Department of Pharmacology and Toxicology Theses and Dissertations

Permanent URI for this collectionhttps://hdl.handle.net/10675.2/321985

This collection contains theses and dissertations submitted by graduate students under the Department of Pharmacology and Toxicology for either a Master of Science degree or a Doctor of Philosophy degree.

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Angiotensin II Signaling Mechanisms Involved in the Elevation of Arginase Activity/Expression and Vascular Dysfunction
(2011-11) Shatanawi, Alia; Department of Pharmacology and Toxicology
Vascular endothelial dysfunction is a major cause of morbidity and mortality in patients with cardiovascular diseases such as hypertension, atherosclerosis and diabetes. Nitric oxide (NO) produced by endothelial nitric oxide synthase (NOS) is needed for normal vascular function. During hypertension, diabetes or atherosclerosis, elevated levels of arginase can compete with NOS for available L-arginine thus reducing vascular NO production. Elevated angiotensin II (Ang II) is a key participant of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. In this study we explored the signaling pathway leading to increased arginase expression/activity in responses to Ang II in bovine aortic endothelial cells (BAEC). Treatment of BAEC with Ang II (10-7 M, 24 hrs) caused a 40±6% increase in arginase activity. This was accompanied by 30±8% decrease in NO production. Our studies indicate involvement of the RhoA/ROCK-p38 mitogen activated protein kinase (MAPK) in Ang II-induced arginase upregulation and reduced NO production, as inhibitors of ROCK or p38 MAPK prevented the Ang II-induced increase in arginase activity. Our studies in mice also show involvement of p38 MAPK in Ang II-induced vascular dysfunction associated with elevated arginase activity and expression. Ang II (42 μg/kg/h) caused impaired EC-dependent vasorelaxation in mouse aorta (55±7% vs. 75±8% for control). This impairment was prevented by treatment with p38 inhibitor SB203580 (5 μg/kg/day). Ang II also caused a 6.2 fold increase in vascular arginase activity/expression that was completely prevented by p38 MAPK inhibition. Additionally, treatment of BAEC with Ang II causes phosphorylation of activating transcription factor-2 (ATF-2) and enhancement of the binding of ATF-2 to arginase promotor through an AP-1 site as evident from electrophoretic mobility shift assay experiments. Transfection of BAEC with ATF-2 siRNA prevents Ang II-induced increases in arginase activity/expression and maintains NO production. These results indicate that ATF-2 is necessary for enhanced expression of arginase by Ang II. Collectively, our results indicate that Ang II increases endothelial arginase activity/expression through a RhoA/ROCK-p38 MAPK-ATF-2 pathway leading to reduced NO production and endothelial dysfunction. Targeting these signaling steps might be therapeutic points for preventing vascular endothelial dysfunction associated with elevated arginase activity/expression.
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Attenuating the Interaction Between Delta Protein Kinase C and the "d" Subunit of FIFo ATp Synthase Protects Against Cardiac Ischemia/Repferusion injury
(6/3/2016) Walker, Matthew; Deparment of Pharmacology and Toxicology
Cardiac ischemia / reperfusion (IR) injury most often results from the thrombotic blockade of the coronary arteries and is the most frequent cause of death in humans. Despite the significant role energy deprivation plays in cardiac IR injury, few studies have targeted the IR-induced impairment of the mitochondrial F1Fo ATP synthase. We have previously demonstrated delta protein kinase C (δPKC) involvement in cardiac myocyte energy deprivation via its interaction with the “d” subunit of F1Fo ATP synthase (dF1Fo) and have developed a peptide inhibitor [NH2YGRKKRQRRMLATRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK- COOH] of this interaction. It targets to the mitochondrial matrix / inner membrane. The inhibitor peptide contains a FLAG epitope which allowed confirmation of its uptake into cardiac mitochondria. Our early studies in neonatal cardiac myocytes (NCMs) led us to the hypothesis that PKC inhibits ATP production in vivo via an interaction with dF1Fo to exacerbate cardiac IR injury. To directly test our hypothesis, we first utilized the Langendorff isolated heart model to show that PKC co-immunoprecipitates (co-IPs) with antisera to dF1Fo in myocardium exposed to simulated IR injury. Administration of the inhibitor peptide to the isolated rat hearts prior to cardiac IR attenuated the co-IP of 􀁇PKC with dF1Fo, improved recovery of contractility, diminished levels of tissue t-carbonyls and 4-hydroxy-2-nonenal (HNE), and reduced myocardial infarct size (as assessed by 2, 3, 5 triphenyltetrazolium chloride (TTC) staining) following simulated IR exposures. Additionally, this peptide enhanced ATP levels 2.1 fold, improved ADP-stimulated mitochondrial respiration, and attenuated Ca++-induced mitochondrial swelling in ischemic myocardium. We next evaluated the inhibitor peptide in an in situ rat coronary ligation model for its ability to protect live rats from cardiac IR injury. A 10 min coronary ligation increased the PKC-dF1Fo co-IP in the region at risk (RAR) by 5-fold which was attenuated by 71% with intravenous infusion of the inhibitor peptide. This response correlated with an enhancement of ATP levels, a 2-fold reduction in oxidative stress markers, improvement in systolic cardiac function, and a reduction in TTC monitored myocardial infarct size in the RAR. These results support further development of this peptide as a first-in-class-translational therapeutic for the treatment of cardiac IR injury.
