Hypertension is a prevalent disease affecting one in three adults in the United States. Approximately 25 % of the adult population is either not receiving therapy for their hypertension or is unable to control their blood pressure with current therapies, making treatment of hypertension an important public health goal. In blood pressure regulation, PGE2 can act in a pro-hypertensive or anti-hypertensive manner. It has been demonstrated that EP2 and EP4 receptors mediate the vasodepressor actions of PGE2 and EP1 and EP3 receptors mediate the vasopressor actions of PGE2. Additionally, PGE2 and the EP1 receptor have been demonstrated to mediate at least part of the actions of angiotensin II. I sought to determine the contribution of EP1 and/or EP3 receptors to hypertensive end-organ damage and diabetic nephropathy. In this dissertation, I utilize mice with genetic disruption of EP1 or EP3 receptors and characterize the outcomes of several models of hypertensive organ damage. In the Nphx/DOCA-NaCl/Ang II model of hypertension, I have demonstrated that disruption of EP1 or EP3 can afford substantial protection from end-organ damage and reduce incidence of mortality. The beneficial effects of EP1 disruption, and likely EP3 disruption, appear to be a result of reduction in MAP in this model. The use of another model involving uninephrectomy and Ang II on a 129S6 background suggests the EP1 receptor plays an important role in hypertensive renal disease independent of blood pressure reduction. Furthermore, genetic disruption of EP1 protected eNOS-/- mice from diabetes-induced proteinuria, independent of blood pressure reduction. In summary, the data presented in this dissertation advances our knowledge of the role of EP1 and EP3 receptors in hypertension and subsequent sequalae and demonstrate a detrimental role of EP1 in this disease. Targeting the EP1 receptor may be a viable pharmaceutical treatment strategy for hypertension and subsequent organ damage.

To study the effects of neurotransmitters on [Ca²⁺]_i and oxidative metabolism of pyruvate, selected adrenergic, serotonergic, purinergic and peptidergic agonists were chosen. Experiments were conducted using the fluorescent calcium indicator, indo-1/AM, to examine changes in [Ca<sup>2+,/sup>]_i and [1-¹⁴C] pyruvate to measure rates of labeled CO₂ formation from pyruvate dehydrogenation. The adrenergic agonist noradrenaline induced an increase in [Ca²⁺]_i via activation of both á₁ and á</sub>2</sub> receptors as both phenylephrine (á₁ agonist) and clonidine (á₂ agonist) caused an elevation in [Ca²⁺]_i level. The increase in [Ca²⁺]_i evoked by either noradrenaline or phenylephrine was inhibited by inclusion of phentolamine (a nonspecific á-antagonist). Yohimbine, an á₂-antagonist, inhibited both noradrenaline- and clonidine-evoked increases in [Ca²⁺]_i, indicating the existence of a population of á₂-adrenoceptors in cultured astrocytes. Noradrenaline also enhanced dehydrogenation of pyruvate to acetyl-CoA. Clonidine exerted a stimulation on pyruvate dehydrogenation and there was a tendency towards a small stimulation by phenylephrine. In contrast, CO₂ formation was not increased when the potassium concentration was raised above 5 mM. Noradrenaline-induced stimulation of flux from pyruvate to acetylCoA was abolished in the absence of extracellular calcium (combined with an elevation of the magnesium concentration or a calcium chelator, EGTA), suggesting that the effect is calcium-dependent. Exposure to dexmedetomidine, a highly specific á₂-adrenergic agonist, led to a biphasic increase in [Ca²⁺]_i and in pyruvate oxidation. The effect was inhibited both by yohimbine and idazoxan. Chronic treatment with 1 mM lithium chloride decreased noradrenaline-induced increase in [Ca<sup>2+,/sup>]_i. This is in agreement with the assumption that lithium impairs the turnover in the inositol phosphate (IP) cycle. Serotonin caused an increase of [Ca²⁺]_i in concentrations between 10 pM and 10 ìM in dibutyryl cyclic adenosine 3',5'-monophosphate (dBcAMP) treated astrocytes by stimulating 5HT_2C and/or 5-HT_2A receptors. Micromolar concentrations of serotonin were required for activation of the 5-HT_2A receptor, whereas low nanomolar concentrations stimulated the 5-HT_2C receptor. Fluoxetine, an antidepressant acting like 5-HT, exerted an agonist effect on [Ca²⁺]_i in astrocytes. Adenosine and guanosine, purinergic agonists, evoked significant increases in [Ca²⁺]_i, an effect that was not blocked by P₁ antagonists. Arginine vasopressin (AVP) induced increases in [Ca²⁺]_i which could be abolished by a V₁-selective antagonist but not by removal of extracellular Ca²⁺. However, AVP failed to affect pyruvate oxidation in astrocytes.

