughout the clamp trial, while MCA Vmean increased during hand-grip (+10.6 ±1.8 cm s–1) and PEMI (+9.2 ± 1.6 cm s–1; P < 0.05 versus rest and control trial). Increases in minute ventilation and mean arterial pressure during hand-grip and PEMI were not different in the control and P ET ,CO2 clamp trials (P > 0.05). These findings indicate that metabolically sensitive skeletal muscle afferents play an important role in the regional increase in cerebral perfusion observed in exercise, but that influence can be masked by a decrease in P ET ,CO2 when they are activated in isolation during PEMI.

Pyridoxamine protects against mechanical defects in cardiac ageing in rats: studies on load dependence of myocardial relaxation

01 November 2014

Our team demonstrated in the past that pyridoxamine attenuated arterial stiffening by targeting the pathogenic formation of glycated collagen cross-links in aged rats. Herein, we examined whether pyridoxamine therapy can protect against mechanical defects in myocardial relaxation by improving arterial wave properties and cardiac contractile performance in senescent animals. Fifteen-month-old male Fisher 344 rats were treated daily with pyridoxamine (1 g l–1 in drinking water) for 5 months and compared with age-matched untreated control animals (20 months old). Arterial wave properties were characterized by wave transit time (w) and wave reflection factor (Rf). We measured the contractile status of the myocardium in an intact heart as the left ventricular (LV) end-systolic elastance (Ees). Myocardial relaxation was described according to the time constant of the LV isovolumic pressure decay (e). Pyridoxamine therapy prevented the age-associated prolongation in LV e and the diminished Ees in senescent rats. The drug also attenuated the age-related augmentation in afterload imposed on the heart, as evidenced by the increased w and decreased Rf. We found that the LV e was significantly influenced by both the arterial w and Rf (e = 16.3902 + 8.3123 x Rf – 0.4739 x w; r = 0.7048, P < 0.005). In the meantime, the LV e and the LV Ees showed a significant inverse linear correlation (e = 13.9807 – 0.0068 x Ees; r = 0.6451, P < 0.0005). All these findings suggested that long-term treatment with pyridoxamine might ameliorate myocardial relaxation rate, at least partly through its ability to enhance myocardial contractile performance, increase wave transit time and decrease wave reflection factor in aged rats.

Cerebral vasomotor reactivity: steady-state versus transient changes in carbon dioxide tension

01 November 2014

Cerebral vasomotor reactivity (CVMR) to changes in arterial carbon dioxide tension (P aCO 2) is assessed during steady-state or transient changes in P aCO 2. This study tested the following two hypotheses: (i) that CVMR during steady-state changes differs from that during transient changes in P aCO 2; and (ii) that CVMR during rebreathing-induced hypercapnia would be blunted when preceded by a period of hyperventilation. For each hypothesis, end-tidal carbon dioxide tension (P ET , CO 2) middle cerebral artery blood velocity (CBFV), cerebrovascular conductance index (CVCI; CBFV/mean arterial pressure) and CVMR (slope of the linear regression between changes in CBFV and CVCI versus P ET , CO 2) were assessed in eight individuals. To address the first hypothesis, measurements were made during the following two conditions (randomized): (i) steady-state increases in P ET , CO 2 of 5 and 10 Torr above baseline; and (ii) rebreathing-induced transient breath-by-breath increases in P ET , CO 2. The linear regression for CBFV versus P ET , CO 2 (P = 0.65) and CVCI versus P ET , CO 2 (P = 0.44) was similar between methods; however, individual variability in CBFV or CVCI responses existed among subjects. To address the second hypothesis, the same measurements were made during the following two conditions (randomized): (i) immediately following a brief period of hypocapnia induced by hyperventilation for 1 min followed by rebreathing; and (ii) during rebreathing only. The slope of the linear regression for CBFV versus P ET , CO 2 (P < 0.01) and CVCI versus P ET , CO 2 (P < 0.01) was reduced during hyperventilation plus rebreathing relative to rebreathing only. These results indicate that cerebral vasomotor reactivity to changes in P aCO 2 is similar regardless of the employed methodology to induce changes in P aCO 2 and that hyperventilation-induced hypocapnia attenuates the cerebral vasodilatory responses during a subsequent period of rebreathing-induced hypercapnia.