Open Access
Biomechanical behavior related to structure in normal and congenitally disordered elastic arteries
(Augusta University, 1992-12) Beall, Arthur C.; Department of Pharmacology and Toxicology
Open Access
Calpain-2 Activates Akt via the TGF~ 1-mTORC2 Pathway in Pulmonary Artery Smooth Muscle Cells
(8/23/2016) Abeyrathna, Prasanna; Deparment of Pharmacology and Toxicology
Calpain is a family of calcium-dependent nonlysosomal neutral cysteine endopeptidases. Akt is a serine/threonine kinase that belongs to the AGC kinases and plays important roles in cell survival, growth, proliferation, angiogenesis, and cell metabolism. Both calpain and Akt are downstream signaling molecules of platelet-derived growth factor (PDGF) and mediate PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. We found that inhibition of calpain-2 using the calpain inhibitor MDL28170 and calpain-2 siRNA attenuated Akt phosphorylation at serine-473 (S473) and threonine-308 (T308) as well as collagen synthesis and cell proliferation ofPASMCs induced by PDGF. Overexpression of calpain-2 in PASMCs induced dramatic increases in Akt phosphorylation at S4 73 and T308. Moreover, knockout of calpain attenuated Akt phosphorylation at S473 and T308 in smooth muscle of pulmonary arterioles of mice with chronic hypoxic pulmonary hypertension. The cell-permeable specific TGF~ receptor inhibitor SB431542 attenuated Akt phosphorylation at both S473 and T308 induced by PDGF and overexpressed calpain- 2 in PASMCs. Moreover, SB-431452 and knock down of ALK5 significantly reduced PDGF-induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGF~l using a cell-impermeable TGF~l neutralizing antibody did not affect PDGF-induced Akt phosphorylation at S473 and T308. Further, inhibition of mTORC2 by knocking down its component protein Rictor prevented Akt phosphorylation at S473 and T308 induced by PDGF and overexpressed calpain-2. These data provide the first evidence supporting that calpain-2 up-regulates PDGF-induced Akt phosphorylation via an intracrine TGF~ 1/mTORC2 mechanism.
Open Access
CaMKIIβ association with F-actin in developing cortical neurons
(2008-08) Lin, Yu-Chih; Department of Pharmacology and Toxicology
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase that is best known for its role in synaptic plasticity and memory .. Although multiple roles of CaMKII have been identified in the hippocampus, its role in the developing cerebral cortex is less well understood. Immunostaining showed Ca~KII~, but not CaMKIIa was expressed in embryonic day 18 (E 18) cortical neurons at 4 days in vitro (DIV) and localized to a F-actin rich cytoskeletal structure we termed "micro spike". Further characterization of micro spikes revealed that micro spikes were composed of bundled actin, and were stable over time. Besides CaMKII~, several actin binding proteins, such as Arp3, cortactiti"and ~1-integrin were also colocalized in microspikes. Fluorescence recovery after photo bleaching (FRAP) analyses showed different dynamics of actin and CaMKII~ in microspikes compared to dendrite spines. The colocalization of CaMKII~ and F-actin in microspikes was dependent on the F-actin binding domain and the oligomerization domain. FRAP analyses confirmed the association of CaMKIIP with F-actin in microspikes was via the F-actin binding domain. This association was altered by the co-expression of CaMKIIa. FRAP analyses with stabilized F-actin using jasplakinolide or cytochalasin-D further indicated CaMKIIP, but not CaMKIIa, had a strong interaction with stable F-actin. Inhibiting calmodulin binding on CaMKII using a CaMKII inhibitor, KN93, dissociated CaMKIIP from stable F-actin. Increasing CaMKIIP activity with KCl or an active form of CaMKIIP, CaMKIIPT287D, also dissociated CaMKIIP from stable F-actin. A calmodulin binding mutant, CaMKIIPA303R, or a kinase dead mutant, CaMKIIPK43R, however, did not recover differently from wildtype CaMKIIp. The differential binding of CaMKIIP with F-actin shown in FRAP analyses correlated with CaMKIIP enrichment in microspikes and the prominence of microspikes. While overexpressed CaMKIIP increased the number of cells with microspikes, knockdown of CaMKIIP with shRNA reduced it. Taken together, these data suggested that CaMKIIP is associated with F-actin in cortical neurons, and this association is regulated by CaMKIIa and calcium signals · contributing to the stability of micro spikes.
