Journal of Physiology

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Nitric oxide synthase deficiency and the pathophysiology of muscular dystrophy

01 November 2014

The secondary loss of neuronal nitric oxide synthase (nNOS) that occurs in dystrophic muscle is the basis of numerous, complex and interacting features of the dystrophic pathology that affect not only muscle itself, but also influence the interaction of muscle with other tissues. Many mechanisms through which nNOS deficiency contributes to misregulation of muscle development, blood flow, fatigue, inflammation and fibrosis in dystrophic muscle have been identified, suggesting that normalization in NO production could greatly attenuate diverse aspects of the pathology of muscular dystrophy through multiple regulatory pathways. However, the relative importance of the loss of nNOS from the sarcolemma versus the importance of loss of total nNOS from dystrophic muscle remains unknown. Although most current evidence indicates that nNOS localization at the sarcolemma is not required to achieve NO-mediated reductions of pathology in muscular dystrophy, the question remains open concerning whether membrane localization would provide a more efficient rescue from features of the dystrophic phenotype.

Catecholamine exocytosis during low frequency stimulation in mouse adrenal chromaffin cells is primarily asynchronous and controlled by the novel mechanism of Ca2+ syntilla suppression

01 November 2014

Adrenal chromaffin cells (ACCs), stimulated by the splanchnic nerve, generate action potentials (APs) at a frequency near 0.5 Hz in the resting physiological state, at times described as ‘rest and digest’. How such low frequency stimulation in turn elicits sufficient catecholamine exocytosis to set basal sympathetic tone is not readily explained by the classical mechanism of stimulus–secretion coupling, where exocytosis is synchronized to AP-induced Ca2+ influx. By using simulated action potentials (sAPs) at 0.5 Hz in isolated patch-clamped mouse ACCs, we show here that less than 10% of all catecholaminergic exocytosis, measured by carbon fibre amperometry, is synchronized to an AP. The asynchronous phase, the dominant phase, of exocytosis does not require Ca2+ influx. Furthermore, increased asynchronous exocytosis is accompanied by an AP-dependent decrease in frequency of Ca2+ syntillas (i.e. transient, focal Ca2+ release from internal stores) and is ryanodine sensitive. We propose a mechanism of disinhibition, wherein APs suppress Ca2+ syntillas, which themselves inhibit exocytosis as they do in the case of spontaneous catecholaminergic exocytosis.

Voltage- and ATP-dependent structural rearrangements of the P2X2 receptor associated with the gating of the pore

01 November 2014

P2X2 is an extracellular ATP-gated cation channel which has a voltage-dependent gating property even though it lacks a canonical voltage sensor. It is a trimer in which each subunit has two transmembrane helices and a large extracellular domain. The three inter-subunit ATP binding sites are linked to the pore forming transmembrane (TM) domains by β-strands. We analysed structural rearrangements of the linker strands between the ATP binding site and TM domains upon ligand binding and voltage change, electrophysiologically in Xenopus oocytes, using mutants carrying engineered thiol-modifiable cysteine residues. (1) We demonstrated that the double mutant D315C&I67C (at β-14 and β-1, respectively) shows a 2- to 4-fold increase in current amplitude after treatment with a reducing reagent, dithiothreitol (DTT). Application of the thiol-reactive metal Cd2+ induced current decline due to bond formation between D315C and I67C. This effect was not observed in wild type (WT) or in single point mutants. (2) Cd2+-induced current decline was analysed in hyperpolarized and depolarized conditions with different pulse protocols, and also in the presence and absence of ATP. (3) Current decline induced by Cd2+ could be clearly observed in the presence of ATP, but was not clear in the absence of ATP, showing a state-dependent modification. (4) In the presence of ATP, Cd2+ modification was significantly faster in hyperpolarized than in depolarized conditions, showing voltage-dependent structural rearrangements of the linker strands. (5) Experiments using tandem trimeric constructs (TTCs) with controlled number and position of mutations in the trimer showed that the bridging by Cd2+ between 315 and 67 was not intra- but inter-subunit. (6) Finally, we performed similar analyses of a pore mutant T339S, which makes the channel activation voltage insensitive. Cd2+ modification rates of T339S were similar in hyperpolarized and depolarized conditions. Taking these results together, we demonstrated that structural rearrangements of the linker region of the P2X2 receptor channel are induced not only by ligand binding but also by membrane potential change.

