Interaction between cysteines introduced into each transmembrane domain of the rat P2X(2) receptor

1. ATP-gated ion channels (P2X receptors) contain two hydrophobic segments that are presumed to span the plasma membrane (TM1 and TM2). Pairs of cysteines were introduced by mutagenesis into the rat P2X(2) receptor, one in TM1 one in TM2, at positions where single substitutions have previously been shown to confer sensitivity to methanethiosulfonates. The receptors were expressed in HEK293 cells; interactions between the cysteines were sought by measuring the effects on ionic currents of dithiothreitol and methanethiosulfonates. 2. Nine pairs gave normally functioning channels: F44C/I328C, F44C/N333C, F44C/L338C, Q37C/I328C, Q37C/N333C, Q37C/T336C, Q37C/L338C, G30C/I328C, G30C/N333C. None formed functionally detectable disulfide bonds. 3. Currents at the F44C/L338C receptor had time course and ATP-sensitivity similar to those for the F44C mutation alone. Methyl-methanethiosulfonate bound to L338C but did not inhibit ionic current. Methyl-methanethiosulfonate inhibited currents at F44C, but not at F44C/L338C. 4. Ethylammonium-methylthiosulfonate inhibited currents at both F44C and L338C, but not at F44C/L338C. It reversed the rapid current deactivation at F44C/L338C. 5. The results suggest that a methanethiosulfonate binding to L338C prevents binding to F44C; this might indicate proximity of these two residues

Subunit arrangement in P2X receptors

ATP-gated ionotropic receptors (P2X receptors) are distributed widely in the nervous system. For example, a hetero-oligomeric receptor containing both P2X2and P2X3subunits is involved in primary afferent sensation. Each subunit has two membrane-spanning domains. We have used disulfide bond formation between engineered cysteines to demonstrate close proximity between the outer ends of the first transmembrane domain of one subunit and the second transmembrane domain of another. After expression in HEK 293 cells of such modified P2X2or P2X4subunits, the disulfide bond formation is evident because an ATP-evoked channel opening requires previous reduction with dithiothreitol. In the hetero-oligomeric P2X2/3receptor the coexpression of doubly substituted subunits with wild-type partners allows us to deduce that the hetero-oligomeric channel contains adjacent P2X3subunits but does not contain adjacent P2X2subunits. The results suggest a “head-to-tail” subunit arrangement in the quaternary structure of P2X receptors and show that a trimeric P2X2/3receptor would have the composition P2X2(P2X3)2.</jats:p

Long-term intensive endurance exercise training is associated to reduced markers of cellular senescence in the colon mucosa of older adults

Regular endurance exercise training is an effective intervention for the maintenance of metabolic health and the prevention of many age-associated chronic diseases. Several metabolic and inflammatory factors are involved in the health-promoting effects of exercise training, but regulatory mechanisms remain poorly understood. Cellular senescence-a state of irreversible growth arrest-is considered a basic mechanism of aging. Senescent cells accumulate over time and promote a variety of age-related pathologies from neurodegenerative disorders to cancer. Whether long-term intensive exercise training affect the accumulation of age-associated cellular senescence is still unclear. Here, we show that the classical senescence markers p16 and IL-6 were markedly higher in the colon mucosa of middle-aged and older overweight adults than in young sedentary individuals, but this upregulation was significantly blunted in age-matched endurance runners. Interestingly, we observe a linear correlation between the level of p16 and the triglycerides to HDL ratio, a marker of colon adenoma risk and cardiometabolic dysfunction. Our data suggest that chronic high-volume high-intensity endurance exercise can play a role in preventing the accumulation of senescent cells in cancer-prone tissues like colon mucosa with age. Future studies are warranted to elucidate if other tissues are also affected, and what are the molecular and cellular mechanisms that mediate the senopreventative effects of different forms of exercise training.</p