Breath-by-breath pulmonary O2 uptake kinetics: effect of data processing on confidence in estimating model parameters

01 November 2014

To improve the signal-to-noise ratio of breath-by-breath pulmonary O2 uptake (VO2p) data, it is common practice to perform multiple step transitions, which are subsequently processed to yield an ensemble-averaged profile. The effect of different data-processing techniques on phase II VO2p kinetic parameter estimates (VO2p amplitude, time delay and phase II time constant (VO2p)] and model confidence [95% confidence interval (CI95)] was examined. Young (n = 9) and older men (n = 9) performed four step transitions from a 20 W baseline to a work rate corresponding to 90% of their estimated lactate threshold on a cycle ergometer. Breath-by-breath VO2p was measured using mass spectrometry and volume turbine. Mono-exponential kinetic modelling of phase II VO2p data was performed on data processed using the following techniques: (A) raw data (trials time aligned, breaths of all trials combined and sorted in time); (B) raw data plus interpolation (trials time aligned, combined, sorted and linearly interpolated to second by second); (C) raw data plus interpolation plus 5 s bin averaged; (D) individual trial interpolation plus ensemble averaged [trials time aligned, linearly interpolated to second by second (technique 1; points joined by straight-line segments), ensemble averaged]; (E) ‘D’ plus 5 s bin averaged; (F) individual trial interpolation plus ensemble averaged [trials time aligned, linearly interpolated to second by second (technique 2; points copied until subsequent point appears), ensemble averaged]; and (G) ‘F’ plus 5 s bin averaged. All of the model parameters were unaffected by data-processing technique; however, the CI95 for VO2p in condition ‘D’ (4 s) was lower (P < 0.05) than the CI95 reported for all other conditions (5–10 s). Data-processing technique had no effect on parameter estimates of the phase II VO2p response. However, the narrowest interval for CI95 occurred when individual trials were linearly interpolated and ensemble averaged.

Reduced hypoxic ventilatory response in newborn mice knocked-out for the progesterone receptor

01 November 2014

Recent studies showed that progesterone stimulates the hypoxic ventilatory response and may reduce apnoea frequency in newborn rats, but so far we still do not know by what mechanisms and whether endogenous progesterone might contribute to respiratory control in neonates. We therefore determined the role of the nuclear progesterone receptor (PR; member of the steroid receptor superfamily) by using wild-type (WT) and PR knock-out (PRKO) mice at postnatal days (P) 1, 4 and 10. We measured the hypoxic ventilatory response (14 and 12% O2, 20 min each) and apnoea frequency in both male and female mice by using whole-body plethysmography. In response to hypoxia, WT male mice had a marked hypoxic ventilatory response at P1 and P10, but not at P4. At P1 and P10, PRKO male mice had a lower hypoxic ventilatory response than WT males. Wild-type female mice had a marked hypoxic ventilatory response at P10, but not at P1 and P4. At P1 and P10, PRKO female mice had a lower hypoxic ventilatory response than WT females. In basal conditions, apnoea frequency was similar in WT and PRKO mice at P1, P4 and P10. During hypoxia, apnoea frequency was higher in WT male mice compared with PRKO male mice and WT female mice at P1. We conclude that PR is a key contributor to the hypoxic ventilatory response in newborn mice, but PR deletion does not increase the frequency of apnoea during normoxia or hypoxia.

Randomized controlled trial using bosentan to enhance the impact of exercise training in subjects with type 2 diabetes mellitus

01 November 2014

In type 2 diabetes patients, endothelin (ET) receptor blockade may enhance blood flow responses to exercise training. The combination of exercise training and ET receptor blockade may represent a more potent stimulus than training alone to improve vascular function, physical fitness and glucose homeostasis. We assessed the effect of an 8 week exercise training programme combined with either ET blockade or placebo on vasculature, fitness and glucose homeostasis in people with type 2 diabetes. In a double-blind randomized controlled trial, brachial endothelium-dependent and -independent dilatation (using flow-mediated dilatation and glyceryl trinitrate, respectively), glucose homeostasis (using Homeostasis Model Assessment for Insulin Resistance (HOMA-IR)) and physical fitness (maximal cycling test) were assessed in 18 men with type 2 diabetes (60 ± 6 years old). Subjects underwent an 8 week exercise training programme, with half of the subjects receiving ET receptor blockade (bosentan) and the other half a placebo, followed by reassessment of the tests above. Exercise training improved physical fitness to a similar extent in both groups, but we did not detect changes in vascular function in either group. This study suggests that there is no adaptation in brachial and femoral artery endothelial function after 8 weeks of training in type 2 diabetes patients. Endothelin receptor blockade combined with exercise training does not additionally alter conduit artery endothelial function or physical fitness in type 2 diabetes.

