P2Y1 and P2Y12 receptor cross-talk in calcium signalling: Evidence from nonstarved and long-term serum-deprived glioma C6 cells

The current work presents results of experiments on the calcium response evoked by the stimulation by extracellular nucleotides occurring in control, nonstarved glioma C6 cells and in cells after long-term (96 h) serum starvation. Three nucleotide receptors were studied: P2Y1, P2Y2 and P2Y12. Two of them, P2Y1 and P2Y2, directly stimulate calcium response. The protein level of the P2Y2 receptor did not change during the serum starvation, while P2Y1 protein level fell dramatically. Observed changes in the calcium response generated by P2Y1 are directly correlated with the receptor protein level as well as with the amount of calcium present in the intracellular calcium stores, partially depleted during starvation process. The third receptor, P2Y12, did not directly evoke calcium response, however it is activated by the same ligand as P2Y1. The experiments with AR-C69941MX, the P2Y12-specific antagonist, indicated that in control and serum-starved cells, calcium response evoked by P2Y1 receptor is potentiated by the activity of P2Y12-dependent signaling pathways. This potentiation may be mediated by P2Y12 inhibitory effect on the plasma membrane calcium pump. The calcium influx enhanced by the cooperation of P2Y1 and P2Y12 receptor activity directly depends on the capacitative calcium entrance mechanism

Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks

The role of nucleotides in intracellular energy provision and nucleic acid synthesis has been known for a long time. In the past decade, evidence has been presented that, in addition to these functions, nucleotides are also autocrine and paracrine messenger molecules that initiate and regulate a large number of biological processes. The actions of extracellular nucleotides are mediated by ionotropic P2X and metabotropic P2Y receptors, while hydrolysis by ecto-enzymes modulates the initial signal. An increasing number of studies have been performed to obtain information on the signal transduction pathways activated by nucleotide receptors. The development of specific and stable purinergic receptor agonists and antagonists with therapeutical potential largely contributed to the identification of receptors responsible for nucleotide-activated pathways. This article reviews the signal transduction pathways activated by P2Y receptors, the involved second messenger systems, GTPases and protein kinases, as well as recent findings concerning P2Y receptor signalling in C6 glioma cells. Besides vertical signal transduction, lateral cross-talks with pathways activated by other G protein-coupled receptors and growth factor receptors are discussed

Modeling and Analysis of the Molecular Basis of Pain in Sensory Neurons

Intracellular calcium dynamics are critical to cellular functions like pain transmission. Extracellular ATP plays an important role in modulating intracellular calcium levels by interacting with the P2 family of surface receptors. In this study, we developed a mechanistic mathematical model of ATP-induced P2 mediated calcium signaling in archetype sensory neurons. The model architecture, which described 90 species connected by 162 interactions, was formulated by aggregating disparate molecular modules from literature. Unlike previous models, only mass action kinetics were used to describe the rate of molecular interactions. Thus, the majority of the 252 unknown model parameters were either association, dissociation or catalytic rate constants. Model parameters were estimated from nine independent data sets taken from multiple laboratories. The training data consisted of both dynamic and steady-state measurements. However, because of the complexity of the calcium network, we were unable to estimate unique model parameters. Instead, we estimated a family or ensemble of probable parameter sets using a multi-objective thermal ensemble method. Each member of the ensemble met an error criterion and was located along or near the optimal trade-off surface between the individual training data sets. The model quantitatively reproduced experimental measurements from dorsal root ganglion neurons as a function of extracellular ATP forcing. Hypothesized architecture linking phosphoinositide regulation with P2X receptor activity explained the inhibition of P2X-mediated current flow by activated metabotropic P2Y receptors. Sensitivity analysis using individual and the whole system outputs suggested which molecular subsystems were most important following P2 activation. Taken together, modeling and analysis of ATP-induced P2 mediated calcium signaling generated qualitative insight into the critical interactions controlling ATP induced calcium dynamics. Understanding these critical interactions may prove useful for the design of the next generation of molecular pain management strategies

Introduction: Connecting Water and Heritage for the Future

Water has served and sustained societies throughout the history of humankind. People have actively shaped its course, form, and function for human settlement and the development of civilizations. Around water, they have created socioeconomic structures, policies, and cultures; a rich world of narratives, laws, and practices; and an extensive tangible network of infrastructure, buildings, and urban form. Today, the complex and diverse systems of the past are necessarily the framework for preservation and reuse as well as for new systems. Through twenty-one chapters in five thematic sections, this book links the practices of the past to a present in which heritage and water are largely two separate disciplinary and professional fields. It describes an alternative emerging present in which policymaking and design work together to recognize and build on traditional knowledge and skills while imagining how such efforts will help us develop sustainable futures for cities, landscapes, and bodies of water.Archaeological Heritage Managemen

Development and psychometric properties of the PROMIS® pediatric fatigue item banks

PURPOSE: This paper reports on the development and psychometric properties of self-reported pediatric fatigue item banks as part of the Patient Reported Outcomes Measurement Information System (PROMIS). METHODS: Candidate items were developed by using PROMIS qualitative methodology. The resulting 39 items (25 tiredness- and 14 energy-related) were field tested in a sample that included 3,048 participants aged 8–17 years. We used confirmatory factor analysis (CFA) to evaluate dimensionality, differential item functioning (DIF) analysis to evaluate parameter stability between genders and by age; we examined residual correlations to evaluate local dependence (LD) among items, and estimated the parameters of item response theory (IRT) models. RESULTS: Of 3,048 participants, 48% were males, 60% were white and 23% had at least one chronic condition. CFA results suggest two moderately correlated factors. Two items were removed due to high LD, and three due to gender-based DIF. Two item banks were calibrated separately using IRT: Tired and (Lack of) Energy, which consisted of 23 and 11 items, respectively; ten- and 8-item short-forms were created. CONCLUSION: The PROMIS assessment of self-reported fatigue in pediatrics includes two item banks: Tired and (Lack of) Energy. Both demonstrated satisfactory psychometric properties and can be used for research settings