Opioid analgesics are the leading class of prescription drugs that cause unintentional overdose deaths via respiratory depression. Poly-drug use is a common cause of death, with ethanol detected in approx.. 50% of heroin-related cases. Tolerance can be reversed by low concentrations of ethanol in locus coeruleus neurones and tolerance in these neurones occurs through a PKC-dependant mechanism. One possible explanation for the correlation between simultaneous opiate and alcohol use, and the likelihood of fatality, is that alcohol has a direct effect on PKC activity causing a reversal of opioid induced tolerance. This thesis aims to elucidate the mechanisms involved in the development and maintenance of opioid tolerance and the potential drug interactions that may affect this phenomenon. The effect of ethanol on PKC activity was investigated using a non-radioactive PKC detection assay and a fluorescent DAG-reporter expressed in HEK293 cells. The results from the in vitro assay did not demonstrate a direct effect of ethanol on PKC activity. In HEK293 cells ethanol reduced the increase in fluorescence associated with increased DAG activity. Therefore ethanol may indirectly inhibit PKC activity by inhibiting DAG. The role of PKC in morphine-induced desensitisation was investigated in brain slices containing LC neurones. PKC was shown to play two different but important role in desensitisation. At the lower basal levels of PKC in ex vivo brain slices, PKC inhibitors enhanced desensitisation, which was subsequently reversed in the presence of both the GRK and PKC inhibitors. Following preactivation of PKC with oxo-M, inhibitors caused reduction in the extent of desensitisation, together suggesting that more than one kinase may be involved in MOPr desensitisation. Using whole body plethysmography, the PKC inhibitor, GF109203x, reversed tolerance to morphine-induced respiratory depression. Tamoxifen (also reported to inhibit PKC activity) also reversed tolerance to respiratory depression. Therefore it is likely that PKC plays a role in opioid-induced tolerance to respiratory depression and this may be important in the increased risk of overdose in polydrug use. Data in this thesis provides evidence that PKC plays a pivotal role in the development of both desensitisation and tolerance. However there may be . additional signalling pathways involved, such as GRK. Furthermore, ethanol may inhibit DAG activation, thus indirectly affecting PKC activity; however a direct ethanol-PKC interaction was not demonstrated. Further elucidating the mechanisms involved in opioid tolerance will allow more effective advice to be given to opioid users at risk.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:684743 |
| Date | January 2015 |
| Creators | Withey, Sarah Louise |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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