Thesis--PlanB (M.S.)--University of Wisconsin--Stout, 2001. / Includes bibliographical references.

Alpha4 is multi-domain protein with a structured N-terminal region that binds to PP2A family catalytic subunits and an unstructured C-terminal domain that binds to E3-ubiquitin ligases. Unlike typical regulatory subunits which are specific for a particular family member, Alpha4 interacts with all members of the PP2A family and regulates stability via an undetermined mechanism. We undertook structural, biochemical, and cell-based approaches to investigating the mechanism by which Alpha4 regulates phosphatase catalytic subunit stability and expression. Based on previous work, we hypothesized that the N-terminal structured region (Alpha4ΔC) would be sufficient to provide protection to PP2Ac. We determined the structure of Alpha4ΔC and found that it is similar to tetratricopeptide repeat proteins (TPRs) that mediate protein-protein interactions, but with a reversed topology in the third TPR motif that increases flexibility. In addition to structural analysis, we investigated the ability of Alpha4ΔC to protect PP2Ac from polyubiquitination and degradation using both in vitro and cell-based assays. Contrary to our hypothesis, we found that Alpha4ΔC was not sufficient for protection, but rather both the PP2Ac binding domain and the C-terminal E3-ubiqutin ligase-binding domain were required for the protective effect of Alpha4 on PP2Ac. To explore the roles of these different domains in regulating phosphatase stability, we created a lentiviral expression system that could simultaneously knockdown endogenous Alpha4, using shRNA targeted to the 3UTR, and express a Flag-tagged ectopic version of the protein, driven by a pCMV promoter, allowing investigation of mutants of Alpha4 in the absence of wild-type protein. We tested the system by creating stable cell lines and assessing the role of Alpha4 knockdown and re-expression of wild-type Flag-Alpha4 in HEK293T cells on the expression levels of PP2A family members. We found that Alpha4 differentially affected the expression of the various PP2A family members and that knockdown of Alpha4 had the greatest effects on PP4c and PP6c expression levels, indicating that these may be the primary targets of Alpha4 in cells.

Mice carrying a targeted disruption of the prostaglandin E2 E-prostanoid receptor 3 (EP3) gene, Ptger3, were fed a high-fat diet (HFD), or a micronutrient matched control diet, to investigate the effects of disrupted PGE2-EP3 signaling on diabetes in a setting of diet induced obesity. While no differences in body weight were seen in mice fed the control diet, when fed a HFD, EP3-/- mice gained more weight relative to EP3+/+ mice. Overall, EP3-/- mice had increased epididymal fat mass and adipocyte size; paradoxically a relative decrease in both epididymal fat pad mass and adipocyte size was observed in the heaviest EP3-/- mice. The EP3-/- mice had increased macrophage infiltration, TNF-α, MCP-1, IL-6 expression, and necrosis in their epididymal fat pads as compared to EP3+/+ animals. Adipocytes isolated from EP3+/+ or EP3-/- mice were assayed for the effect of PGE2-evoked inhibition of lipolysis. Adipocytes isolated from EP3-/- mice lacked PGE2-evoked inhibition of isoproterenol stimulated lipolysis compared to EP3+/+. EP3-/- mice fed HFD had exaggerated ectopic lipid accumulation in skeletal muscle and liver, with evidence of hepatic steatosis. Both blood glucose and plasma insulin levels were similar between genotypes on a control diet, but when fed HFD, EP3-/- mice became hyperglycemic and hyperinsulinemic when compared to EP3+/+ fed HFD, demonstrating a more severe insulin resistance phenotype in EP3-/-. These results demonstrate that when fed a HFD, EP3-/- mice have abnormal lipid distribution, developing excessive ectopic lipid accumulation and associated insulin resistance.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have the potential to be an important tool for cardiovascular disease modeling, pre-clinical cardiotoxicity evaluation, and drug discovery. However, detailed studies of their excitation-contraction (EC) coupling are limited by the lack of appropriate methodology. Here, I set out to investigate the EC coupling of normal and diseased hiPSC-CMs and compare the results to that of adult ventricular CMs (i.e., rabbit and mouse) under identical experimental conditions. I found that hiPSC-CMs display relatively mature EC coupling properties (i.e., electrophysiology, Ca handling and contractility). To assess their contractile properties I have developed a novel culture method that enables robust contractile measurements of single hiPSC-CMs. I discovered that hiPSC-CMs display contractile properties comparable to that of adult rabbit CMs, including comparable contraction kinetics. Moreover, EC coupling properties were comparable across hiPSC-CM lines generated at different institutions and post recovery from cryopreservation. Using disease specific hiPSC-CMs I revealed that HCM MYH7 R633H hiPSC-CMs display contractile abnormalities, and I provided evidence supporting an enhanced myofilament Ca sensitivity mechanism. Furthermore, I demonstrated hiPSC-CMs display a robust response to pharmacological stimuli including the myofilament Ca sensitizer EMD57033. These findings will aid functional studies of disease specific hiPSC-CMs as well as the effects of novel and known pharmacological agents.