Na+ current properties in islet {alpha}- and {beta}-cells reflect cell-specific Scn3a and Scn9a expression

01 November 2014

Mouse pancreatic β- and α-cells are equipped with voltage-gated Na+ currents that inactivate over widely different membrane potentials (half-maximal inactivation (V0.5) at –100 mV and –50 mV in β- and α-cells, respectively). Single-cell PCR analyses show that both α- and β-cells have Nav1.3 (Scn3) and Nav1.7 (Scn9a) α subunits, but their relative proportions differ: β-cells principally express Nav1.7 and α-cells Nav1.3. In α-cells, genetically ablating Scn3a reduces the Na+ current by 80%. In β-cells, knockout of Scn9a lowers the Na+ current by >85%, unveiling a small Scn3a-dependent component. Glucagon and insulin secretion are inhibited in Scn3a–/– islets but unaffected in Scn9a-deficient islets. Thus, Nav1.3 is the functionally important Na+ channel α subunit in both α- and β-cells because Nav1.7 is largely inactive at physiological membrane potentials due to its unusually negative voltage dependence of inactivation. Interestingly, the Nav1.7 sequence in brain and islets is identical and yet the V0.5 for inactivation is >30 mV more negative in β-cells. This may indicate the presence of an intracellular factor that modulates the voltage dependence of inactivation.

Human lymphatic vessel contractile activity is inhibited in vitro but not in vivo by the calcium channel blocker nifedipine

01 November 2014

Calcium channel blockers (CCB) are widely prescribed anti-hypertensive agents. The commonest side-effect, peripheral oedema, is attributed to a larger arterial than venous dilatation causing increased fluid filtration. Whether CCB treatment is detrimental to human lymphatic vessel function and thereby exacerbates oedema formation is unknown. We observed that spontaneous lymphatic contractions in isolated human vessels (thoracic duct and mesenteric lymphatics) maintained under isometric conditions were inhibited by therapeutic concentrations (nanomolar) of the CCB nifedipine while higher than therapeutic concentrations of verapamil (micromolar) were necessary to inhibit activity. Nifedipine also inhibited spontaneous action potentials measured by sharp microelectrodes. Furthermore, noradrenaline did not elicit normal increases in lymphatic vessel tone when maximal constriction was reduced to 29.4 ± 4.9% of control in the presence of 20 nmol l–1 nifedipine. Transcripts for the L-type calcium channel gene CACNA1C were consistently detected from human thoracic duct samples examined and the CaV1.2 protein was localized by immunoreactivity to lymphatic smooth muscle cells. While human lymphatics ex vivo were highly sensitive to nifedipine, this was not apparent in vivo when nifedipine was compared to placebo in a randomized, double-blinded clinical trial: conversely, lymphatic vessel contraction frequency was increased and refill time was faster despite all subjects achieving target nifedipine plasma concentrations. We conclude that human lymphatic vessels are highly sensitive to nifedipine in vitro but that care must be taken when extrapolating in vitro observations of lymphatic vessel function to the clinical situation, as similar changes in lymphatic function were not evident in our clinical trial comparing nifedipine treatment to placebo.

Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart

01 November 2014

Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l–1 NaCl (as control) or 0.7 mmol l–1 NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics.

Spontaneous transient hyperpolarizations in the rabbit small intestine

01 November 2014

Four types of electrical activity were recorded and related to cell structure by intracellular recording and dye injection into impaled cells in muscles of rabbit small intestine. The specific cell types from which recordings were made were longitudinal smooth muscle cells (LSMCs), circular smooth muscle cells (CSMCs), interstitial cells of Cajal distributed in the myenteric region (ICC-MY) and fibroblast-like cells (FLCs). Slow waves (slow wavesSMC) were recorded from LSMCs and CSMCs. Slow waves (slow wavesICC) were of greatest amplitude (>50 mV) and highest maximum rate of rise (>10 V s–1) in ICC-MY. The dominant activity in FLCs was spontaneous transient hyperpolarizations (STHs), with maximum amplitudes above 30 mV. STHs were often superimposed upon small amplitude slow waves (slow wavesFLC). STHs displayed a cyclical pattern of discharge irrespective of background slow wave activity. STHs were inhibited by MRS2500 (3 μm), a P2Y1 antagonist, and abolished by apamin (0.3 μm), a blocker of small conductance Ca2+-activated K+ channels. Small amplitude STHs (<15 mV) were detected in smooth muscle layers, whereas STHs were not resolved in cells identified as ICC-MY. Electrical field stimulation evoked purinergic inhibitory junction potentials (IJPs) in CSMCs. Purinergic IJPs were not recorded from ICC-MY. These results suggest that FLCs may regulate smooth muscle excitability in the rabbit small intestine via generation of rhythmic apamin-sensitive STHs. Stimulation of P2Y1 receptors modulates the amplitudes of STHs. Our results also suggest that purinergic inhibitory motor neurons regulate the motility of the rabbit small intestine by causing IJPs in FLCs that conduct to CSMCs.

