Utilisation of Nitric Oxide Donors for the Relief of Mechanical Allodynia in a Rat Model of Painful Diabetic Neuropathy

Lillian Huang (2011). Utilisation of Nitric Oxide Donors for the Relief of Mechanical Allodynia in a Rat Model of Painful Diabetic Neuropathy PhD Thesis, School of Pharmacy, The University of Queensland.

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Author Lillian Huang
Thesis Title Utilisation of Nitric Oxide Donors for the Relief of Mechanical Allodynia in a Rat Model of Painful Diabetic Neuropathy
School, Centre or Institute School of Pharmacy
Institution The University of Queensland
Publication date 2011-03
Thesis type PhD Thesis
Supervisor Professor Maree T Smith
Dr Bruce Wyse
Total pages 300
Total colour pages 74
Total black and white pages 226
Subjects 11 Medical and Health Sciences
Abstract/Summary Painful diabetic neuropathy (PDN) is a long-term microvascular complication of diabetes mellitus, giving rise to primary afferent nerve injury and degeneration particularly of the long nerves in a ‘glove and stocking’ distribution. Although abundant evidence implicates elevated levels of nitric oxide (NO) as an important factor in the cascade of events that lead to neuropathic pain following peripheral nerve injury, impaired nitrergic neurotransmission due to selective degeneration of nitrergic nerves has been implicated in the pathogenesis of diabetic autonomic neuropathy. Hence the possibility of reduced, rather than elevated NO levels contributing to the pathogenesis of PDN, a sensory form of diabetic neuropathy, was investigated. The furoxan NO donor, PRG150 (3-methylfuroxan-4-carbaldehyde), was assessed as a potential compound for the relief of mechanical allodynia in a rodent model of PDN as a means of replenishing the loss of NO bioactivity that occurs as a result of diabetes. Previous studies undertaken by Dr Debbie Tsui in our laboratory showed that single subcutaneous (s.c.) bolus doses of PRG150 evoked dose-dependent anti-allodynia in the hindpaws of STZ-diabetic rats with a marked temporal increase in the dosing requirements as the severity of diabetes progressed in these animals. This increase in the magnitude of the PRG150 doses required to fully relieve mechanical allodynia from 14-wks post-streptozotocin (STZ) administration onwards coincided with the development of morphine hyposensitivity in the same animals, implicating the possibility of a common mechanistic pathway involving the μ-opioid receptor (MOP-R). In Chapters 2 and 3 herein, possible mechanisms through which PRG150 may produce its pain-relieving effects were investigated. In Chapter 2, in vitro studies were carried out in HEK293 cells stably expressing the murine MOP-, δ (DOP)- and κ (KOP)-R as well as in native non-transfected HEK293 cell lines. Results from functional assays demonstrated that PRG150 evoked bimodal, concentration-dependent, naloxone-insensitive inhibitory, mildly stimulatory or no effects on forskolin-stimulated cAMP formation by a mechanism dependent on the MOP-R and pertussis toxin (PTX)-sensitive G-proteins. The modulatory effects of PRG150 on cAMP responses in HEK-MOP cells did not appear to be mediated through the classic NO/soluble guanylyl cyclase (sGC)/cGMP signalling pathway, nor through binding to the naloxone-sensitive binding site on the MOP-R. Additionally, the cell signalling pathway by which PRG150 produced its effects appeared to involve transduction molecules residing within membrane rafts/caveolae in cell membranes. In Chapter 3, the STZ-diabetic rat model of PDN was utilised to further elucidate the effects of PRG150. It appeared that there was progressive loss of NO bioactivity as evidenced by the marked increase in dosing requirements for PRG150 to produce anti-allodynia in the hindpaws of STZ-diabetic rats with diabetes duration, consistent with previous findings from our laboratory. In 14-18 wk post-STZ diabetic Wistar rats, the anti-allodynic effects of PRG150 were naloxone-insensitive but partially reversed by pretreatment with the specific sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), suggesting involvement of both cGMP-dependent and -independent mechanisms. Subsequent investigation (Chapter 4) showed that by increasing exposure of HEK-MOP cells 6-fold from 30 min to 3 h, there was a significant increase in cGMP formation in an ODQ-sensitive manner. Additionally, it is also