Diabetic retinopathy (DR) is disease of microvascular complications and is a leading cause of blindness in working age adults. DR consists of two main stages: the early non-proliferative stage is driven primarily by inflammation while the later proliferative stage is characterized by pre-retinal neovascularization. The peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is a transcription factor with known functions in lipid metabolism and glucose homeostasis. The role of PPARβ/δ in inflammation and angiogenesis, however, is not yet defined. To determine the action of PPARβ/δ in processes related to DR, rna-sequencing was first used to evaluate the effect of the PPARβ/δ antagonist GSK0660 on TNFα-induced inflammation in retinal endothelial cells. It was discovered that GSK0660 regulates TNFα-induced chemokine expression and subsequent leukocyte adhesion to endothelial monolayers via a mechanism involving inhibition of both ERK activation and NF-κB translocation. Further studies determined activation of PPARβ/δ with the agonist GW0742 increased expression of the angiogenic protein ANGPTL4 from retinal cells, increased endothelial cell tube formation, and promoted retinal neovascularization. GSK0660 was anti-angiogenic as it reduced expression of ANGPTL4 from retinal cells, inhibited endothelial cell proliferation and tube formation, and ultimately mitigated retinal neovascularization. Taken together, inhibition of PPARβ/δ was both anti-inflammatory and anti-angiogenic, resulting in reduced chemokine secretion, leukostasis, and retinal angiogenesis.

PHARMACOLOGY Pharmacological and Genetic Rescue of Idiopathic Epilepsies Lyndsey Leigh Anderson Dissertation under the direction of Professor Alfred George, Jr. Epilepsy is a common neurological disorder affecting approximately 1% of the population worldwide. Many epilepsy patients achieve complete seizure control with current antiepileptic drugs; however, these medications fail to control seizures in 30% of patients, highlighting the need for novel treatments and for research into the underlying molecular mechanisms of epilepsy. Mutations within voltage-gated sodium channels have been identified in association with epilepsy and several mouse models have been generated to understand how these disease-associated mutations manifest in epilepsy development. The genetically engineered mouse line, Scn2aQ54, expresses a transgene encoding an inactivation-impaired neuronal Nav1.2 channel. Mice expressing the Scn2aQ54 transgene exhibit a severe epilepsy phenotype correlated with increased persistent sodium current in hippocampal neurons. We investigated the antiepileptic potential of preferential persistent sodium current inhibition using ranolazine and the novel compound, GS967. We observed that both ranolazine and GS967 reduced seizure frequency in Scn2aQ54 mice, and GS967 inhibited spontaneous action potential firing in neurons isolated from Scn2aQ54 mice. GS967 was also effective at protecting against seizures in the maximal electroshock model. Additionally, we found that long-term treatment with GS967 improved survival, prevented neuron loss and suppressed mossy fiber sprouting in Scn2aQ54 mice. Heterozygous Scn1a knockout (Scn1a+/-) mice recapitulate the phenotype of Dravet syndrome such as spontaneous seizures and premature lethality. Electrophysiological studies in dissociated hippocampal neurons from Scn1a+/- mice have shown a reduced sodium current density and impaired excitability in GABAergic interneurons suggesting that impaired GABA-mediated inhibition underlies the pathophysiology of Dravet syndrome. We generated a mouse line in which SCN1A is selectively expressed in GABAergic interneurons to directly test the hypothesis that the epilepsy phenotype in Scn1a+/- mice can be rescued by restoring SCN1A in GABAergic interneurons. Utilizing this genetic approach, we were not able to attenuate the reduced lifespan of Scn1a+/- mice by restoring SCN1A selectively in interneurons, suggesting that additional mechanisms may contribute to the reduced survival of Dravet syndrome. However, pharmacological intervention with GS967 was able to successfully rescue the lifespan of Scn1a+/- mice. These results suggest that GS967 is an effective novel antiepileptic drug that can be utilized to probe the pathophysiology of epilepsy.