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Characterization of the Physical and Functional Interaction between a Novel Protein CRIPla and the CBj Cannabinoid Receptor
(2006-10) Liu, Yunguang; Department of Pharmacology and Toxicology
CBi receptors modulate synaptic transmission and play important roles in analgesia, appetite and neuroprotection. However, little is known about how CBi activity is regulated. The possibility that a novel protein CRIPla interacts with CBj was studied to determine whether CBt functions are modulated by such an interaction. CRIPla specifically interacted with the C-terminus of CBi in a GST pull-down assay and co-precipitated with CBi oligomer in HEK293 cells, demonstrating that CRIPla interacts with CB^ Moreover, CRIPla and CBj co-localized when heterologously expressed in HEK293 cells and in rat superior cervical ganglion (SCG) neurons. The functional CBr CRIPla interaction was investigated using whole-cell voltage-clamp recordings of N-type Ca2+ channels in SCG neurons heterologously expressing CBi with or without CRIPla. The electrophysiological data demonstrated that CRIPla significantly reduced the ability of the CB! inverse agonist SR141716 to enhance the Ca2+ current but did not affect the ability of the CBi agonist WIN 55,212- 2 to inhibit the Ca2+ current. In addition, CRIPla significantly decreased Ca2+ current basal facilitation ratio. Since CRIPla did not alter CB! expression pattern or the EC50 response to WIN 55,212-2 in neurons co-expressing CRIPla and CBi; it is unlikely that the attenuated SR141716 response or the reduced Ca2+ current basal facilitation ratio resulted from decreased surface expression of CB,. Our data indicate that CRIPla inhibits the constitutive activity of CB! receptors. Deletion of the last nine amino acids of the CBi receptor abolished the interaction with CRIPla in a GST pull-down assay, indicating that these residues of the CBi receptor constitute the CRIPla interaction domain. In SCG neurons, the CBiA465-473 receptor missing the CRIPla interaction domain was constitutively active but the constitutive activity was not affected by CRIPla, demonstrating that deletion of the CRIPla interaction domain from CB! receptors reversed the ability of CRIPla to inhibit CBi constitutive activity. Taken together, our data suggest that CRIPla controls CBi constitutive activity by interacting with the last nine amino acids of the CBi receptor.
Open Access
Circadian Clock in Angiotensin II Induced Hypertension and Vascular Disease
(2015) Pati, Paramita; Department of Pharmacology and Toxicology
Hypertension remains a major risk factor for cardiovascular disease and death. While clinical studies and guideline recommendations underscore the benefits of reducing sodium intake in the treatment of high blood pressure, recent human data suggest that underlying conditions of disease may confound these positive effects of low salt diets. Herein, we examined the influence of circadian dysfunction during experimental hypertension caused by angiotensin II (Ang II), a key peptide in blood pressure regulation. While a low salt diet caused the expected decrease in blood pressure in wild type (WT) mice, mice with disruption of the circadian clock exhibited a paradoxical response to low salt. Mice with disruption in the circadian clock component Period (Period-knockout/KO mice), were abolished in blood pressure rhythm due to an increase in daytime blood pressure. This impairment in blood pressure rhythm in Per-KO mice on the low salt diet was restored to rhythmic oscillation by the angiotensin receptor blocker losartan. Similarly, exogenous administration of Ang Il caused a non-dipping blood pressure phenotype in the Per-KO mice on a normal salt diet, which resulted in pathological thickening of the vasculature indicative of vascular disease. These effects were related to circadian rhythm as impairment in blood pressure caused by low salt was recapitulated in WT mice induced to circadian derangement by a shortened light cycle. Further thickening of the vasculature and increased renin levels were observed in Per-KO mice on a chronic low salt diet but not in WT mice. Moreover, disruption of the Period gene altered ATI receptor expression and other components of the renin-angiotensin system. These data suggest that circadian dysfunction may compromise the benefits of a low salt diet and support recent clinical data that raise caution to sodium restriction as a therapy for hypertension.
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Control of Retinal Pericyte Excitability: Exploring the Role of Glucose, PGI2 and BKCa Channels
(2006-06) Burnette, Jason O.; Department of Pharmacology and Toxicology
The retinal pericyte plays an important role in regulation of retinal capillary blood flow. Retinal pericytes express the large conductance, voltage and calcium-gated potassium channel (B K Ca), which exerts important control over membrane potential and excitability. To date, very little is known about the regulation of B K c a channel activity in retinal pericytes. Our findings indicate that high glucose (25mM) increases B K Ca channel activity and is consistent with previous studies demonstrating similar results. However, the cellular/molecular basis of this phenomenon is still undetermined. Our findings suggest that high glucose (25mM) induces expression of COX-2 in retinal pericytes, with subsequent production of P G I2. Furthermore, glucose may also exert its effects on pericyte excitability by altering the expression of B K c a channel subunits, thus influencing voltage and calcium gating of this ion channel. It has been shown that prostacyclin (PGI2 ) is the predominant prostanoid produced by the retinal pericyte, and this prostanoid can regulate pericyte contractility. However, the signaling pathway(s) governing this effect have yet to be elucidated. Our findings demonstrate that PGI2 increases B K c a channel activity. Furthermore, this activity can be reproduced using a cell permeable cyclic nucleotide, chlorophenylthio (CPT)-cAMP. We have further discovered that PGI2 as well as an increase in intracellular cAMP, regulates pericyte excitability by opening of BKCa channels via a process involving the cGMP-dependent protein kinase (PKG). Taken together, these findings suggest that high glucose can stimulate BKCa channel activity by inducing COX-2 expression and increasing PGI2 in the retinal pericyte, as well as by increasing BKCa 3-subunit expression. Furthermore we have demonstrated a novel signaling pathway for PGI2-stimulated BKCa channel activation: cAMP- dependent cross-activation of PKG.