Angiotensin II mobilizes intracellular calcium and activates pannexin-1 channels in rat carotid body type II cells via AT1 receptors

01 November 2014
Key points

  • A locally generating, angiotensin II (ANG II) system is present in the rat carotid body (CB) and up-regulation of this system occurs in certain pathophysiological situations, enhancing sympathetic activity.

  • Here, we show that, similar to chemoreceptor type I cells, glial-like type II cells also express functional AT1Rs, stimulation of which causes release of Ca2+ from intracellular stores.

  • ANG II–AT1R signalling in type II cells activates an inward current carried by pannexin-1 (Panx-1) channels which are known to act as conduits for release of ATP, a key CB excitatory neurotransmitter.

  • Combined effects of ANG II and ATP, which also activates Panx-1 currents via P2Y2 receptors, were synergistic; chelating intracellular Ca2+ with BAPTA prevented Panx-1 current activation.

  • We propose that the excitatory function of ANG II in the CB involves dual actions at both type I and type II cells.

  • Abstract

    A local angiotensin-generating system is present in the carotid body (CB) and increased angiotensin II (ANG II) signalling contributes to enhanced CB excitation in chronic heart failure (CHF) and after chronic or intermittent hypoxia. ANG II actions have thus far been attributed solely to stimulation of AT1 receptors (AT1Rs) on chemoreceptor type I cells. Here, we show that in dissociated rat CB cultures, ANG II also stimulates glial-like type II cells, identified by P2Y2-receptor-induced intracellular Ca2+ elevation ([Ca2+]i). ANG II induced a dose-dependent (EC50 ~8 nm), robust [Ca2+]i in type II cells that was reversibly abolished by the AT1R blocker losartan (1 μm). The ANG II-induced [Ca2+]i persisted in Ca2+-free medium but was sensitive to store depletion with cyclopiazonic acid (1 μm). Similar to P2Y2 receptor agonists, ANG II (20–1000 nm) activated pannexin-1 (Panx-1) current that was reversibly abolished by carbenoxolone (5 μm). This current arose with a variable delay and was reversibly inhibited by losartan. Repeated application of ANG II often led to current run-down, attributable to AT1R desensitization. When applied to the same cell the combined actions of ANG II and ATP on Panx-1 current were synergistic. Current induced by either ligand was inhibited by BAPTA-AM (1 μm), suggesting that intracellular Ca2+ signalling contributed to Panx-1 channel activation. Because open Panx-1 channels release ATP, a key CB excitatory neurotransmitter, it is plausible that paracrine stimulation of type II cells by ANG II contributes to enhanced CB excitability, especially in pathophysiological conditions such as CHF and sleep apnoea.

    Healthy humans with a narrow upper airway maintain patency during quiet breathing by dilating the airway during inspiration

    01 November 2014

    A patent upper airway is essential for survival. Increased age, obesity and some upper airway anatomical features are associated with failure to maintain upper airway patency during sleep, leading to obstructive sleep apnoea. However, many healthy subjects with these risk factors do not develop this condition. The aim of this study was to determine how anatomical factors and active dilator muscle contraction contribute to upper airway patency in healthy volunteers across a broad range of age and body mass index (BMI). A ‘tagged’ magnetic resonance imaging technique quantified respiratory-related motion of the anterior and lateral walls of the upper airway during quiet breathing in the supine position. Fifty-two subjects aged 22–68 years with BMI from 17.5 to 40.1 kg m–2 were studied. Higher BMI was associated with smaller airway cross-sectional area at the level of soft palate (P < 0.05). The genioglossus moved anteriorly to dilate the upper airway during inspiration. This movement increased with increasing BMI, increasing age, a smaller airway area, and steeper tongue-base angle (all P < 0.05). Motion of the lateral upper airway at the soft-palate level was variable and less strongly linked to anatomical features of the upper airway. Multiple regression indicated that anterior genioglossus motion decreased with increasing airway area (P = 0.03) and with increasing tongue-base angle (P = 0.02). These data suggest that healthy humans, including those whose anatomy places them at increased risk of airway closure, can maintain upper airway patency by dynamically dilating the airway during inspiration.