The macromolecular assembly of protein serine/threonine phosphatasekinase complexes mechanistically enhances the speed and fidelity of cell signaling events. These signaling modules allow for acute regulation of substrates common to both enzymes as well as intramolecular regulation of kinase activity by the associated phosphatase or vice versa. Protein serine/threonine phosphatase 2A (PP2A) interacts with calcium/calmodulin-dependent protein kinase IV (CaMKIV) and negatively regulates the activity of the kinase. A phospho-Thr200-CaMKIV antibody was utilized to monitor ionomycin-induced signal output of endogenous CaMKIVPP2A complexes. The PP2A canonical regulatory Balpha and Bdelta subunits recruit the catalytic subunit of PP2A (PP2AC) to the kinase but do not promote dephosphorylation of the activating phospho-Thr200 site. Balpha and Bdelta subunits also recruit PP2AC to the serine/threonine kinase Raf1. In contrast to CaMKIV, PP2A positively regulates Raf1-MEK-ERK signaling by dephosphorylating the inhibitory phospho-Ser259-Raf1 residue. To better understand the regulatory mechanisms controlling ABalpha/deltaC-Raf1 interactions in response to stimuli, we coupled Reversible Cross-Link Immuno-Precipitation (ReCLIP) with mass spectrometry. Tandem affinity-purified ABdeltaCRaf1 complexes were isolated from cells treated with a chemical crosslinking reagent to stabilize transient protein-protein interactions. Proteomic analysis of the PP2ARaf1 complex identified several putative interacting proteins that may play a role in the regulation of this phosphatasekinase complex. Raf1 signaling is inactivated following the dephosphorylation of phospho-Ser338 by protein serine/threonine phosphatase 5 (PP5). We monitored the binding of PP2A and PP5 to Raf1 using co-immunoprecipitation assays and found that both phosphatases concurrently associate with Raf1, which acts as the scaffold in this multiprotein complex. We also identify several novel PP5kinase complexes whose stable interactions are facilitated by HSP90. Notably, PP5 interacts with multiple ERKs. Our studies support a novel role for PP5ERK complexes in regulating the feedback phosphorylation of Raf1 at Ser-289, Ser-296, and/or Ser-301 and show that these PP5ERK complexes are regulated by small G proteins. Whereas constitutively active Rac1 promotes the assembly of PP5ERK1/2 complexes, oncogenic HRas has no effect on PP5-ERK1 binding but selectively decreases the PP5-ERK2 interaction, in a manner that is independent of PP5 and MEK activity, yet paradoxically requires ERK2 activity.

In 1948 a program was instituted at Ste Anne's Veterans' Hospital, Ste. Anne de Bellevue, P.Q., to investigate the effects of prefrontal lobotomy on schizophrenic patients. Prefrontal lobotomy is an operation involving bilateral subcortical section of the anterior portion of each frontal lobe. Patients selected to undergo this operation were those who were unresponsive to all other available treatments, and in whom the disease process appeared intractable. [...]