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Cytoprotective Actions of Nicotine: The Increased Expression of a7 Nicotinic Receptors and NGF/TrkA Receptors
(2001-07) Jonnala, Ramamohana R.; Department of Pharmacology and Toxicology
Certain epidemiological studies have reported a negative correlation between smoking and neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease, reflecting perhaps the neurotrophic actions of nicotine. In recent years there has been intense interest in the development of new nicotinic acetylcholine receptor (nAChR) agonists. These agents have the potential to be used in the treatment of patients with AD. However, the mechanism for the neuroprotective action of the nicotine is not yet known, indeed, it is not yet clear as to which subtype of nAChR mediates the response. In neuronal cell lines, the induction of cytoprotection often requires exposure to nicotine for up to 24 hr to produce a full neuroprotective effect and this chronic exposure of nicotine is also known to increase nAChR receptors and cell surface nerve growth factor (NGF) receptors. The purpose of this study is to determine which subtype of nAChRs are involved in nicotine’s neuroprotective actions and also to determine whether nicotine’s neuroprotective actions are related to its ability to increase cell surface nAChRs and NGF receptors. Preincubation of differentiated PC 12 cells with nicotine for 24 hr protected the cells from growth factor withdrawal-induced toxicity in a time and concentration-dependent manner. Nicotine’s cytoprotective actions were completely blocked by non-selective nAChR antagonist mecamylamine, and the cc7nAChR preferring antagonist methyllcaconitine (MLA) indicating that the response was primarily mediated by the subtype of a7 nAChR receptors. The acetylcholine precursor, choline is a very selective and full agonist at a7 receptors. Among five choline analogs tested for neuroprotection potential, acetylpyrrolidinecholine and pyrrolidinecholine were found to be more potent and more efficacious than their parent compound, choline. The rank order of the six compounds tested for their cytoprotective ability is as follows: acetylpyrrolidinecholine = pyrrolidinecholine > choline = monoethylcholine = diethylcholine = triethylcholine. Further, to confirm the above structure activity relationships with respect to their binding affinities at a7nAChR, [I25I]a-bungarotoxin (BGT) displacement binding studies were performed using differentiated PC 12 cells. Choline was only 50 fold less potent than nicotine in displacing [ I]a-BGT binding. Pyrrolidinecholine, the most active analog, fully displaced [l25I]a-BGT binding and it exhibited a slightly greater affinity for the site than did choline. Next we compared the ability of seven different nAChR agonists with varying activities at a7 receptors for their ability to produce cytoprotection. Among the seven compounds tested, nicotine was the most effective and the most potent followed in order of potency by 40H-GTS21, epibatidine, methylcarbamylcholine, l,l-Dimethyl-4-phenyl-piperazinum, cytisine and tetraethylammonium. Since, epibatidine and cytisine were less efficacious than nicotine despite their greater affinity for a7 receptors and because short-term exposure of cells to nicotine did not produce cytoprotective actions, we next compared the ability of these compounds to upregulate cell surface a7 receptors. After, incubation of cells for 2 hr with either nicotine or cytisine, the number o f [I25I]a-BGT binding sites on differentiated PC 12 cells were measured. Nicotine, the most efficacious compound increased the [l2SI]a- BGT binding sites by ~40% over the untreated control cells. In contrast, cytisine, the least effective compound failed to do so, indicating that the ability to upregulate a7 receptors may provide one possible mechanism for neuroprotective actions of nicotine. Further, we confirmed that these additional populations of receptors were functional and that they mediate an enhanced neuroprotective response to subsequent nicotine stimulation. Earlier studies had shown that prolonged exposure to nicotine increases NGF receptors on differentiated PC12 cells. In the present study, high affinity NGF receptors (TrkA) were shown to increase with nicotine treatment in a time-and concentrationdependent manner. This effect was blocked by co-treatment with mecamylamine and with MLA, but not a low concentration of dihydro-P-erythroidine (selective for P2 and p4 containing receptors), indicating that the response was mediated by predominantly a7 nAChRs. Next, we measured the TrkA protein levels in rat hippocampal and cortical tissues after nicotine treatment. Chronic