Fluoroquinolone-induced motor changes in the guinea-pig isolated ileum

The effects of norfloxacin and enoxacin were examined on spontaneous motor activity in the guinea-pig isolated ileum. Micromolar concentrations of both compounds caused a biphasic response consisting of relaxation followed by transient contraction. Relaxation to norfloxacin, which was unaffected by phentolamine, propranolol and hyoscine (each at 1 microM), was partially sensitive to tetrodotoxin (1 microM). This indicates that the response is partly mediated by non-adrenergic non-cholinergic (NANC) inhibitory nerves, and partly related to a direct action on the smooth muscle. Apamin (0.1 microM) and suramin (300 microM) inhibited norfloxacin-induced relaxations to an extent similar to that of tetrodotoxin. Conversely, NG-nitro-L-arginine (300 microM) was ineffective. In the presence of theophylline (100 microM) and 3-isobutyl-1-methylxanthine (10 microM), norfloxacin caused relaxation less effective than when added alone. Based on this observation, the NANC component of the relaxation apparently depends on ATP release, whereas the direct component might be due, at least in part, to phosphodiesterase inhibition. Norfloxacin-induced contractions were neurogenic and cholinergic in nature. They were reduced by indomethacin or S-ketoprofen (both at 0.01-1 microM) and suramin (300 microM), suggesting involvement of local prostaglandin production probably induced by ATP release. Previous findings revealed that norfloxacin acted as a non-competitive antagonist at enteric GABAA receptors. In this study the same property was shared by enoxacin against the contractile response to 3-aminopropane sulphonic acid (3-APS), a GABAA receptor agonist. In conclusion, fluoroquinolones exert inhibitory and excitatory effects in the guinea-pig ileum. These are mediated by ATP, prostaglandin and acetylcholine release that might underlie, at least in part, the alterations of gastrointestinal motility observed after fluoroquinolone administration. Furthermore, isolated intestinal preparations might be useful to predict the GABAA-antagonist potential of this class of compounds

Effects of protein restriction on insulin-like growth factor (IGF)-1 in men with prostate cancer: results from a randomized clinical trial

Abstract Background Insulin-like growth factor (IGF)-1 and its binding proteins are important in cancer growth, especially in prostate cancer. Observational studies suggest that protein restriction can lower IGF-1 levels. However, it is unclear whether an isocaloric protein-restricted diet affects IGF-1 and IGFBPs in men with prostate cancer. Methods In this academic, single-center, parallel-group, prospective, randomized, open-label, blinded end-point trial, 38 consenting overweight (BMI 30.5 ± 5.5 kg/m2) men with localized prostate cancer, aged 43–72 years, were randomized (1:1) with permuted blocks to 4–6 weeks of customized isocaloric PR diets (0.8 g protein/kg lean body mass) or their usual diet. Biomarkers influencing cancer biology, including serum IGF-1 and its binding proteins were measured longitudinally. Results Contrary to our hypothesis, feeding individuals an isocaloric protein-restricted diet did not result in a significant reduction in serum IGF-1. Moreover, there was no observed increase in serum IGFBP-1 or IGFBP-3 concentration. Conclusion These findings demonstrate that protein restriction without calorie restriction does not reduce serum IGF-1 concentration or increase IGFBP-1 and IGFBP-3 in men with localized prostate cancer. Further research is needed to identify dietary interventions for safely and effectively reducing IGF-1 in this patient group

ATP analogues with modified phosphate chains and their selectivity for rat P2X(2) and P2X(2/3) receptors

1. Heteromeric P2X(2/3) receptors are much more sensitive than homomeric P2X(2) receptors to αβ-methylene-ATP, and this ATP analogue is widely used to discriminate the two receptors on sensory neurons and other cells. 2. We sought to determine the structural basis for this selectivity by synthesising ADP and ATP analogues in which the αβ and/or βγ oxygen atoms were replaced by other moieties (including –CH(2)–, –CHF–, –CHCl–, –CHBr–, –CF(2)–, –CCl(2)–, –CBr(2)–, –CHSO(3)–, –CHPO(3)–, –CFPO(3)–, –CClPO(3)–, –CH(2)–CH(2)–, –C≡C–, –NH–, –CHCOOH–). 3. We tested their actions as agonists or antagonists by whole-cell recording from human embryonic kidney cells expressing P2X(2) subunits alone (homomeric P2X(2) receptors), or cells expressing both P2X(2) and P2X(3) subunits, in which the current through heteromeric P2X(2/3) receptors was isolated. 4. ADP analogues had no agonist or antagonist effect at either P2X(2) or P2X(2/3) receptors. All the ATP analogues tested were without agonist or antagonist activity at homomeric P2X(2) receptors, except βγ-difluoromethylene-ATP, which was a weak agonist. 5. At P2X(2/3) receptors, βγ-imido-ATP, βγ-methylene-ATP, and βγ-acetylene-ATP were weak agonists, whereas αβ,βγ- and βγ,γδ-bismethylene-AP(4) were potent full agonists. βγ-Carboxymethylene-ATP and βγ-chlorophosphonomethylene-ATP were weak antagonists at P2X(2/3) receptors (IC(50) about 10 μM). 6. The results indicate (a) that the homomeric P2X(2) receptor presents very stringent structural requirements with respect to its activation by ATP; (b) that the heteromeric P2X(2/3) receptor is much more tolerant of αβ and βγ substitution; and (c) that a P2X(2/3)-selective antagonist can be obtained by introduction of additional negativity at the βγ-methylene