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Journal of Physiology
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Antioxidant treatment improves neonatal survival and prevents impaired cardiac function at adulthood following neonatal glucocorticoid therapy
Glucocorticoids are widely used to treat chronic lung disease in premature infants but their longer-term adverse effects on the cardiovascular system raise concerns. We reported that neonatal dexamethasone treatment in rats induced in the short term molecular indices of cardiac oxidative stress and cardiovascular tissue remodelling at weaning, and that neonatal combined antioxidant and dexamethasone treatment was protective at this time. In this study, we investigated whether such effects of neonatal dexamethasone have adverse consequences for NO bioavailability and cardiovascular function at adulthood, and whether neonatal combined antioxidant and dexamethasone treatment is protective in the adult. Newborn rat pups received daily i.p. injections of a human-relevant tapering dose of dexamethasone (D; n = 8; 0.5, 0.3, 0.1 g g–1) or D with vitamins C and E (DCE; n = 8; 200 and 100 mg kg–1, respectively) on postnatal days 1–3 (P1–3); vitamins were continued from P4 to P6. Controls received equal volumes of vehicle from P1 to P6 (C; n = 8). A fourth group received vitamins alone (CCE; n = 8). At P100, plasma NO metabolites (NOx) was measured and isolated hearts were assessed under both Working and Langendorff preparations. Relative to controls, neonatal dexamethasone therapy increased mortality by 18% (P < 0.05). Surviving D pups at adulthood had lower plasma NOx concentrations (10.6 ± 0.8 vs. 28.0 ± 1.5 m), an increased relative left ventricular (LV) mass (70 ± 2 vs. 63 ± 1%), enhanced LV end-diastolic pressure (14 ± 2 vs. 8 ± 1 mmHg) and these hearts failed to adapt output with increased preload (cardiac output: 2.9 ± 2.0 vs. 10.6 ± 1.2 ml min–1) or afterload (cardiac output: –5.3 ± 2.0 vs.1.4 ± 1.2 ml min–1); all P < 0.05. Combined neonatal dexamethasone with antioxidant vitamins improved postnatal survival, restored plasma NOx and protected against cardiac dysfunction at adulthood. In conclusion, neonatal dexamethasone therapy promotes cardiac dysfunction at adulthood. Combined neonatal treatment with antioxidant vitamins is an effective intervention.
Although myogenic mechanisms have been hypothesized to play a role in cerebrovascular regulation, previous data from both animals and humans have not provided an unequivocal answer. However, cerebral autoregulation is explicitly non-linear and most prior work relied on simple linear approaches for assessment, potentially missing important changes in autoregulatory characteristics. Therefore, we examined cerebral blood flow responses to augmented arterial pressure oscillations with and without calcium channel blockade (nicardipine) during blood pressure fluctuations (oscillatory lower body negative pressure, OLBNP) across a range of frequencies in 16 healthy subjects. Autoregulation was characterized via a robust non-linear method (projection pursuit regression, PPR). Blockade resulted in significant tachycardia, a modest but significant elevation in mean arterial pressure, and reductions in mean cerebral blood flow and end-tidal CO2 during OLBNP. The reductions in flow were directly related to the reductions in CO2 (r = 0.57). While linear cross-spectral analysis showed that the relationship between pressure–flow fluctuations was preserved after blockade, PPR showed that blockade significantly altered the non-linearity between pressure and flow, particularly at the slowest fluctuations. At 0.03 Hz, blockade reduced the range of pressure fluctuations that can be buffered (7.5 ± 1.0 vs. 3.7 ± 0.8 mmHg) while increasing the autoregulatory slope (0.10 ± 0.05 vs. 0.24 ± 0.08 cm s–1 mmHg–1). Furthermore, the same rate of change in pressure elicited a change in flow more than twice as large as at baseline. Thus, our results show that myogenic mechanisms play a significant role in cerebrovascular regulation but this may not be appreciated without adequately characterizing the non-linearities inherent in cerebrovascular regulation.