nicotine infusion increased TrkA protein levels within the hippocampus, and this effect was blocked by co-treatment with mecamylamine. In contrast, TrkA protein levels in cortical tissues were not altered. Since the majority of nAChRs in hippocampus are of the a7 subtype and whereas in cortex consist largely of the a4p2 subtype, it is reasonable to conclude that the differences observed in TrkA receptor expression in hippocampus and cortex were due to presence of a7nAChR. and non-a7nAChR. To determine whether the neuroprotective actions of nicotine were due to enhanced NGF trophic activity during the drug incubation period, the ability of nicotine to protect cells from trophic factor withdrawal in the presence and in the absence of NGF were compared. Nicotine was found to be effective in both experimental paradigms. However, nicotine was found to be 10 fold more effective when it was incubated with cells in the presence of NGF. When cells were treated with nicotine and k252a or nicotine and anti-TrkA antibody, nicotine was only partially effective as a neuroprotective agent, indicating that mechanisms apart from enhanced NGF mediated trophic activity during drug incubation period were involved in nicotine’s cytoprotective actions. One consistent finding with regard to in AD pathology is the selective loss of basal forebrain cholinergic neurons (BFC). The survival and maintenance of these neurons depended on the availability of NGF from target tissues. Evidence from previous studies suggests that impairment in NGF support could be an initial insult in AD pathology. Previous studies have shown that much of the oxidative damage in AD tissue was mediated by peroxynitrite. Breif exposure of undifferentiated PC 12 cells to 3- morpholinosydnonmine (SIN-1, peroxynitrite generator) was sufficient to inhibit an NGF mediated cellular response by 67% of that measured in control cells. This inhibition of the NGF cellular response by SIN-1 was not related to generalized cellular toxicity. In fact, the peroxynitrite scavenger uric acid significantly attenuated the inhibitory actions of SIN-1. Pretreatment with SIN-1 also resulted in a decrease in the NGF-induced phosphorylation of TrkA protein. Furthermore, SIN-1 treatment reduced the activity of mitogen activated protein kinase, a downstream kinase activated by TrkA receptor stimulation'. These data suggest that SIN-1 treatment inhibits NGF signaling by inactivating TrkA receptors through the formation of nitrotyrosine residues on the receptor. The inactivation of TrkA receptors may contribute to the initial insult that eventually ieads to neuronal cell death.
Open Access
A Data-Mining Strategy That Identifies Drugs and Genes Associated With Anti-Cancer Drug Sensitivity
(2017) Schleifer, Robert John; Department of Pharmacology and Toxicology
The success of cancer therapy for patients often hinges on the responsiveness of the cancer cells to therapeutics. Drug resistance to anti-cancer therapeutics, both intrinsic and acquired, has important clinical and scientific significance. Identification of drug resistance genes using traditional methodologies and translation of those findings to the clinic has proven challenging. We developed a predictive data mining-based bioinformatic framework using public patient data and high-throughput cancer cell drug screening data. This information was used for genome-wide rankings of putative drug resistance genes. Prominent drug resistance genes (e.g. ABCB1, EGFR, and AXL) were successfully identified by the pipeline, additional genes hypothesized to be novel drug resistance genes were then investigated. Experimental confirmation of the novel genes using knockdown technologies indicated a propensity for of decreased proliferation/viability of cancer cells and increased sensitivity for anticancer compounds after knockdown much like known drug resistance genes. We then assessed the potential of each gene as an anti-cancer therapeutic target by exploring how gene knockdown behaved with clinical anticancer compounds. A second arm of the data-mining pharmaco-genomic strategy involved identification of candidate compounds that decrease expression of drug resistance genes. Using the drug resistance gene AXL as a proof-of-concept, three compounds were identified that decreased AXL expression at sub-micromolar concentrations. These compounds were characterized using microarray and cell signaling studies and found to decrease cell cycle signaling as well as activity of the Akt, mTOR, and ERK pathways. This study illustrates a novel approach for rapid and efficient identification of drug sensitivity genes or gene expression altering compounds utilizing bioinformatic data-mining.