plausible that PRG150 may be biotransformed in vivo to metabolites that modulate the NO/sGC/cGMP signalling cascade to produce effects that were not observed in vitro. Importantly however, there were no significant effects of PRG150 on blood pressure in normotensive non-diabetic Wistar rats in doses that were up to 10,000-fold higher than the doses required to fully alleviate hindpaw sensitivity in STZ-diabetic rats with advanced diabetes. In Chapter 4, the in vitro and in vivo effects of PRG150 were compared with the respective effects evoked by other NO donors of the furoxan and sydnonimine classes. Interestingly, there were significant between-NO donor differences in their pharmacological profiles in STZ-diabetic rats, as single s.c. bolus doses of the furoxan NO donor, 4-phenylfuroxan-3-carbaldehyde (PFC), as well as the sydnonimine NO donor, 3-morpholinosydnonimine (SIN-1) and its prodrug, molsidomine, were found to be ineffective for the relief of mechanical allodynia in 16-wk post-STZ diabetic rats. In addition, in contrast to PRG150, SIN-1, molsidomine and PFC did not modulate forskolin-stimulated cAMP formation and resulted in greater release of NO as well as stimulation of cGMP formation in HEK-MOP cells. Collectively these results demonstrated that the effects produced by PRG150 are not generalisable to other NO donors and that NO donors with low NO/sGC/cGMP stimulatory activity, combined with modulatory effects on forskolin-stimulated cAMP responses in vitro, may be more effective in relieving mechanical allodynia in the STZ-diabetic rat model of PDN. In Chapter 5, live cell imaging was used to gain further insight into the cellular mechanisms of PRG150. These experiments provided additional evidence that the MOP-R is integral to PRG150’s cellular actions, as PRG150 evoked MOP-R internalisation in a manner analogous to the MOP-R agonist, DAMGO in HEK-MOP cells. However in contrast to DAMGO, PRG150 appeared to produce its effects via cGMP-independent S-nitrosylation of the MOP-R and/or its downstream effector proteins to activate Src-dependent pathways to facilitate MOP-R internalisation and modulate forskolin-stimulated cAMP responses. In Chapter 6, the role of the MOP-R in mediating the effects of PRG150 was further investigated using a Oprm1 knockout (MOP-KO) mouse model. Interestingly, in the mouse PRG150 evoked primarily pronociceptive, rather than antinociceptive effects, in contrast to the antinociceptive effects observed in the rat. These findings suggest that there may be between-species differences that are potentially underpinned by pharmacokinetic and/or pharmacodynamic factors but further investigation is required to ascertain their relative contributions. Nevertheless, the extent and duration of pronociception was found to be attenuated in homozygous (-/-) MOP-KO relative to that observed in their heterozygous (+/-) and wild-type (+/+) counterparts, further implicating a role for the MOP-R in transducing the pharmacological effects of PRG150 in vivo. In summary, my doctoral research studies described in this thesis, implicate a loss of NO bioactivity in the development of mechanical allodynia and morphine hyposensitivity, two defining symptoms of PDN. Additionally, my findings show that the furoxan NO donor, PRG150, produces anti-allodynia in STZ-diabetic rats by both cGMP-dependent and -independent mechanisms. Cell-based studies show that the latter mechanism requires the MOP-R, inhibitory G-proteins and membrane rafts and appears to involve S-nitrosylation of the MOP-R and/or its effector proteins to activate Src-dependent intracellular signalling and modulate MOP-R function. Together these findings indicate that PRG150 is worthy of further investigation as a potential novel therapeutic agent for the symptomatic relief of PDN.
Keyword Painful diabetic neuropathy
Streptozotocin-diabetic rat
Nitric oxide
Opioid receptor
Mechanical allodynia
Additional Notes Colour Pages: 38, 45, 53, 55, 65, 81, 87, 94, 110, 112, 114, 115, 117-119, 121, 138-141, 143-145, 147, 148, 150-152, 155-157, 175-178, 180, 181, 183, 184, 186-188, 208, 210-212, 214-217, 219-222, 225-227, 239-244, 246-251, 296-300 Landscape Pages: 41, 45, 59, 65, 71, 72, 78, 84, 85, 94, 148, 180, 247

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Created: Thu, 16 Jun 2011, 23:25:23 EST by Miss Lillian Huang on behalf of Library - Information Access Service