Exchange protein activated by cAMP (Epac) induces vascular relaxation by activating Ca2+-sensitive K+ channels in rat mesenteric artery
Vasodilator-induced elevation of intracellular cyclic AMP (cAMP) is a central mechanism governing arterial relaxation but is incompletely understood due to the diversity of cAMP effectors. Here we investigate the role of the novel cAMP effector exchange protein directly activated by cAMP (Epac) in mediating vasorelaxation in rat mesenteric arteries. In myography experiments, the Epac-selective cAMP analogue 8-pCPT-2-O-Me-cAMP-AM (5 m, subsequently referred to as 8-pCPT-AM) elicited a 77.6 ± 7.1% relaxation of phenylephrine-contracted arteries over a 5 min period (mean ± SEM; n = 6). 8-pCPT-AM induced only a 16.7 ± 2.4% relaxation in arteries pre-contracted with high extracellular K+ over the same time period (n = 10), suggesting that some of Epac's relaxant effect relies upon vascular cell hyperpolarization. This involves Ca2+-sensitive, large-conductance K+ (BKCa) channel opening as iberiotoxin (100 nm) significantly reduced the ability of 8-pCPT-AM to reverse phenylephrine-induced contraction (arteries relaxed by only 35.0 ± 8.5% over a 5 min exposure to 8-pCPT-AM, n = 5; P < 0.05). 8-pCPT-AM increased Ca2+ spark frequency in Fluo-4-AM-loaded mesenteric myocytes from 0.045 ± 0.008 to 0.103 ± 0.022 sparks s-1 m-1 (P < 0.05) and reversibly increased both the frequency (0.94 ± 0.25 to 2.30 ± 0.72 s–1) and amplitude (23.9 ± 3.3 to 35.8 ± 7.7 pA) of spontaneous transient outward currents (STOCs) recorded in isolated mesenteric myocytes (n = 7; P < 0.05). 8-pCPT-AM-activated STOCs were sensitive to iberiotoxin (100 nm) and to ryanodine (30 m). Current clamp recordings of isolated myocytes showed a 7.9 ± 1.0 mV (n = 10) hyperpolarization in response to 8-pCPT-AM that was sensitive to iberiotoxin (n = 5). Endothelial disruption suppressed 8-pCPT-AM-mediated relaxation in phenylephrine-contracted arteries (24.8 ± 4.9% relaxation after 5 min of exposure, n = 5; P < 0.05), as did apamin and TRAM-34, blockers of Ca2+-sensitive, small- and intermediate-conductance K+ (SKCa and IKCa) channels, respectively, and NG-nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase (NOS). In Fluo-4-AM-loaded mesenteric endothelial cells, 8-pCPT-AM induced a sustained increase in global Ca2+. Our data suggest that Epac hyperpolarizes smooth muscle by (1) increasing localized Ca2+ release from ryanodine receptors (Ca2+ sparks) to activate BKCa channels, and (2) endothelial-dependent mechanisms involving the activation of SKCa/IKCa channels and NOS. Epac-mediated smooth muscle hyperpolarization will limit Ca2+ entry via voltage-sensitive Ca2+ channels and represents a novel mechanism of arterial relaxation.
Properties of myenteric neurones and mucosal functions in the distal colon of diet-induced obese mice
Colonic transit and mucosal integrity are believed to be impaired in obesity. However, a comprehensive assessment of altered colonic functions, inflammatory changes and neuronal signalling of obese animals is missing. In mice, we studied the impact of diet-induced obesity (DIO) on: (i) in vivo colonic transit; (ii) signalling in the myenteric plexus by recording responses to nicotine and 2-methyl-5-hydroxytryptamine (2-methyl-5-HT), together with the expression of tryptophan hydroxylase (TPH) 1 and 2, serotonin reuptake transporter, choline acetyltransferase and the paired box gene 4; and (iii) expression of proinflammatory cytokines, epithelial permeability and density of macrophages, mast cells and enterochromaffin cells. Compared with controls, colon transit and neuronal sensitivity to nicotine and 2-methyl-5-HT were enhanced in DIO mice fed for 12 weeks. This was associated with increased tissue acetylcholine and 5-hydroxytryptamine (5-HT) content, and increased expression of TPH1 and TPH2. In DIO mice, upregulation of proinflammatory cytokines was found in fat tissue, but not in the gut wall. Accordingly, mucosal permeability or integrity was unaltered without signs of immune cell infiltration in the gut wall. Body weight showed positive correlations with adipocyte markers, tissue levels of 5-HT and acetylcholine, and the degree of neuronal sensitization. DIO mice fed for 4 weeks showed no neuronal sensitization, had no signs of gut wall inflammation and showed a smaller increase in leptin, interleukin-6 and monocyte chemoattractant protein 1 expression in fat tissue. DIO is associated with faster colonic transit and impacts on acetylcholine and 5-HT metabolism with enhanced responsiveness of enteric neurones to both mediators after 12 weeks of feeding. Our study demonstrates neuronal plasticity in DIO prior to the development of a pathological histology or abnormal mucosal functions. This questions the common assumption that increased mucosal inflammation and permeability initiate functional disorders in obesity.
