ItemExamination of the Effects of Valproic Acid Exposure or P300 Protein Inhibition on the Balance of Apoptosis and Proliferation in P19 Embryonal Carcinoma Cells(2016-08-26) Bricker, Jordan; Pharmacology and Toxicology; Winn, Louise M.Valproic acid (VPA), a commonly-used anticonvulsant drug, is associated with increased risk of fetal malformations, including neural tube defects (NTDs). Previous in vivo studies determined that VPA-exposed embryos with a NTD had altered expression of several proteins regulated by p300, a histone acetyltransferase (HAT) protein. p300 is capable of acetylating histones and non-histone proteins through its HAT activity, allowing it to transcriptionally regulate genes as well as modulate the stability and activity of specific proteins. NFκB, Stat3 and Egr1, all of which function as transcription factors, are regulated by p300 through its HAT activity. Together, these proteins all play an important role in maintaining the balance of apoptosis, proliferation and differentiation, the regulation of which is extremely important for proper embryonic development. The studies in this thesis utilized P19 embryonal carcinoma (EC) cells in order to determine the effects of VPA exposure on the expression of p300 and the aforementioned transcription factors, as well as apoptosis and proliferation, in vitro. P19 EC cells were exposed to C646, a selective p300 inhibitor, in order to assess whether the effects observed as a result of VPA exposure were due to p300 protein degradation. It was found that VPA exposure for 24 hours in P19 EC cells in vitro resulted in a significant decrease in p300 protein expression. VPA exposure also significantly decreased NFκB protein expression, while resulting in increased Stat3 protein expression. However, Stat3 acetylation and phosphorylation, which both contribute to Stat3 activation, were significantly decreased as a result of VPA exposure. p300 inhibition resulted in a significant decrease in NFκB, similar to what was observed as a result of VPA exposure, which suggests that VPA-mediated degradation of p300 may play a role in reduced NFκB protein expression following VPA exposure. Conversely, Stat3 protein expression, acetylation and phosphorylation were not significantly changed as a result of p300 inhibition, suggesting that p300 degradation does not play a role in VPA’s effects on Stat3 protein expression and activation. VPA exposure also resulted in a significant increase in apoptosis, while p300 inhibition did not significantly increase apoptosis. These data suggest that p300 degradation plays a role in VPA-mediated teratogenicity, and that VPA may target other cellular mechanisms in order to exert its teratogenic effects. ItemAttenuation of morphine tolerance, reward, and spinal gliosis in neuropathic pain by ultra-low dose alpha2-adrenergic antagonists(2016-04-28) Grenier, Patrick; Pharmacology and Toxicology; Cahill, Catherine M.; Olmstead, Mary C.Aims: To determine the effects of ULD α2-AR antagonists on: i) sensory responses in morphine tolerant and nerve injured rats, ii) chronic morphine and nerve-injury induced spinal gliosis and neuronal activation, iii) morphine conditioned place preference (CPP), a paradigm that assesses the affective or emotional component of pain processing. Methods & Results: In a model of opioid tolerance, ULD atipamezole attenuated the loss of morphine antinociception in pain naïve rats, consistent with the literature. Next, a model of neuropathic pain (chronic constriction injury (CCI)) was employed and changes in responses to mechanical and thermal nociceptive stimuli were tracked over time. This was the first study to show positive effects of ULD α2-AR antagonists in alleviating pain hypersensitivity associated with nerve injury and attenuation of morphine tolerance in neuropathic animals. Using immunohistochemistry, tissue collected from all the animals was labeled to determine if molecular changes correlated with the behavior induced by ULD atipamezole. Morphine and CCI-induced gliosis in the spinal dorsal horn were attenuated in animals chronically administered ULD atipamezole. Neuronal activity inferred by c-Fos cell counts was