Editorial Board

01 October 2014

Alterations in atrial perfusion during atrial fibrillation

01 October 2014

Left atrial (LA) perfusion during disease states has been a topic of much interest, because the clinical implications and detrimental effects of lack of blood flow to the atria are numerous. In the chronic setting, changes in perfusion may lead to LA ischaemia and structural remodelling, a factor implicated in the self-perpetuation of chronic atrial fibrillation (AF). The association between AF and altered LA perfusion has been studied, but a direct causal association between perfusion changes and AF has not been established. A comprehensive literature search of Medline, Embase and Google Scholar databases was conducted from 1960 to February 2014. We systematically analysed reference lists of physiological articles and reviews for other possibly relevant studies. The aim of this review is to provide a comprehensive discussion of the AF-mediated changes in LA perfusion and the potential mechanisms underlying the alterations in coronary flow to the LA in this setting. In addition, we discuss the clinical contexts in which changes in LA perfusion may be relevant. Finally, this article highlights the need for longitudinal studies of AF that would elucidate the changes in LA perfusion resulting from chronic AF and lead to advancements in effective treatments to prevent progression of this disease.

Calcium in the heart: from physiology to disease

01 October 2014

Contraction of the heart results from an increase of cytoplasmic Ca2+ concentration ([Ca2+]i), the so-called systolic Ca2+ transient. Most of this results from the release of Ca2+ from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR). In turn, the amplitude of this Ca2+ transient determines the contractility of the heart. In this lecture, I consider the factors which govern the size and stability of this Ca2+ release. The amplitude of the Ca2+ transient is a steep function of SR Ca, resulting in a requirement for very precise beat-to-beat regulation of SR Ca content. This is achieved by a simple negative feedback mechanism, in which an increase of SR Ca content increases the size of the Ca2+ transient, resulting in a decrease of Ca2+ influx on the L-type Ca2+ current and an increase of efflux through Na+–Ca2+ exchange. Changing the activity of any of the Ca2+-cycling proteins will change the steady-state SR Ca content. This feedback mechanism has many consequences, including the fact that a change of RyR open probability has a only a temporary effect on the amplitude of the Ca2+ transient due to a compensating change of SR Ca content. The remainder of the article considers the link between intracellular Ca2+ waves and arrhythmias. This is done in the context of catecholaminergic polymorphic ventricular tachycardia, which is an inherited arrhythmia syndrome, in many cases due to a RyR mutation, where arrhythmias occur during exercise as a result of β-adrenergic stimulation. Calcium waves occur only when the SR Ca content exceeds a threshold level. Our data show that the threshold is reduced by the RyR mutation and that the adrenergic stimulation increases SR Ca content.

Inhale your blood volume!

01 October 2014

Respiratory modulation of muscle sympathetic nerve activity in obstructive sleep apnoea

01 October 2014
New Findings

  • What is the central question of this study?

    Muscle sympathetic nerve activity (MSNA) is increased in obstructive sleep apnoea (OSA), leading to hypertension. Is this due to an increase in respiratory–sympathetic coupling, as has been demonstrated in the spontaneously hypertensive rat?

  • What is the main finding and its importance?

    Using direct microelectrode recordings of MSNA in hypertensive OSA patients and normotensive control subjects, we show that the magnitude of respiratory modulation is not increased in OSA, arguing against an amplified respiratory–sympathetic coupling as the underlying cause of the neurogenic hypertension, although the temporal coupling of MSNA to respiration was stronger in OSA.