Contraction-induced lipolysis is not impaired by inhibition of hormone-sensitive lipase in skeletal muscle
In skeletal muscle hormone-sensitive lipase (HSL) has long been accepted to be the principal enzyme responsible for lipolysis of intramyocellular triacylglycerol (IMTG) during contractions. However, this notion is based on in vitro lipase activity data, which may not reflect the in vivo lipolytic activity. We investigated lipolysis of IMTG in soleus muscles electrically stimulated to contract ex vivo during acute pharmacological inhibition of HSL in rat muscles and in muscles from HSL knockout (HSL-KO) mice. Measurements of IMTG are complicated by the presence of adipocytes located between the muscle fibres. To circumvent the problem with this contamination we analysed intramyocellular lipid droplet content histochemically. At maximal inhibition of HSL in rat muscles, contraction-induced breakdown of IMTG was identical to that seen in control muscles (P < 0.001). In response to contractions IMTG staining decreased significantly in both HSL-KO and WT muscles (P < 0.05). In vitro TG hydrolase activity data revealed that adipose triglyceride lipase (ATGL) and HSL collectively account for ~98% of the TG hydrolase activity in mouse skeletal muscle, other TG lipases accordingly being of negligible importance for lipolysis of IMTG. The present study is the first to demonstrate that contraction-induced lipolysis of IMTG occurs in the absence of HSL activity in rat and mouse skeletal muscle. Furthermore, the results suggest that ATGL is activated and plays a major role in lipolysis of IMTG during muscle contractions.
Temporal response of positive and negative regulators in response to acute and chronic exercise training in mice
Angiogenesis is controlled by a balance between positive and negative angiogenic factors, but temporal protein expression of many key angiogenic regulators in response to exercise are still poorly defined. In C57BL/6 mice, we evaluated the temporal protein expression of several pro-angiogenic and anti-angiogenic factors in response to (1) a single acute bout of exercise and (2) chronic exercise training resulting from 3, 5, 7, 14 and 28 days of voluntary wheel running. Following acute exercise, protein levels of vascular endothelial growth factor-A (VEGF), endostatin and nucleolin were increased at 2–4 h (P < 0.05), whereas matrix metalloproteinase (MMP)-2 was elevated within a 12–24 h window (P < 0.05). Training increased muscle capillarity 11%, 15% and 22% starting with 7, 14 and 28 days of training, respectively (P < 0.01). Basal VEGF and MMP-2 were increased by 31% and 22%, respectively, compared to controls (P < 0.05) after 7 days (7d) training, but decreased to back to baseline after 14d training. After 28d training VEGF fell 49% below baseline control (P < 0.01). Basal muscle expression of thrombospondin 1 (TSP-1) was ~900% greater in 14d- and 28d-trained mice compared to either 5d- and 7d-trained mice (P < 0.05), and tended to increase by ~180–258% compared to basal control levels (P < 0.10). The acute responsiveness of VEGF to exercise in untrained mice (i.e. 161% increase, P < 0.001) was lost with capillary adaptation occurring after 7, 14 and 28d training. Taken together, these data support the notion that skeletal muscle angiogenesis is controlled by a balance between positive and negative mitogens, and reveals a complex, highly-coordinated, temporal scheme whereby these factors can differentially influence capillary growth in response to acute versus chronic exercise.
