Long-term (trophic) purinergic signalling: purinoceptors control cell proliferation, differentiation and death

The purinergic signalling system, which uses purines and pyrimidines as chemical transmitters, and purinoceptors as effectors, is deeply rooted in evolution and development and is a pivotal factor in cell communication. The ATP and its derivatives function as a 'danger signal' in the most primitive forms of life. Purinoceptors are extraordinarily widely distributed in all cell types and tissues and they are involved in the regulation of an even more extraordinary number of biological processes. In addition to fast purinergic signalling in neurotransmission, neuromodulation and secretion, there is long-term (trophic) purinergic signalling involving cell proliferation, differentiation, motility and death in the development and regeneration of most systems of the body. In this article, we focus on the latter in the immune/defence system, in stratified epithelia in visceral organs and skin, embryological development, bone formation and resorption, as well as in cancer. Cell Death and Disease (2010) 1, e9; doi:10.1038/cddis.2009.11; published online 14 January 201

Gene duplications and evolution of vertebrate voltage-gated sodium channels

Author Posting. © The Author(s), 2006. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Journal of Molecular Evolution 63 (2006): 208-221, doi:10.1007/s00239-005-0287-9.Voltage-gated sodium channels underlie action potential generation in excitable tissue. To establish the evolutionary mechanisms that shaped the vertebrate sodium channel a-subunit (SCNA) gene family and their encoded Nav1 proteins, we identified all SCNA genes in several teleost species. Molecular cloning revealed that teleosts have eight SCNA genes, comparable to the number in another vertebrate lineage, mammals. Prior phylogenetic analyses had indicated that teleosts and tetrapods share four monophyletic groups of SCNA genes and that tandem duplications selectively expanded the number of genes in two of the four mammalian groups. However, the number of genes in each group varies between teleosts and tetrapods suggesting different evolutionary histories in the two vertebrate lineages. Our findings from phylogenetic analysis and chromosomal mapping of Danio rerio genes indicate that tandem duplications are an unlikely mechanism for generation of the extant teleost SCNA genes. Instead, analysis of other closely mapped genes in D. rerio supports the hypothesis that a whole genome duplication was involved in expansion of the SCNA gene family in teleosts. Interestingly, despite their different evolutionary histories, mRNA analyses demonstrated a conservation of expression patterns for SCNA orthologues in teleosts and tetrapods, suggesting functional conservation.The authors’ work was supported by NIH grants (NS 38937; AEN, ADT and ABR, NS 25513; HHZ and YL and NSF IBN 0236147; MCJ)

P2X receptor-mediated purinergic sensory pathways to the spinal cord dorsal horn

P2X receptors are expressed on different functional groups of primary afferent fibers. P2X receptor-mediated sensory inputs can be either innocuous or nociceptive, depending on which dorsal horn regions receive these inputs. We provide a brief review of P2X receptor-mediated purinergic sensory pathways to different regions in the dorsal horn. These P2X purinergic pathways are identified in normal animals, which provides insights into their physiological functions. Future studies on P2X purinergic pathways in animal models of pathological conditions may provide insights on how P2X receptors play a role in pathological pain states

Two-Photon Imaging of Calcium in Virally Transfected Striate Cortical Neurons of Behaving Monkey

Two-photon scanning microscopy has advanced our understanding of neural signaling in non-mammalian species and mammals. Various developments are needed to perform two-photon scanning microscopy over prolonged periods in non-human primates performing a behavioral task. In striate cortex in two macaque monkeys, cortical neurons were transfected with a genetically encoded fluorescent calcium sensor, memTNXL, using AAV1 as a viral vector. By constructing an extremely rigid and stable apparatus holding both the two-photon scanning microscope and the monkey's head, single neurons were imaged at high magnification for prolonged periods with minimal motion artifacts for up to ten months. Structural images of single neurons were obtained at high magnification. Changes in calcium during visual stimulation were measured as the monkeys performed a fixation task. Overall, functional responses and orientation tuning curves were obtained in 18.8% of the 234 labeled and imaged neurons. This demonstrated that the two-photon scanning microscopy can be successfully obtained in behaving primates

Danger- and pathogen-associated molecular patterns recognition by pattern-recognition receptors and ion channels of the transient receptor potential family triggers the inflammasome activation in immune cells and sensory neurons.