likewise attenuated in neuropathic animals. Finally, through the use of the CPP paradigm, it was shown ULD atipamezole is neither rewarding nor aversive on its own, but disrupts the development of morphine CPP in CCI animals, but not the sham or pain naïve animals. Currently, experimental evidence suggests a reduction in opioid reward in the neuropathic rats. Conclusion: ULD α2-AR antagonist atipamezole inhibits morphine tolerance and enhances opioid analgesia in pain naïve and chronic pain states, alleviates the mechanical hypersensitivity following nerve injury, inhibits chronic morphine and nerve injury-induced glial and neuronal activation in the spinal dorsal horn, and disrupts opioid reward in chronic pain states. ItemHeme-Oxygenase Inhibitors: a Novel Strategy For Treating Chronic and Persistent Pain(2015-12-16) Philbrook, Michael J. P.; Pharmacology and Toxicology; Cahill, Catherine M.; Nakatsu, KanjiIt is well recognized that there is a lack of effective therapies for treating chronic pain. Therefore, development of novel analgesics with minimal side effects and the absence of addiction potential are essential. The present study assessed the effectiveness of a novel class of selective heme oxygenase (HO) inhibitors in models of inflammatory and neuropathic pain in rats. A research collaboration of Dr. Kanji Nakatsu (Pharmacology and Toxicology) and Dr. Walter Szarek (Chemistry) has designed and synthesized a series of azole-based compounds with a range of activity as HO inhibitors. This series has been dubbed as QC-xx. QC-15 selectively inhibits the HO-1 isoform, while QC-10 inhibits both HO-1 and HO-2. Intraplantar injection of 1% formalin produces a biphasic nociceptive response characterized by licking and flinching of the affected hind paw. Intraperitoneal (IP) administration of QC-15 (100μmol/kg) attenuated nociceptive behaviors to formalin, which were not blocked by the opioid receptor antagonist, naloxone (1 mg/kg, IP). Similarly, IP delivery of QC-10 (100μmol/kg) decreased formalin-induced nociceptive behavior. To determine whether the effects of the drug were peripheral or spinally mediated, compounds were delivered either locally or spinally. Intrathecal (IT) administration of QC-15 (0.3 μmol) attenuated pain behavior, whereas local injection into the paw had no effect. The effects of QC-15 were also assessed in a model of neuropathic pain, induced by constriction of the common sciatic nerve using 4 chromic sutures. Mechanical withdrawal thresholds (von Frey filaments) were used to assess mechanical allodynia before and after IP administration of QC-15. Animals treated with the HO-1 inhibitor had significantly higher thresholds (indicating decreased mechanical allodynia) on both day 7 and 14 compared to pre-drug baselines. These observations suggest that HO-1 may also play a role in neuropathic pain. Finally we were able to demonstrate that QC-15 had no effect on motor coordination that would interfere with behavioral testing. Animals treated with the 100μmol/kg dose of QC-15 were able to maintain equal performance as controls treated with saline. In conclusion, behavioral results indicate that the inducible form of HO (HO-1) may play an important role in persistent and chronic pain states. ItemImpacts of Placental Growth Factor Deficiency and of Preeclampsia on Brain Development and Function(2015-10-03) Ratsep, Matthew; Pharmacology and Toxicology; Croy, B. Anne; Adams, Michael A.Preeclampsia (PE) is a significant gestational disorder affecting 3-5% of all human pregnancies. In many PE pregnancies, maternal plasma is low in the placentally-produced angiokine “placental growth factor” (PGF). Offspring of PE (PE-F1) compared to uncomplicated pregnancies have higher risks for hypertension, cognitive impairment, and stroke. However, mechanisms explaining these risks are poorly understood. This thesis aimed to explore the mechanistic links between deficient gestational PGF expression, PE, and brain structural and functional development in PE-F1s. It was hypothesized that PGF deficiency, which often manifests in PE, diminishes brain vascular development, leading to impaired cognition and elevated stroke risk