A membrane glucocorticoid receptor mediates the rapid/non-genomic actions of glucocorticoids in mammalian skeletal muscle fibres
Glucocorticoids (GCs) are steroid hormones released from the adrenal gland in response to stress. They are also some of the most potent anti-inflammatory and immunosuppressive drugs currently in clinical use. They exert most of their physiological and pharmacological actions through the classical/genomic pathway. However, they also have rapid/non-genomic actions whose physiological and pharmacological functions are still poorly understood. Therefore, the primary aim of this study was to investigate the rapid/non-genomic effects of two widely prescribed glucocorticoids, beclomethasone dipropionate (BDP) and prednisolone acetate (PDNA), on force production in isolated, intact, mouse skeletal muscle fibre bundles. The results show that the effects of both GCs on maximum isometric force (Po) were fibre-type dependent. Thus, they increased Po in the slow-twitch fibre bundles without significantly affecting that of the fast-twitch fibre bundles. The increase in Po occurred within 10 min and was insensitive to the transcriptional inhibitor actinomycin D. Also, it was maximal at ~250 nm and was blocked by the glucocorticoid receptor (GCR) inhibitor RU486 and a monoclonal anti-GCR, suggesting that it was mediated by a membrane (m) GCR. Both muscle fibre types expressed a cytosolic GCR. However, a mGCR was present only in the slow-twitch fibres. The receptor was more abundant in oxidative than in glycolytic fibres and was confined mainly to the periphery of the fibres where it co-localised with laminin. From these findings we conclude that the rapid/non-genomic actions of GCs are mediated by a mGCR and that they are physiologically/therapeutically beneficial, especially in slow-twitch muscle fibres.
The working stroke of the myosin II motor in muscle is not tightly coupled to release of orthophosphate from its active site
Skeletal muscle shortens faster against a lower load. This force–velocity relationship is the fundamental determinant of muscle performance in vivo and is due to ATP-driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half-sarcomere. Crystallographic studies suggest that the working stroke is associated with the release of phosphate (Pi) and consists of 70 deg tilting of a light-chain domain that connects the catalytic domain of the myosin motor to the myosin tail and filament. However, the coupling of the working stroke with Pi release is still an unsolved question. Using nanometre–microsecond mechanics on skinned muscle fibres, we impose stepwise drops in force on an otherwise isometric contraction and record the isotonic velocity transient, to measure the mechanical manifestation of the working stroke of myosin motors and the rate of its regeneration in relation to the half-sarcomere load and [Pi]. We show that the rate constant of the working stroke is unaffected by [Pi], while the subsequent transition to steady velocity shortening is accelerated. We propose a new chemo-mechanical model that reproduces the transient and steady state responses by assuming that: (i) the release of Pi from the catalytic site of a myosin motor can occur at any stage of the working stroke, and (ii) a myosin motor, in an intermediate state of the working stroke, can slip to the next actin monomer during filament sliding. This model explains the efficient action of muscle molecular motors working as an ensemble in the half-sarcomere.
AMP-activated protein kinase regulates nicotinamide phosphoribosyl transferase expression in skeletal muscle
Deacetylases such as sirtuins (SIRTs) convert NAD to nicotinamide (NAM). Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme in the NAD salvage pathway responsible for converting NAM to NAD to maintain cellular redox state. Activation of AMP-activated protein kinase (AMPK) increases SIRT activity by elevating NAD levels. As NAM directly inhibits SIRTs, increased Nampt activation or expression could be a metabolic stress response. Evidence suggests that AMPK regulates Nampt mRNA content, but whether repeated AMPK activation is necessary for increasing Nampt protein levels is unknown. To this end, we assessed whether exercise training- or 5-amino-1--d-ribofuranosyl-imidazole-4-carboxamide (AICAR)-mediated increases in skeletal muscle Nampt abundance are AMPK dependent. One-legged knee-extensor exercise training in humans increased Nampt protein by 16% (P < 0.05) in the trained, but not the untrained leg. Moreover, increases in Nampt mRNA following acute exercise or AICAR treatment (P < 0.05 for both) were maintained in mouse skeletal muscle lacking a functional AMPK 2 subunit. Nampt protein was reduced in skeletal muscle of sedentary AMPK 2 kinase dead (KD), but 6.5 weeks of endurance exercise training increased skeletal muscle Nampt protein to a similar extent in both wild-type (WT) (24%) and AMPK 2 KD (18%) mice. In contrast, 4 weeks of daily AICAR treatment increased Nampt protein in skeletal muscle in WT mice (27%), but this effect did not occur in AMPK 2 KD mice. In conclusion, functional 2-containing AMPK heterotrimers are required for elevation of skeletal muscle Nampt protein, but not mRNA induction. These findings suggest AMPK plays a post-translational role in the regulation of skeletal muscle Nampt protein abundance, and further indicate that the regulation of cellular energy charge and nutrient sensing is mechanistically related.
