The Journal of physiology
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The Journal of physiology · Jun 2016
Randomized Controlled TrialCarbon dioxide-mediated vasomotion of extra-cranial cerebral arteries in humans: a role for prostaglandins?
Cerebral blood flow increases during hypercapnia and decreases during hypocapnia; it is unknown if vasomotion of the internal carotid artery is implicated in these responses. Indomethacin, a non-selective cyclooxygenase inhibitor (used to inhibit prostaglandin synthesis), has a unique ability to blunt cerebrovascular carbon dioxide reactivity, while other cyclooxygenase inhibitors have no effect. We show significant dilatation and constriction of the internal carotid artery during hypercapnia and hypocapnia, respectively. Indomethacin, but not ketorolac or naproxen, reduced the dilatatory response of the internal carotid artery to hypercapnia The differential effect of indomethacin compared to ketorolac and naproxen suggests that indomethacin inhibits vasomotion of the internal carotid artery independent of prostaglandin synthesis inhibition. ⋯ Extra-cranial cerebral blood vessels are implicated in the regulation of cerebral blood flow during changes in arterial CO2 ; however, the mechanisms governing CO2 -mediated vasomotion of these vessels in humans remain unclear. We determined if cyclooxygenase inhibition with indomethacin (INDO) reduces the vasomotor response of the internal carotid artery (ICA) to changes in end-tidal CO2 (P ETC O2). Using a randomized single-blinded placebo-controlled study, participants (n = 10) were tested on two occasions, before and 90 min following oral INDO (1.2 mg kg(-1) ) or placebo. Concurrent measurements of beat-by-beat velocity, diameter and blood flow of the ICA were made at rest and during steady-state stages (4 min) of iso-oxic hypercapnia (+3, +6, +9 mmHg P ETC O2) and hypocapnia (-3, -6, -9 mmHg P ETC O2). To examine if INDO affects ICA vasomotion independent of cyclooxygenase inhibition, two participant subsets (each n = 5) were tested before and following oral ketorolac (post 45 min, 0.25 mg kg(-1) ) or naproxen (post 90 min, 4.2 mg kg(-1) ). During pre-drug testing in the INDO trial, the ICA dilatated during hypercapnia at +6 mmHg (4.72 ± 0.45 vs. 4.95 ± 0.51 mm; P < 0.001) and +9 mmHg (4.72 ± 0.45 mm vs. 5.12 ± 0.47 mm; P < 0.001), and constricted during hypocapnia at -6 mmHg (4.95 ± 0.33 vs. 4.88 ± 0.27 mm; P < 0.05) and -9 mmHg (4.95 ± 0.33 vs. 4.82 ± 0.27 mm; P < 0.001). Following INDO, vasomotor responsiveness of the ICA to hypercapnia was reduced by 67 ± 28% (0.045 ± 0.015 vs. 0.015 ± 0.012 mm mmHg P ETC O2(-1) ). There was no effect of the drug in the ketorolac and naproxen trials. We conclude that: (1) INDO markedly reduces the vasomotor response of the ICA to changes in P ETC O2; and (2) INDO may be reducing CO2 -mediated vasomotion via a mechanism(s) independent of cyclooxygenase inhibition.
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The Journal of physiology · Jun 2016
On the mechanism of gating defects caused by the R117H mutation in cystic fibrosis transmembrane conductance regulator.
