P2X receptors: epithelial ion channels and regulators of salt and water transport.

When the results from electrophysiological studies of renal epithelial cells are combined with data from in vivo tubule microperfusion experiments and immunohistochemical surveys of the nephron, the accumulated evidence suggests that ATP-gated ion channels, P2X receptors, play a specialized role in the regulation of ion and water movement across the renal tubule and are integral to electrolyte and fluid homeostasis. In this short review, we discuss the concept of P2X receptors as regulators of salt and water salvage pathways, as well as acknowledging their accepted role as ATP-gated ion channels

Biphasic effect of extracellular ATP on human and rat airways is due to multiple P2 purinoceptor activation

BACKGROUND: Extracellular ATP may modulate airway responsiveness. Studies on ATP-induced contraction and [Ca(2+)](i )signalling in airway smooth muscle are rather controversial and discrepancies exist regarding both ATP effects and signalling pathways. We compared the effect of extracellular ATP on rat trachea and extrapulmonary bronchi (EPB) and both human and rat intrapulmonary bronchi (IPB), and investigated the implicated signalling pathways. METHODS: Isometric contraction was measured on rat trachea, EPB and IPB isolated rings and human IPB isolated rings. [Ca(2+)](i )was monitored fluorimetrically using indo 1 in freshly isolated and cultured tracheal myocytes. Statistical comparisons were done with ANOVA or Student's t tests for quantitative variables and χ(2 )tests for qualitative variables. Results were considered significant at P < 0.05. RESULTS: In rat airways, extracellular ATP (10(-6)–10(-3 )M) induced an epithelium-independent and concentration-dependent contraction, which amplitude increased from trachea to IPB. The response was transient and returned to baseline within minutes. Similar responses were obtained with the non-hydrolysable ATP analogous ATP-γ-S. Successive stimulations at 15 min-intervals decreased the contractile response. In human IPB, the contraction was similar to that of rat IPB but the time needed for the return to baseline was longer. In isolated myocytes, ATP induced a concentration-dependent [Ca(2+)](i )response. The contractile response was not reduced by thapsigargin and RB2, a P2Y receptor inhibitor, except in rat and human IPB. By contrast, removal of external Ca(2+), external Na(+ )and treatment with D600 decreased the ATP-induced response. The contraction induced by α-β-methylene ATP, a P2X agonist, was similar to that induced by ATP, except in IPB where it was lower. Indomethacin and H-89, a PKA inhibitor, delayed the return to baseline in extrapulmonary airways. CONCLUSION: Extracellular ATP induces a transient contractile response in human and rat airways, mainly due to P2X receptors and extracellular Ca(2+ )influx in addition with, in IPB, P2Y receptors stimulation and Ca(2+ )release from intracellular Ca(2+ )stores. Extracellular Ca(2+ )influx occurs through L-type voltage-dependent channels activated by external Na(+ )entrance through P2X receptors. The transience of the response cannot be attributed to ATP degradation but to purinoceptor desensitization and, in extrapulmonary airways, prostaglandin-dependent PKA activation

The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanellaoneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O2 deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research

Improving the outcome of infants born at <30 weeks' gestation - a randomized controlled trial of preventative care at home

Background: Early developmental interventions to prevent the high rate of neurodevelopmental problems in very preterm children, including cognitive, motor and behavioral impairments, are urgently needed. These interventions should be multi-faceted and include modules for caregivers given their high rates of mental health problems

Energy metabolism, altered proteins, sirtuins and ageing: converging mechanisms?

The predominant molecular symptom of ageing is the accumulation of altered gene products. Nutritional studies show that ageing in animals can be significantly influenced by dietary restriction. Genetics has revealed that ageing may be controlled by changes in intracellular NAD/NADH ratio regulating sirtuin activity. Physiological and other approaches indicate that mitochondria may also regulate ageing. A mechanism is proposed which links diet, exercise and mitochondria-dependent changes in NAD/NADH ratio to intracellular generation of altered proteins. It is suggested that ad libitum feeding conditions decrease NAD availability which also decreases metabolism of the triose phosphate glycolytic intermediates, glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate, which can spontaneously decompose into methylglyoxal (MG). MG is a highly toxic glycating agent and a major source of protein advanced-glycosylation end-products (AGEs). MG and AGEs can induce mitochondrial dysfunction and formation of reactive oxygen species (ROS), as well as affect gene expression and intracellular signalling. In dietary restriction–induced fasting, NADH would be oxidised and NAD regenerated via mitochondrial action. This would not only activate sirtuins and extend lifespan but also suppress MG formation. This proposal can also explain the apparent paradox whereby increased aerobic activity suppresses formation of glycoxidized proteins and extends lifespan. Variation in mitochondrial DNA composition and consequent mutation rate, arising from dietary-controlled differences in DNA precursor ratios, could also contribute to tissue differences in age-related mitochondrial dysfunction

The S4–S5 Linker Acts as a Signal Integrator for hERG K+ Channel Activation and Deactivation Gating

Human ether-à-go-go-related gene (hERG) K+ channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4–S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4–S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4–S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4–S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4–S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4–S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel

TRPV1 enhances the afferent response to P2X receptor activation in the mouse urinary bladder

Both TRPV1 and P2X receptors present on bladder sensory nerve fibres have been implicated in mechanosensation during bladder filling. The aim of this study was to determine possible interactions between these receptors in modulating afferent nerve activity. In wildtype (TRPV1+/+) and TRPV1 knockout (TRPV1−/−) mice, bladder afferent nerve activity, intravesical pressure, and luminal ATP and acetylcholine levels were determined and also intracellular calcium responses of dissociated pelvic DRG neurones and primary mouse urothelial cells (PMUCs). Bladder afferent nerve responses to the purinergic agonist αβMethylene-ATP were depressed in TRPV1−/− mice (p ≤ 0.001) and also in TRPV1+/+ mice treated with the TRPV1-antagonist capsazepine (10 µM; p ≤ 0.001). These effects were independent of changes in bladder compliance or contractility. Responses of DRG neuron to αβMethylene-ATP (30 µM) were unchanged in the TRPV1−/− mice, but the proportion of responsive neurones was reduced (p ≤ 0.01). Although the TRPV1 agonist capsaicin (1 µM) did not evoke intracellular responses in PMUCs from TRPV1+/+ mice, luminal ATP levels were reduced in the TRPV1−/− mice (p ≤ 0.001) compared to wildtype. TRPV1 modulates P2X mediated afferent responses and provides a mechanistic basis for the decrease in sensory symptoms observed following resiniferatoxin and capsaicin treatment for lower urinary tract symptoms