    Role of {alpha}-adrenergic vasoconstriction in regulating skeletal muscle blood flow and vascular conductance during forearm exercise in ageing humans

    01 November 2014

    In healthy humans, ageing is typically associated with reduced skeletal muscle blood flow and vascular conductance during exercise. Further, there is a marked increase in resting sympathetic nervous system (SNS) activity with age, yet whether augmented SNS-mediated α-adrenergic vasoconstriction contributes to the age-associated impairment in exercising muscle blood flow and vascular tone in humans is unknown. We tested the hypothesis that SNS-mediated vasoconstriction is greater in older than young adults and limits muscle (forearm) blood flow (FBF) during graded handgrip exercise (5, 15, 25% maximal voluntary contraction (MVC)). FBF was measured (Doppler ultrasound) and forearm vascular conductance (FVC) was calculated in 11 young (21 ± 1 years) and 12 older (62 ± 2 years) adults in control conditions and during combined local α- and β-adrenoreceptor blockade via intra-arterial infusions of phentolamine and propranolol, respectively. Under control conditions, older adults exhibited significantly lower FBF and FVC at 15% MVC exercise (22.6 ± 1.3 vs. 29 ± 3.3 ml min–1 100 g forearm fat-free mass (FFM)–1 and 21.7 ± 1.2 vs. 33.6 ± 4.0 ml min–1 100 g FFM–1 100 mmHg–1; P < 0.05) and 25% MVC exercise (37.4 ± 1.4 vs. 46.0 ± 4.9 ml min–1 100 g FFM–1 and 33.7 ± 1.4 vs. 49.0 ± 5.7 ml min–1 100 g FFM–1 100 mmHg–1; P < 0.05), whereas there was no age group difference at 5% MVC exercise. Local adrenoreceptor blockade increased FBF and FVC at rest and during exercise in both groups, although the increase in FBF and FVC from rest to steady-state exercise was similar in young and older adults across exercise intensities, and thus the age-associated impairment in FBF and FVC persisted. Our data indicate that during graded intensity handgrip exercise, the reduced FVC and subsequently lower skeletal muscle blood flow in older healthy adults is not due to augmented sympathetic vasoconstriction, but rather due to impairments in local signalling or structural limitations in the peripheral vasculature with advancing age.

    Exercise training augments neuronal nitric oxide synthase-mediated inhibition of sympathetic vasoconstriction in contracting skeletal muscle of rats

    01 November 2014

    We tested the hypothesis that exercise training would increase neuronal nitric oxide synthase (nNOS)-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle. Sprague–Dawley rats (n = 18) were randomized to sedentary or exercise-trained (40 m min–1, 5° grade; 5 days week–1 for 4 weeks) groups. Following completion of sedentary behaviour or exercise training, rats were anaesthetized and instrumented with a brachial artery catheter, femoral artery flow probe and stimulating electrodes on the lumbar sympathetic chain. The percentage change of femoral vascular conductance (%FVC) in response to sympathetic chain stimulations delivered at 2 and 5 Hz was determined at rest and during triceps surae muscle contraction before (control) and after selective nNOS blockade with S-methyl-l-thiocitrulline (SMTC, 0.6 mg kg–1, i.v.) and subsequent non-selective NOS blockade with l-NAME (5 mg kg–1, i.v.; SMTC + l-NAME). At rest, sympathetic vasoconstrictor responsiveness was greater (P < 0.05) in exercise-trained compared to sedentary rats in control, SMTC and SMTC + l-NAME conditions. During contraction, the constrictor response was not different (P > 0.05) between exercise trained (2 Hz: –11 ± 4%FVC; 5 Hz: –21 ± 5%FVC) and sedentary rats (2 Hz: –7 ± 6%FVC; 5 Hz: –18 ± 10%FVC) in control conditions. SMTC augmented (P < 0.05) sympathetic vasoconstriction in sedentary and exercise-trained rats; however, sympathetic vasoconstrictor responsiveness was greater (P < 0.05) in exercise-trained (2 Hz: –27 ± 5%FVC; 5 Hz: –39 ± 5%FVC) compared to sedentary (2 Hz: –17 ± 6%FVC; 5 Hz: –27 ± 8%FVC) rats during selective nNOS inhibition. SMTC + l-NAME further augmented (P < 0.05) sympathetic vasoconstrictor responsiveness by a similar magnitude (P > 0.05) in exercise-trained and sedentary rats. These data demonstrate that exercise training augmented nNOS-mediated inhibition of sympathetic vasoconstriction in contracting muscle.