postpartum. Uteroplacental angiogenesis was assessed in pregnant mice expressing or lacking maternal and/or conceptus derived PGF by whole-mount immunohistochemistry or paraffin histology. Pgf-/- and Pgf+/+ adult mouse brain vasculature and structural anatomy were examined by arterial polymer casting and magnetic resonance imaging (MRI), respectively. Cognition and behaviour were assessed in these mice by standard paradigms that tested depression, spatial learning, short and long term memory, activity and anxiety. PE-F1 and control children aged 7-10 were assessed for cognitive functions through psychometric testing and eye tracking of saccadic eye movements. Brain structural and vascular anatomy were assessed in the same children through MRI. Pgf-/- mice displayed reduced and aberrant uteroplacental vascular structure, particularly when conceptus-derived PGF was absent. Pgf-/- brain vasculature was deficient and abnormally patterned compared to Pgf+/+ controls. Cognitive and behavioural testing revealed numerous impairments in Pgf-/- mice, with sexually dimorphic differences. MRI revealed structural anatomic differences between the brains of Pgf-/- and Pgf+/+ mice, again with sexually dimorphic differences. In children, PE-F1s displayed deficits in working memory and oculomotor control compared to controls. PE-F1s additionally exhibited altered brain structural and vascular anatomy compared to controls. This work has uncovered a previously unknown link between deficient PGF expression, PE, and brain development in mice and humans. These results have implications for the clinical management of women with PE, as well as their offspring, and underscore the importance of PE prevention. ItemThe Effect of Leukotriene Modifiers (LTMs) on Organic Anion Transport by Multidrug Resistance Proteins (MRPs)(2015-08-13) Csandl, Mark; Pharmacology and Toxicology; Cole, Susan P. C.The multidrug resistance proteins (MRPs) are plasma membrane efflux transporters that transport a diverse array of compounds. MRP1, MRP2, MRP3 and MRP4 are considered the most pharmacologically relevant drug-transporting MRPs and have broad substrate specificities that include many xenobiotics (e.g. pharmacological agents, environmental toxins), endobiotics, and their metabolites. Although MRP1-4 have distinct substrate profiles, they can all transport glucuronide conjugated estradiol (E217βG). MK-571 was originally designed as a type 1 cysteinyl leukotriene receptor (CysLT1R) antagonist to treat asthma; it is also the most popular MRP1 inhibitor. However, MK-571 is non-selective and inhibits all MRP homologues as well as at least one solute carrier transporter, limiting its usefulness as an experimental tool. Additional leukotriene modifiers (LTMs) selective for either CysLT1R or the type 2 cysteinyl leukotriene receptor (CysLT2R) have been developed but little is known of their ability to modulate MRP1 and its homologues. In this study, seven LTMs selective for either CysLT1R or CysLT2R were examined for their ability to modulate E217βG uptake into MRP1, MRP2, MRP3, or MRP4-enriched membrane vesicles. Their effect on the uptake of a second physiological organic anion was also measured for MRP1 (leukotriene C¬4; LTC¬4) and MRP4 (prostaglandin E2; PGE2). For E217βG, the IC50 values of the 7 LTMs tested ranged from 1.2 to 26.9 μM and the IC50 values for MRP1 and MRP4 were the most similar. In contrast, some LTMs stimulated MRP2 and MRP3 activity. Thus, LY171883 stimulated MRP2 and MRP3-mediated E217βG uptake by 2 to 4-fold while montelukast and pranlukast modulated MRP2 activity in a biphasic manner. CysLT1R-selective LTMs were generally less potent as modulators of MRP2 and MRP3 activity although CysLTR selectivity did not correlate with LTM potency for MRP1 and MRP4. The rank order of LTM inhibitory potencies for E217βG versus LTC4 uptake by MRP1 and E217βG versus PGE2 uptake by MRP4 were similar. These data suggest that, like MK-571, LTMs are generally non-selective modulators of MRP transport activity despite their CysLTR selectivity, and should therefore be used with caution in MRP-related research because of their potential to confound data interpretation.