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The effect of GPCR crosstalk on intracellular Ca2+ responses and downstream signalling

By Caroline Jane Hall


This thesis studies crosstalk between G q/11-coupled muscarinic M3 receptors and G s-coupled\ud 2-adrenoceptors leading to a synergistic increase in intracellular Ca2+. The aims were to\ud generally understand the integration of signalling and specifically the potential interactions\ud between these receptors in physiological and pathological settings, for example in asthma and\ud chronic obstructive pulmonary disease (COPD). This study demonstrated that only in the\ud presence of a muscarinic agonist did stimulation of 2-adrenoceptors elicit a Ca2+ response.\ud Both full and partial agonists of the muscarinic M3 receptor were used to examine differences\ud in the pattern of Ca2+ signalling both mediated by the muscarinic M3 receptor and the 2-\ud adrenoceptor. Crosstalk was dependent on the concentration of the muscarinic agonist, being\ud more robust and consistent in the presence of a partial rather than a full muscarinic agonist.\ud Under crosstalk conditions, single cell imaging highlighted pronounced alterations in the\ud pattern of Ca2+ signalling, such as oscillatory frequency. This Ca2+ signal was independent of\ud extracellular Ca2+ but was dependent on thapsigargin-sensitive stores. Investigation of the\ud potential mechanism indicated that exchange proteins directly activated by cAMP (Epac) and\ud protein kinase A (PKA) were not involved. An alternative mechanism was suggested, with\ud localised increases in cAMP directly sensitising Ins(1,4,5)P3 receptors. Downstream signalling\ud events regulated by Ca2+, including extracellular signal-regulated kinases (ERK) and nuclear\ud factor-kappaB (NF-kB) were examined to determine if their regulation altered in the presence\ud of crosstalk. Although crosstalk experiments were unsuccessful in rat tracheal smooth muscle\ud cells, due to loss of muscarinic M3 receptor expression, it is still possible that this crosstalk\ud could alter the regulation of cell function and be an important factor in treating diseases such as\ud asthma and COPD

Publisher: University of Leicester
Year: 2011
OAI identifier: oai:lra.le.ac.uk:2381/9528

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