    Corrigendum

    01 November 2014

    Increased cardiac output, not pulmonary artery systolic pressure, increases intrapulmonary shunt in healthy humans breathing room air and 40% O2

    15 October 2014

    Blood flow through intrapulmonary arteriovenous anastomoses (IPAVAs) has been demonstrated to increase in healthy humans during a variety of conditions; however, whether or not this blood flow represents a source of venous admixture (Q VA /QT) that impairs pulmonary gas exchange efficiency (i.e. increases the alveolar-to-arterial PO2 difference (A–aDO2)) remains controversial and unknown. We hypothesized that blood flow through IPAVAs does provide a source of Q VA /QT. To test this, blood flow through IPAVAs was increased in healthy humans at rest breathing room air and 40% O2: (1) during intravenous adrenaline (epinephrine) infusion at 320 ng kg–1 min–1 (320 ADR), and (2) with vagal blockade (2 mg atropine), before and during intravenous adrenaline infusion at 80 ng kg–1 min–1 (ATR + 80 ADR). When breathing room air the A–aDO2 increased by 6 ± 2 mmHg during 320 ADR and by 5 ± 2 mmHg during ATR + 80 ADR, and the change in calculated Q VA /QT was +2% in both conditions. When breathing 40% O2, which minimizes contributions from diffusion limitation and alveolar ventilation-to-perfusion inequality, the A–aDO2 increased by 12 ± 7 mmHg during 320 ADR, and by 9 ± 6 mmHg during ATR + 80 ADR, and the change in calculated Q VA /QT was +2% in both conditions. During 320 ADR cardiac output (QT) and pulmonary artery systolic pressure (PASP) were significantly increased; however, during ATR + 80 ADR only QT was significantly increased, yet blood flow through IPAVAs as detected with saline contrast echocardiography was not different between conditions. Accordingly, we suggest that blood flow through IPAVAs provides a source of intrapulmonary shunt, and is mediated primarily by increases in QT rather than PASP.

    Muscle disuse alters skeletal muscle contractile function at the molecular and cellular levels in older adult humans in a sex-specific manner

    15 October 2014
    Key points

  • Muscle disuse that accompanies ageing and chronic disease may hasten physical disability by impairing skeletal muscle contractility.

  • We compared skeletal muscle contractile function at the various anatomic levels between two groups of older men and women matched for sex, health status and body size, of which one group was habitually active and the other inactive.

  • Muscle disuse reduced force generation, power output and contractile velocity in single muscle fibres, with differential adaptations in some parameters in men and women. Sex-specific cellular phenotypes were explained by differential adaptations in molecular muscle function. Moreover, aspects of the molecular functional phenotype apparent in inactive women could be recapitulated in vitro by chemical modification of protein thiols.

  • Our results identify molecular and cellular contractile dysfunction in skeletal muscle that may contribute to reduced physical function with muscle disuse, with sex-specific differences that may explain a greater disposition towards disability in women.