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Effect of chronic oral treatment with risperidone or quetiapine on cognitive performance and neurotrophin-related signaling molecules in rats
(8/7/2018) Poddar, Indrani; Department of Pharmacology and Toxicology
Antipsychotic (APs) drugs are among the top selling pharmaceuticals in the world and they have a variety of important therapeutic applications for neuropsychiatric disorders. However, there are a number of controversies related to this class of agents and many of the relevant questions are difficult to prospectively address in the clinical trial environment. For example, there have been multiple clinical trials for pro-cognitive agents in schizophrenia that have failed; however, the question of how chronic prior treatment with APs might influence the response to a pro-cognitive agent was not addressed. Moreover, there is clinical evidence that chronic treatment with some APs may lead to impairments in cognition, however, this issue and the potential molecular mechanisms of the deleterious effects have been not been prospectively addressed. Accordingly, the purpose of the work described in this dissertation was to prospectively address each of these issues in animals (specifically rats) were environmental conditions can be rigorously controlled. In each of the manuscripts included in this dissertation, two of the most commonly prescribed APs, risperidone and quetiapine were evaluated. In the work conducted in Manuscript 1, we established a therapeutic relevant dosing approach for rats (oral administration in drinking water) and reinforced the argument that these two APs are not pro-cognitive agents. Moreover, we determined that alpha-7 nicotinic acetylcholine receptor (nAChR) ligand like tropisetron has potential as an adjunctive medication in schizophrenia since the pro-cognitive effect was maintained in the presence of chronic AP treatment. In Manuscript 2, we concluded that chronic treatment with risperidone or quetiapine in rats can lead to impairments in a domain of cognition (recognition memory) that is commonly altered in neuropsychiatric disorders. Moreover, the negative effects of the APs appeared to be exacerbated over time. In Manuscript 3, we concluded that risperidone and quetiapine when administered chronically to rats have the potential to adversely affect neurotrophin-related signaling molecules that support synaptic plasticity and cognitive function. These data would suggest that the extensive prescribing of these APs across multiple conditions in patients ranging in age from the very young to the very old should be carefully reexamined. Key Words: antipsychotic, cognition, brain volume, schizophrenia, neurotrophin
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Effect of Homocysteine on Bovine Aortic Endothelial Cell Function
(2001-05) Jin, Liming; Department of Pharmacology and Toxicology
Hypothesis: In hyperhomocyst(e)inemic states, oxidative stress resulting from elevation of homocyste(e)ine levels leads to a decrease in L-arginine transport activity and a decrease in intracellular L-arginine availability. This will in turn limit NO production and increase superoxide production from eNOS. Therefore, the oxidative stress will be increased in endothelial cells, which may eventually lead to endothelial dysfunction and predispose to atherothrombosis. SPECIFIC AIMS 1. Characterize the cationic amino acid transport systems in bovine aortic endothelial cells (BAECs). 2. Examine the effect of HCY on L-arginine transport over time. 3. Determine the influence of oxidative stress, which is produced by HCY, on L-arginine transport. 4. Determine the effect of HCY on endothelial cell membrane potential. 5. Determine effect of HCY on expression of the transport protein CAT-1. 6. Determine the effect of HCY on eNOS activity, eNOS protein levels and NO formation. 7. Determine the effect of HCY on the production of 3-nitro-tyrosine, a marker for production of peroxynitrite. 8 . Determine the effect of HCY on vascular responses to acetylcholine.
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Effects of Parturition on Estrogen Signaling in Resistance Arteries
(2011-04) Royal, Crista Ruth; Department of Pharmacology and Toxicology
Few studies have examined the potential effects of childbirth on the responses of the female vasculature – especially the resistance microvasculature of non-reproductive tissues. We investigated the response of mesenteric microvascular resistance vessels to 17β-estradiol (E2), an important vasoactive hormone. Vessels were obtained from either nulliparous or postpartum female Sprague-Dawley rats, and isometric tension studies were performed. We found that E2 induced a concentration-dependent, endotheliumindependent relaxation of microvessels precontracted with 10-5M phenylephrine; however, E2-induced relaxation was reduced by nearly half in vessels from postpartum animals compared to nulliparous controls. Inhibiting nitric oxide synthase activity with 10-4M L-NMMA attenuated the relaxation effect of E2 on arteries from nulliparous animals. In contrast, L-NPA (which exhibits selectivity for type 1 or nNOS) had little effect on arteries from postpartum animals, suggesting a reduced influence of nNOS after parturition. Moreover, expression of nNOS protein in microvessels was decreased 39% in the postpartum state compared to arteries from nulliparous animals. We propose that the impaired E2-induced relaxation response of microvessels from postpartum animals reflects a downregulation of NO production due to lower nNOS expressed in vascular smooth muscle cells. We measured a 73% decrease in serum E2 levels in the postpartum state compared to nulliparous animals. Because E2 has been shown to increase nNOS protein expression, we propose that lower E2 levels after parturition decrease expression of nNOS, leading to a reduced vasodilatory capacity of resistance microvessels. Impaired E2-induced relaxation in resistance arteries from postpartum rats could also be restored by inhibiting COX with indomethacin (E2 300 nM 23.5±8.9% n=5) and even more profoundly with COX-2 inhibitor celecoxib (E2 300 nM 33.9±5.5% n=8). However, many women artificially increase estrogen levels soon after giving birth by taking oral contraceptives. Little is known regarding how parturition affects estrogen signaling, especially in resistance arteries, which can contribute to blood pressure regulation. We found that inhibiting COX-2 restored E2-induced vasodilation in arteries from postpartum rats.