  • Obstructive sleep apnoea (OSA) is associated with elevated muscle sympathetic nerve activity (MSNA) during normoxic daytime wakefulness, leading to hypertension. We tested the hypothesis that respiratory–sympathetic coupling, postulated to be the underlying cause of neurogenic hypertension, is increased in OSA. Muscle sympathetic nerve activity, blood pressure, ECG and respiration were recorded in 21 normotensive control subjects and 21 newly diagnosed patients with OSA before and after 6 and 12 months of treatment with continuous positive airway pressure. Muscle sympathetic nerve activity was recorded via tungsten microelectrodes inserted percutaneously into the peroneal nerve. Cardiac and respiratory modulation of MSNA was quantified from the cross-correlation histograms constructed between the sympathetic spikes and either ECG or respiration. Muscle sympathetic nerve activity was significantly elevated in newly diagnosed OSA patients compared with control subjects (53 ± 2 versus 28 ± 2 bursts min–1). There was a significant fall in MSNA after 6 months of continuous positive airway pressure (37 ± 2 bursts min–1), with no further change after 12 months (37 ± 2 bursts min–1). There were no significant differences in the magnitude of respiratory modulation of MSNA between the OSA patients and control subjects (40 ± 3.1 versus 39 ± 3.4%). However, when considering the normalized temporal profile there were changes in the respiratory patterning of MSNA in OSA, with more activity occurring in postinspiration and less in inspiration and expiration. This was largely reversed following long-term continuous positive airway pressure.

    Neuronal activation by mucosal biopsy supernatants from irritable bowel syndrome patients is linked to visceral sensitivity

    01 October 2014

    Based on the discomfort/pain threshold during rectal distension, irritable bowel syndrome (IBS) patients may be subtyped as normo- or hypersensitive. We previously showed that mucosal biopsy supernatants from IBS patients activated enteric and visceral afferent neurons. We tested the hypothesis that visceral sensitivity is linked to the degree of neuronal activation. Normo- and hypersensitive IBS patients were distinguished by their discomfort/pain threshold to rectal balloon distension with a barostat. Using potentiometric and Ca2+ dye imaging, we recorded the response of guinea-pig enteric submucous and mouse dorsal root ganglion (DRG) neurons, respectively, to mucosal biopsy supernatants from normosensitive (n = 12 tested in enteric neurons, n = 9 tested in DRG) and hypersensitive IBS patients (n = 9, tested in both types of neurons). In addition, we analysed the association between neuronal activation and individual discomfort/pain pressure thresholds. The IBS supernatants evoked Ca2+ transients in DRG neurons and spike discharge in submucous neurons. Submucous and DRG neurons showed significantly stronger responses to supernatants from hypersensitive IBS patients as reflected by higher spike frequency or stronger [Ca2+]i transients in a larger proportion of neurons. The neuroindex as a product of spike frequency or [Ca2+]i transients and proportion of responding neurons correlated significantly with the individual discomfort/pain thresholds of the IBS patients. Supernatants from hypersensitive IBS patients caused stronger activation of enteric and DRG neurons. The level of activation correlated with the individual discomfort/pain threshold pressure values. These findings support our hypothesis that visceral sensitivity is linked to activation of peripheral neurons by biopsy supernatants.

    Increased cardiac output contributes to the development of chronic intermittent hypoxia-induced hypertension

    01 October 2014

    Chronic intermittent hypoxia (CIH) in animal models has been shown to result in hypertension and elevation of sympathetic nervous system activity. Sympathetically mediated vasoconstriction is believed to be the primary mechanism underpinning CIH-induced hypertension; however, the potential contribution of the heart is largely overlooked. We sought to determine the contribution of cardiac output (CO) and lumbar sympathetic control of the hindlimb circulation to CIH-induced hypertension. Male Wistar rats (n = 64) were exposed to 2 weeks of CIH [cycles of 90 s hypoxia (5% O2 nadir) and 210 s normoxia] or normoxia for 8 h day–1. Under urethane anaesthesia, CIH-treated animals developed hypertension (81.4 ± 2.2 versus 91.6 ± 2.4 mmHg; P < 0.001), tachycardia (397 ± 8 versus 445 ± 7 beats min–1; P < 0.001) and an increased haematocrit (42.4 ± 0.4 versus 45.0 ± 0.4%; P < 0.001). Echocardiography revealed that CIH exposure increased the CO [19.3 ± 1.7 versus 25.8 ± 2.6 ml min–1 (100 g)–1; P = 0.027] with no change in total peripheral resistance (4.93 ± 0.49 versus 4.17 ± 0.34 mmHg ml–1 min–1; P = 0.123). Sympathetic ganglionic blockade revealed that sympathetic control over blood pressure was not different (–27.7 ± 1.6 versus –32.3 ± 2.9 mmHg; P = 0.095), and no chronic vasoconstriction was found in the hindlimb circulation of CIH-treated animals (39.4 ± 2.5 versus 38.0 ± 2.4 μl min–1 mmHg–1; P = 0.336). Lumbar sympathetic control over the hindlimb circulation was unchanged in CIH-treated animals (P = 0.761), although hindlimb arterial sympathetic density was increased (P = 0.012) and vascular sensitivity to phenylephrine was blunted (P = 0.049). We conclude that increased CO is sufficient to explain the development of CIH-induced hypertension, which may be an early adaptive response to raise O2 flow. We propose that sustained elevated cardiac work may ultimately lead to heart failure.