  • Abstract

    Physical inactivity that accompanies ageing and disease may hasten disability by reducing skeletal muscle contractility. To characterize skeletal muscle functional adaptations to muscle disuse, we compared contractile performance at the molecular, cellular and whole-muscle levels in healthy active older men and women (n = 15) and inactive older men and women with advanced-stage, symptomatic knee osteoarthritis (OA) (n = 16). OA patients showed reduced (P < 0.01) knee extensor function. At the cellular level, single muscle fibre force production was reduced in OA patients in myosin heavy chain (MHC) I and IIA fibres (both P < 0.05) and differences in IIA fibres persisted after adjustments for fibre cross-sectional area (P < 0.05). Although no group differences in contractile velocity or power output were found for any fibre type, sex was found to modify the effect of OA, with a reduction in MHC IIA power output and a trend towards reduced shortening velocity in women, but increases in both variables in men (P < 0.05 and P = 0.07, respectively). At the molecular level, these adaptations in MHC IIA fibre function were explained by sex-specific differences (P ≤ 0.05) in myosin–actin cross-bridge kinetics. Additionally, cross-bridge kinetics were slowed in MHC I fibres in OA patients (P < 0.01), attributable entirely to reductions in women with knee OA (P < 0.05), a phenotype that could be reproduced in vitro by chemical modification of protein thiol residues. Our results identify molecular and cellular functional adaptations in skeletal muscle that may contribute to reduced physical function with knee OA-associated muscle disuse, with sex-specific differences that may explain a greater disposition towards disability in women.

    PGC1-{alpha} over-expression prevents metabolic alterations and soleus muscle atrophy in hindlimb unloaded mice

    15 October 2014

    Prolonged skeletal muscle inactivity causes muscle fibre atrophy. Redox imbalance has been considered one of the major triggers of skeletal muscle disuse atrophy, but whether redox imbalance is actually the major cause or simply a consequence of muscle disuse remains of debate. Here we hypothesized that a metabolic stress mediated by PGC-1α down-regulation plays a major role in disuse atrophy. First we studied the adaptations of soleus to mice hindlimb unloading (HU) in the early phase of disuse (3 and 7 days of HU) with and without antioxidant treatment (trolox). HU caused a reduction in cross-sectional area, redox status alteration (NRF2, SOD1 and catalase up-regulation), and induction of the ubiquitin proteasome system (MuRF-1 and atrogin-1 mRNA up-regulation) and autophagy (Beclin1 and p62 mRNA up-regulation). Trolox completely prevented the induction of NRF2, SOD1 and catalase mRNAs, but not atrophy or induction of catabolic systems in unloaded muscles, suggesting that oxidative stress is not a major cause of disuse atrophy. HU mice showed a marked alteration of oxidative metabolism. PGC-1α and mitochondrial complexes were down-regulated and DRP1 was up-regulated. To define the link between mitochondrial dysfunction and disuse muscle atrophy we unloaded mice overexpressing PGC-1α. Transgenic PGC-1α animals did not show metabolic alteration during unloading, preserving muscle size through the reduction of autophagy and proteasome degradation. Our results indicate that mitochondrial dysfunction plays a major role in disuse atrophy and that compounds inducing PGC-1α expression could be useful to treat/prevent muscle atrophy.

    Plasticity in the brainstem vagal circuits controlling gastric motor function triggered by corticotropin releasing factor

    15 October 2014

    Stress impairs gastric emptying, reduces stomach compliance and induces early satiety via vagal actions. We have shown recently that the ability of the anti-stress neuropeptide oxytocin (OXT) to modulate vagal brainstem circuits undergoes short-term plasticity via alterations in cAMP levels subsequent to vagal afferent fibre-dependent activation of metabotropic glutamate receptors. The aim of the present study was to test the hypothesis that the OXT-induced gastric response undergoes plastic changes in the presence of the prototypical stress hormone, corticotropin releasing factor (CRF). Whole cell patch clamp recordings showed that CRF increased inhibitory GABAergic synaptic transmission to identified corpus-projecting dorsal motor nucleus of the vagus (DMV) neurones. In naive brainstem slices, OXT perfusion had no effect on inhibitory synaptic transmission; following exposure to CRF (and recovery from its actions), however, re-application of OXT inhibited GABAergic transmission in the majority of neurones tested. This uncovering of the OXT response was antagonized by pretreatment with protein kinase A or adenylate cyclase inhibitors, H89 and di-deoxyadenosine, respectively, indicating a cAMP-mediated mechanism. In naive animals, OXT microinjection in the dorsal vagal complex induced a NO-mediated corpus relaxation. Following CRF pretreatment, however, microinjection of OXT attenuated or, at times reversed, the gastric relaxation which was insensitive to l-NAME but was antagonized by pretreatment with a VIP antagonist. Immunohistochemical analyses of vagal motoneurones showed an increased number of oxytocin receptors present on GABAergic terminals of CRF-treated or stressed vs. naive rats. These results indicate that CRF alters vagal inhibitory circuits that uncover the ability of OXT to modulate GABAergic currents and modifies the gastric corpus motility response to OXT.