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The effects of retinoic acid-induced differentiation on neurotransmitter receptor content and signal transduction in a human neuroblastoma cell line
(Augusta University, 01/23/1993) Baumgartner, Melissa K.; Department of Pharmacology & Toxicology
The purpose of the present study was to establish the effects of retinoic acidindttced differentiation on muscarinic receptor populations and signal transduction pathways in the human neurroblastoma Sk-N-SH cells. The human neuroblastoma cell line Sk-N-SH was induced to differentiate by treatment with 1 uM retinoic acid for 7 days. Differentiation was characterized by profuse neurite outgrowth, a decrease in cell growth, and a 2~3 fold increase in the protein content of each cell. Muscarinic receptors were labelled-using [3H]N-methyl scopolamine. Muscarinic receptor density increased by approximately 36% after treatment for 7 days with retinoic acid (Bmax, control = 126 ± 13 fmol/mgprotein; Bmax, retinoic acid-treated= 170 ± 17 fmol/mg protein; p<0.05), corresponding to a 170% increase in receptor content per cell. The affinity of [3H]NMS for the receptors was somewhat lower in the differentiated cells (KD, control = 0.14 ± 0.04 nM; KD, retinoic acid-treated = 0.25 ± 0.0.4 nM; p<0.05). The guanine nucleotide sensitivity of agonist (carbamylcholine) binding to Sk-N-SH muscarinic receptors Was slightly decreased by differentiation. Reverse transcriptase/polymerase chain reaction (PCR) analysis using muscarinic receptor subtype specific primers revealed that the undifferentiatied Sk-N-SH cells transcribed mRNA for all 5 receptor subtypes; this pattern was not affected by differentiation. [3H]NMS displacement curves with subtype- selective receptor ligands (pirenzepine, m1; AFDX-116, m2; 4-DAMP, m3) indicated the predominant expression of m1 and m3 receptor subtypes, and differentiation did not affect the pharmacological profile of the expressed muscarinic receptor populations. Differentiation did not affect basal G protein GTPase activity. However, acetylcholine (100 uM) stimulation of G protein GTPase activity was decreased in differentiated cells (18 ± 1.8 pmol/min/mgprotein) compared to the undifferentiatied cells (23 ± 1 .0 pmol/ min/ mg protein) (p<0.05). Inhibition of acetylcholine--stimulated GTPase activity with selective muscarinic receptor antagonists indicated that the m3 antagonist (4-DAMP) was as effective as atropine in inhibiting activity by 80-100%. Selective m1 and m2 antagonists were less effective (30-40%) at inhibiting stimulated GTPase activity. There were no differences in inhibition of stimulated GTPase activity after differentiation. Immunoblots of control and retinoic acid-treated cells revealed no change in Goa, Gsa or Gp content after differentiation; however, 0.1% ethanol and retinoic acid-treated cells displayed a 30% decrease in expression of Gia3, and Gqa. Muscarine (0.1-100 uM) stimulated 45Ca influx into Sk-N-SH cells, and this uptake was inhibited by preincubation with atropine. The magnitude of the muscarinic receptor-mediated uptake was 50-60% lower in the differentiatied cells. Basal adenylate cyclase activity was depressed in the differentiated cells (2.5 pmol / min / mg protein) compared to the undifferentiated cells (8.4 pmol / min / mg protein) (p< 0.05). Forskolin (5 - 50 uM)-stimulated adenylate cyclase activity was not altered, however fractional stimulation was significantly (p<0.0001) increased in the differentiated cells. Differentiated cells displayed a slightly greater receptor-mediated inhibition of the adenylate cyclase activity by carbamylcholine (1 uM- 1 mM). It is demonstrated that in Sk-N-SH cells, retinoic acid-induced differentiation: 1) increases the size of the muscarinic receptor population (Bmax) while decreasing [3H]NMS binding affinity, 2) does not alter muscarinic receptor pharmacology, or the expression of. muscarinic receptor subtypes, 3) decreases muscarinic receptor-stimulated 45Ca flux 50-60% compared to undifferentiated cells, 4) depresses basal adenylate cyclase activity, increases fractional stimulation of forskolin-stimulated activity of adenylate cyclase, and may increase muscarinic receptor-mediated inhibition of adenylate cyclase activity, 5) does not alter basal G protein GTPase activity but depresses muscarinic receptor-stimulated high affinity GTPase activity suggesting muscarinic receptor-G protein coupling is altered, and 6) does not alter expression of Goa, Gsa and Gp content while Gia3 and Gqa are depressed in differentiated as well as in 0.1% e.thanol treated cells.
