Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) receptors are heterodimeric complexes composed of the calcitonin receptor-like receptor (CLR) and a receptor activity-modifying protein (RAMP). Association with RAMP1 gives a high affinity CGRP receptor, whereas association with RAMP2 or RAMP3 gives high affinity ADM receptors. CGRP and ADM are widely distributed throughout the body and play important roles and are implicated in many diseases including migraine, heart failure and sepsis. Recently, CGRP has been shown to promote nitric oxide (NO) production and inducible NO synthase (iNOS) expression in trigeminal ganglion glial cells via ERK activation. CGRP is known to induce iNOS/NO production in thoracic artery smooth muscle cells (TA-SMC) pretreated with interleukin-1b. However, the molecular mechanism of CGRP-induced iNOS/NO production in TA-SMC is unknown. Therefore, in order to determine if CGRP induces iNOS/NO production via ERK activation, I first investigated the exact mechanisms through which CGRP activates ERK1-2 in HEK cells. By using different inhibitors I showed that CGRPinduced ERK activation is mainly activated through two major pathways. I showed for the first time that CGRP induces ERK activation through transactivation of ErbB1 and as expected through the cAMP/PKA pathway. Then, in order to characterise a suitable model to study CGRP-induced iNOS expression, I used primary TA-SMC and I showed that CGRP induces iNOS upregulation, which is reduced when cells are incubated with U0126, a MEK inhibitor. Thus, these results suggest that CGRP induces iNOS expression via ERK activation in TA-SMC. However, further experimentation is required to determine the exact ERK pathway responsible for iNOS induction. Compared to CLR•RAMP1 and CLR•RAMP3, little is known about the postendocytic sorting of CLR•RAMP2. Using HEK cells stably expressing CLR•RAMP2, I investigated the molecular mechanisms regulating the ADM receptor. I first showed that, unlike CLR•RAMP1, even transient stimulation of CLR•RAMP2 with ADM promotes degradation of both CLR and RAMP2, indicating that this ADM receptor does not recycle to the cell-surface. Moreover, I showed that CLR, not RAMP2, is constitutively ubiquitinated, which was further enhanced upon ADM stimulation. In order to elucidate the role of ADM-mediated ubiquitination of CLR, I made a lysine-less mutant of CLR, named CLRD9KR. I showed that ubiquitination of CLR did not affect ADM-induced trafficking of CLR•RAMP2 to lysosomes, nor did it affect the degradation or the ERK signalling of CLR•RAMP2. However, I showed that ubiquitination of CLR regulated the rate of degradation of the receptor. Together, these results indicate that CLR•RAMP2 does not recycle and is degraded via a molecular mechanism that is accelerated by ADM-induced ubiquitination of CLR.

Neuronal-glial interactions play an important role in the development of neuropathic (NP) pain states. Earlier studies in our laboratory suggest a role for activated glia in morphine-induced delta opioid receptor (DOR) trafficking by altering DOR functional competence. Thus, chronic treatment with the glial inhibitor, propentofylline (PF) blocks the anti-allodynic and anti-hyperalgesic effects of the DOR agonist deltorphin II. The present study aimed to determine whether NP pain-induced changes in DOR function and trafficking are dependent on glial activation. The first global aim of this study was to determine the molecular and behavioural effects of glial activation by two glial inhibitors, PF and PJ34 in a model of neuropathic pain. Glial activation was assessed via changes in specific proteins using fluorescent immunohistochemistry (IHC). Neuropathy-induced c-Fos activation was assessed by IHC and pain hypersensitivity was assessed, including mechanical allodynia and spontaneous pain. The second global aim determined the role of activated glia in changes in neuropathy-induced increases in DOR function and DOR subcellular localization using immunogold IHC and transmission electron microscopy (EM). Chronic PJ34 attenuated chronic constriction injury (CCI)-induced mircoglial, but not astrocyte activation. Chronic administration of either PF or PJ34 attenuated the CCI-induced increase in c-Fos immunoreactive expression. However, neither drug attenuated CCI-induced mechanical allodynia or spontaneous pain. Both chronic PF and PJ34 administration in NP animals attenuated the anti-allodynic effects of the DOR-selective agonist deltorphin II, suggesting glial inhibition blocks DOR function. However, chronic PF, but not PJ34, blocked the anti-allodynic effects of another DOR agonist, SNC80. These data suggest that SNC80 might be targeting a different DOR molecular species that is not affected by factors released from microglia. Finally, EM experiments revealed that chronic PF treatment prevented the CCI-induced increase in DOR trafficking providing a positive correlation between behaviour and receptor localization. This study suggests that activated glia contribute to changes in DOR function and trafficking in NP pain states. It also suggests that there is a dissociation between glial inhibition and pain hypersensitivity. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2010-08-31 14:45:47.888