    Vasopressin regulation of epithelial colonic proliferation and permeability is mediated by pericryptal platelet-derived growth factor A

    01 October 2014

    Arginine vasopressin (AVP) has trophic effects on the rat distal colon, increasing the growth of pericryptal myofibroblasts and reducing the colonic crypt wall permeability. This study aimed to reproduce in vitro the effects of AVP observed in vivo using cultures of human CCD-18Co myofibroblasts and T84 colonic epithelial cells. Proliferation of myofibroblasts was quantified by bromodeoxyuridine incorporation; the expression of platelet-derived growth factor A (PDGFA), platelet-derived growth factor B, epidermal growth factor, transforming growth factor-β and vascular endothelial growth factor was measured by PCR and the expression of epithelial junction proteins by Western blot. Arginine vasopressin stimulated myofibroblast proliferation and the expression of PDGFA without affecting the expression of platelet-derived growth factor B, epidermal growth factor, transforming growth factor-β or vascular endothelial growth factor. These effects were prevented when AVP receptor inhibitors were present in the medium. Pre-incubation of CCD-18Co cells with anti-PDGF antibody or with an inhibitor of the PDGF receptor abolished the effects of AVP. When colonocytes were incubated with medium obtained from myofibroblasts incubated with AVP, both cell proliferation and the expression of epithelial junction proteins increased; however, direct incubation of colonocytes with AVP did not modify these variables. These results demonstrate that AVP stimulates myofibroblast proliferation and induces PDGFA secretion, implying that PDGFA mediates local myofibroblast proliferation by an autocrine feedback loop and regulates epithelial proliferation and permeability by a paracrine mechanism.

    Characterization of butyrate transport across the luminal membranes of equine large intestine

    01 October 2014

    The diet of the horse, pasture forage (grass), is fermented by the equine colonic microbiota to short-chain fatty acids, notably acetate, propionate and butyrate. Short-chain fatty acids provide a major source of energy for the horse and contribute to many vital physiological processes. We aimed to determine both the mechanism of butyrate uptake across the luminal membrane of equine colon and the nature of the protein involved. To this end, we isolated equine colonic luminal membrane vesicles. The abundance and activity of cysteine-sensitive alkaline phosphatase and villin, intestinal luminal membrane markers, were significantly enriched in membrane vesicles compared with the original homogenates. In contrast, the abundance of GLUT2 protein and the activity of Na+–K+-ATPase, known markers of the intestinal basolateral membrane, were hardly detectable. We demonstrated, by immunohistochemistry, that monocarboxylate transporter 1 (MCT1) protein is expressed on the luminal membrane of equine colonocytes. We showed that butyrate transport into luminal membrane vesicles is energized by a pH gradient (out < in) and is not Na+ dependent. Moreover, butyrate uptake is time and concentration dependent, with a Michaelis–Menten constant of 5.6 ± 0.45 mm and maximal velocity of 614 ± 55 pmol s–1 (mg protein)–1. Butyrate transport is significantly inhibited by p-chloromercuribenzoate, phloretin and α-cyano-4-hydroxycinnamic acid, all potent inhibitors of MCT1. Moreover, acetate and propionate, as well as the monocarboxylates pyruvate and lactate, also inhibit butyrate uptake. Data presented here support the conclusion that transport of butyrate across the equine colonic luminal membrane is predominantly accomplished by MCT1.