Short-term exercise training augments 2-adrenoreceptor-mediated sympathetic vasoconstriction in resting and contracting skeletal muscle
We hypothesized that exercise training (ET) would alter 2-adrenoreceptor-mediated sympathetic vasoconstriction. Sprague-Dawley rats (n = 30) were randomized to sedentary (S), mild- (M) or heavy-intensity (H) treadmill ET groups (5 days per week for 4 weeks). Following the ET component of the study, rats were anaesthetized, and instrumented for lumbar sympathetic chain stimulation, triceps surae muscle contraction and measurement of femoral vascular conductance (FVC). The percentage change of FVC in response to sympathetic stimulation was determined at rest and during contraction in control, 2 blockade (yohimbine) and combined 2 + nitric oxide (NO) synthase (NOS) blockade (N-nitro-l-arginine methyl ester hydrochloride, l-NAME) conditions. ET augmented (P < 0.05) sympathetic vasoconstrictor responses at rest and during contraction. Yohimbine reduced (P < 0.05) the vasoconstrictor response in ET rats at rest (M: 2 Hz: 8 ± 2%, 5 Hz: 9 ± 4%; H: 2 Hz: 14 ± 5%, 5 Hz: 11 ± 6%) and during contraction (M: 2 Hz: 9 ± 2%, 5 Hz: 9 ± 5%; H: 2 Hz: 8 ± 3%, 5 Hz: 6 ± 6%) but did not change the response in S rats. The addition of l-NAME caused a larger increase (P < 0.05) in the vasoconstrictor response in ET than in S rats at rest (2 Hz: S: 8 ± 2%, M: 15 ± 3%, H: 23 ± 7%; 5 Hz: S: 8 ± 5%, M: 15 ± 3%, H: 17 ± 5%) and during contraction (2 Hz: S: 9 ± 3%, M: 18 ± 3%, H: 22 ± 6%; 5 Hz: S: 9 ± 5%, M: 22 ± 4%, H:26 ± 9%). Sympatholysis was greater (P < 0.05) in ET than in S rats. Blockade of 2-adrenoreceptors and NOS reduced (P < 0.05) sympatholysis in ET rats, but had no effect on sympatholysis in S rats. In conclusion, ET increased 2-mediated vasoconstriction at rest and during contraction.
Ranolazine, an anti-anginal compound, has been shown to significantly improve glycaemic control in large-scale clinical trials, and short-term ranolazine treatment is associated with an improvement in myocardial blood flow. As microvascular perfusion plays critical roles in insulin delivery and action, we aimed to determine if ranolazine could improve muscle microvascular blood flow, thereby increasing muscle insulin delivery and glucose use. Overnight-fasted, anaesthetized Sprague-Dawley rats were used to determine the effects of ranolazine on microvascular recruitment using contrast-enhanced ultrasound, insulin action with euglycaemic hyperinsulinaemic clamp, and muscle insulin uptake using 125I-insulin. Ranolazine's effects on endothelial nitric oxide synthase (eNOS) phosphorylation, cAMP generation and endothelial insulin uptake were determined in cultured endothelial cells. Ranolazine-induced myographical changes in tension were determined in isolated distal saphenous artery. Ranolazine at therapeutically effective dose significantly recruited muscle microvasculature by increasing muscle microvascular blood volume (~2-fold, P < 0.05) and increased insulin-mediated whole body glucose disposal (~30%, P = 0.02). These were associated with an increased insulin delivery into the muscle (P < 0.04). In cultured endothelial cells, ranolazine increased eNOS phosphorylation and cAMP production without affecting endothelial insulin uptake. In ex vivo studies, ranolazine exerted a potent vasodilatatory effect on phenylephrine pre-constricted arterial rings, which was partially abolished by endothelium denudement. In conclusion, ranolazine treatment vasodilatates pre-capillary arterioles and increases microvascular perfusion, which are partially mediated by endothelium, leading to expanded microvascular endothelial surface area available for nutrient and hormone exchanges and resulting in increased muscle delivery and action of insulin. Whether these actions contribute to improved glycaemic control in patients with insulin resistance warrants further investigation.