An increasing number of studies show that the activation of the innate immune system and inflammatory mechanisms play an important role in the pathogenesis of numerous diseases. The innate immune system is present in almost all multicellular organisms and its activation occurs in response to pathogens or tissue injury via pattern-recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Intracellular pathways, linking immune and inflammatory response to ion channel expression and function, have been recently identified. Among ion channels, the transient receptor potential (TRP) channels are a major family of non-selective cation-permeable channels that function as polymodal cellular sensors involved in many physiological and pathological processes.In this review, we summarize current knowledge of interactions between immune cells and PRRs and ion channels of TRP families with PAMPs and DAMPs to provide new insights into the pathogenesis of inflammatory diseases. TRP channels have been found to interfere with innate immunity via both nuclear factor-kB and procaspase-1 activation to generate the mature caspase-1 that cleaves pro-interleukin-1ß cytokine into the mature interleukin-1ß.Sensory neurons are also adapted to recognize dangers by virtue of their sensitivity to intense mechanical, thermal and irritant chemical stimuli. As immune cells, they possess many of the same molecular recognition pathways for danger. Thus, they express PRRs including Toll-like receptors 3, 4, 7, and 9, and stimulation by Toll-like receptor ligands leads to induction of inward currents and sensitization in TRPs. In addition, the expression of inflammasomes in neurons and the involvement of TRPs in central nervous system diseases strongly support a role of TRPs in inflammasome-mediated neurodegenerative pathologies. This field is still at its beginning and further studies may be required.Overall, these studies highlight the therapeutic potential of targeting the inflammasomes in proinflammatory, autoinflammatory and metabolic disorders associated with undesirable activation of the inflammasome by using specific TRP antagonists, anti-human TRP monoclonal antibody or different molecules able to abrogate the TRP channel-mediated inflammatory signals

Illuminating the life of GPCRs

The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. G-protein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented

In pursuit of P2X3 antagonists: novel therapeutics for chronic pain and afferent sensitization

Treating pain by inhibiting ATP activation of P2X3-containing receptors heralds an exciting new approach to pain management, and Afferent's program marks the vanguard in a new class of drugs poised to explore this approach to meet the significant unmet needs in pain management. P2X3 receptor subunits are expressed predominately and selectively in so-called C- and Aδ-fiber primary afferent neurons in most tissues and organ systems, including skin, joints, and hollow organs, suggesting a high degree of specificity to the pain sensing system in the human body. P2X3 antagonists block the activation of these fibers by ATP and stand to offer an alternative approach to the management of pain and discomfort. In addition, P2X3 is expressed pre-synaptically at central terminals of C-fiber afferent neurons, where ATP further sensitizes transmission of painful signals. As a result of the selectivity of the expression of P2X3, there is a lower likelihood of adverse effects in the brain, gastrointestinal, or cardiovascular tissues, effects which remain limiting factors for many existing pain therapeutics. In the periphery, ATP (the factor that triggers P2X3 receptor activation) can be released from various cells as a result of tissue inflammation, injury or stress, as well as visceral organ distension, and stimulate these local nociceptors. The P2X3 receptor rationale has aroused a formidable level of investigation producing many reports that clarify the potential role of ATP as a pain mediator, in chronic sensitized states in particular, and has piqued the interest of pharmaceutical companies. P2X receptor-mediated afferent activation has been implicated in inflammatory, visceral, and neuropathic pain states, as well as in airways hyperreactivity, migraine, itch, and cancer pain. It is well appreciated that oftentimes new mechanisms translate poorly from models into clinical efficacy and effectiveness; however, the breadth of activity seen from P2X3 inhibition in models offers a realistic chance that this novel mechanism to inhibit afferent nerve sensitization may find its place in the sun and bring some merciful relief to the torment of persistent discomfort and pain. The development philosophy at Afferent is to conduct proof of concept patient studies and best identify target patient groups that may benefit from this new intervention