Two functional abnormalities of cystic fibrosis transmembrane conductance regulator (CFTR), a 25% reduction of the single-channel conductance (g) and a ∼13-fold lower open probability (Po ), were found with the R117H mutation that is associated with mild forms of cystic fibrosis. Characterizations of the gating defects of R117H-CFTR led to the conclusion that the mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains (TMDs) without altering the function of the nucleotide binding domains (NBDs). Nonetheless, gating of the R117H-CFTR can be improved by a variety of pharmacological reagents supposedly acting on NBDs such as ATP analogues, or TMDs (e.g. VX-770 or nitrate). These reagents potentiate synergistically R117H-CFTR gating to a level that allows accurate assessments of its gating deficits. Our studies not only elucidate the mechanism underpinning gating dysfunction of R117H-CFTR, but also provide a mechanistic understanding of how VX-770 ameliorates the gating defects in the R117H mutant. ⋯ Cystic fibrosis (CF) is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a phosphorylation-activated, but ATP-gated chloride channel. In the current study, we investigated the mechanism responsible for the gating defects manifested in R117H-CFTR, an arginine-to-histidine substitution at position 117 of CFTR that is associated with mild forms of CF. We confirmed previous findings of a 25% decrease of the single-channel conductance (g) in R117H-CFTR, but found a ∼13-fold lower open probability (Po ). This dramatic gating deficit is not due to dysfunctional nucleotide binding domains (NBDs) as the mutation does not alter the apparent affinity for ATP, and the mutant channels respond to ATP analogues in a similar manner as wild-type CFTR. Furthermore, once ATP hydrolysis is abolished, the R117H mutant can be trapped in a prolonged 'burst opening' conformation that is proposed to be equipped with a stable NBD dimer. On the other hand, our results support the conclusion that the R117H mutation decreases Po by perturbing the gating conformational changes in CFTR's transmembrane domains as even when NBDs are kept at a dimerized configuration, Po is reduced by ∼10-fold. Moreover, our data demonstrate that a synergistic improvement of R117H-CFTR function can be accomplished with a combined regiment of VX-770 (Ivacaftor), nitrate ion (NO3 (-) ) and N(6) -(2-phenylethyl)-2'-deoxy-ATP (d-PATP), which almost completely rectifies the gating defect of R117H-CFTR. Clinical implications of our results are discussed.
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The Journal of physiology · Jun 2016
Increasing taurine intake and taurine synthesis improves skeletal muscle function in the mdx mouse model for Duchenne muscular dystrophy.
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation, oxidative stress and myofibre necrosis. Cysteine precursor antioxidants such as N-acetyl cysteine (NAC) and l-2-oxothiazolidine-4-carboxylate (OTC) reduce dystropathology in the mdx mouse model for DMD, and we propose this is via increased synthesis of the amino acid taurine. We compared the capacity of OTC and taurine treatment to increase taurine content of mdx muscle, as well as effects on in vivo and ex vivo muscle function, inflammation and oxidative stress. Both treatments increased taurine in muscles, and improved many aspects of muscle function and reduced inflammation. Taurine treatment also reduced protein thiol oxidation and was overall more effective, as OTC treatment reduced body and muscle weight, suggesting some adverse effects of this drug. These data suggest that increasing dietary taurine is a better candidate for a therapeutic intervention for DMD. ⋯ Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease for which there is no widely available cure. Whilst the mechanism of loss of muscle function in DMD and the mdx mouse model are not fully understood, disruptions in intracellular calcium homeostasis, inflammation and oxidative stress are implicated. We have shown that protein thiol oxidation is increased in mdx muscle, and that the indirect thiol antioxidant l-2-oxothiazolidine-4-carboxylate (OTC), which increases cysteine availability, decreases pathology and increases in vivo strength. We propose that the protective effects of OTC are a consequence of conversion of cysteine to taurine, which has itself been shown to be beneficial to mdx pathology. This study compares the efficacy of taurine with OTC in decreasing dystropathology in mdx mice by measuring in vivo and ex vivo contractile function and measurements of inflammation and protein thiol oxidation. Increasing the taurine content of mdx muscle improved both in vivo and ex vivo muscle strength and function, potentially via anti-inflammatory and antioxidant effects of taurine. OTC treatment increased taurine synthesis in the liver and taurine content of mdx muscle, improved muscle function and decreased inflammation. However, OTC was less effective than taurine treatment, with OTC also decreasing body and EDL muscle weights, suggesting that OTC had some detrimental effects. These data support continued research into the use of taurine as a therapeutic intervention for DMD, and suggest that increasing dietary taurine is the better strategy for increasing taurine content and decreasing severity of dystropathology.