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Epigenetic Regulation of Adipogenic Differentiation And Lipid Metabolism: Role of Enhancer of Zeste Homolog 2 (EZH2)
(2017) YIEW, KAN HUI; Department of Pharmacology and Toxicology
Adipose tissue expansion in obesity promotes cardiometabolic disease, for which there is an urgent need to elucidate disease mechanisms and develop novel and effective medical therapies. In obesity, adipose tissues can potentially expand through adipocyte hypertrophy and/or hyperplasia, with the latter being a healthier mechanism of fat expansion. Adipocyte hyperplasia (via adipogenic differentiation) is inexplicably restrained in diet-induced obesity (DIO), however, and hypertrophy ensues, leading to inflammation, insulin resistance, and dysregulated adipose tissue lipid metabolism, which together contribute to cardiometabolic disease. The mechanisms of impaired adipogenic differentiation and lipid metabolism during DIO are unclear, with prior studies suggesting epigenetic dysregulation of histone deacetylase 9 (HDAC9, an endogenous repressor of adipogenic differentiation). The overall goal of my dissertation project is to investigate the role of Enhancer of Zeste Homolog 2 (EZH2), a histone methyltransferase, in the aforementioned processes. In Aim 1, we hypothesized that EZH2 promotes adipogenic differentiation by repressing HDAC9. EZH2 recruitment and histone 3 lysine 27 trimethylation (H3K27me3) modification were elevated at the HDAC9 gene promoter (p<0.05) concurrent with dramatic downregulation of HDAC9 mRNA levels (p<0.05) during adipogenic differentiation of primary human preadipocytes. This suggested a role for EZH2 in silencing HDAC9 gene expression. Counterintuitively, a highly selective EZH2 pharmacological inhibitor (GSK126), at a concentration that effectively blocked H3K27me3, led to increased lipid accumulation (p<0.05) in human adipocytes, without inhibiting adipocyte marker gene expression. Consistently, mice with adipose-specific EZH2 deletion (cKO) displayed significantly elevated body weight, adipose tissue mass, and adipocyte cell size. These phenotypic alterations could not be explained by differences in feeding behavior, locomotor activity, or metabolic energy expenditure, thereby suggesting that EZH2 regulates lipid metabolism. This hypothesis was explored in Aim 2. Human adipocytes treated with either an EZH2 inhibitor or vehicle exhibited comparable rates of de novo lipogenesis (DNL), fatty acid (FA) uptake, and basal/stimulated lipolysis. Consistently, cKO and littermate control mice displayed comparable in vivo and ex vivo basal/stimulated adipose lipolysis. EZH2’s function in other important metabolic pathways such as glycolysis and β-oxidation remain to be investigated. Collectively, our findings suggest a potential role of EZH2 in regulating adipocyte lipid metabolism.
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Impaired Cognition in Spontaneously Hypertensive Rats: Role of Central Cholinergic Receptors
(1996-09) Gattu, Mahanandeeshwar; Department of Pharmacology and Toxicology
The cognitive impairment exhibited by the spontaneously hypertensive strain of rat is due to a reduction in the expression fo central cholinergic receptors. Therefore, the specific aims of this study are: 1. To determine whether the hypertensive state present in SHR contributes to impaired performance on spatial memory tasks. 2. To determine whether SHR exhibit altered expression of cholinergic receptor subtypes in brain regions important for memory function 3. To determine whether the decreased expression of central nicotinic-cholinergic receptors observed in SHR have functional significance.
Open Access
Involvement of arginase upregulation in diabetes- and angiotensin II-induced vascular dysfunction
(2015) Bhatta, Anil; Department of Pharmacology and Toxicology
Cardiovascular disease (CVD) is the number 1 killer of men and women in the United States and the world. Diabetes, hypertension, obesity, and aging are some of the risk factors for CVD. A major cause of morbidity and mortality in CVD is vascular dysfunction, which progresses rapidly as the risk factors progress. Vascular dysfunction is characterized by a constellation of blood flow reducing pathologies, including impaired vasorelaxation and elevated arterial stiffening. The mechanisms leading to these vascular abnormalities are not well understood. We tested the hypothesis that arginase, an enzyme in the urea cycle, mediates vascular dysfunction in hypertension and obesity related diabetes. Arginase (ARG) can compete with nitric oxide (NO) synthase for their common substrate, L-arginine. Increased arginase can also provide more ornithine for synthesis of polyamines via ornithine decarboxylase (ODC) and proline/collagen via ornithine aminotransferase (OAT), leading to vascular cell proliferation and collagen formation, respectively. We hypothesized that elevated arginase activity is involved in Ang II-induced vascular dysfunction and that limiting its activity can prevent these changes. We tested this by studies in C57BL/6J mice lacking one copy of the ARG1 gene that were treated with Ang II (1 mg/kg/day, 4 weeks). We demonstrated that Ang II induces smooth muscle cell proliferation, collagen synthesis, and arterial fibrosis and stiffness via a mechanism involving increased arginase activity. Furthermore, we examined the role of arginase in vascular dysfunctions and pathologies associated with obesity-related type 2 diabetes in mice fed with high-fat/high-sucrose (HFHS) diet for 6 months. This model produced a clinical presentation and pathophysiological relevance to the human condition in obesity related type 2 diabetes. We demonstrated that HFHS diet impaired endothelial dependent vasorelaxation and increased arterial stiffness in WT mice, but not in mice treated with arginase inhibitor ABH. Endothelial cell specific knockout of ARG1 (EC-A1-/-) in mice also prevented HFHS induced vascular dysfunctions. Aortic perivascular collagen deposition was significantly higher in HFHS mice compared to normal diet. Furthermore, marked increase in vascular cell adhesion molecule expression and macrophage infiltration into the aortic walls was observed with HFHS diet. Additionally, plasma lipid peroxidase activity, a measure of systemic oxidative stress, was also markedly increased in HFHS mice. These changes were prevented in ABH treated mice and EC-A1-/- mice. These studies suggest that enhanced ARG1 activity promotes vascular dysfunctions associated with elevated Ang II levels or obesity related diabetes.
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Modulation of BKCa Channels by Protein Kinase C in Idiopathic Pulmonary Arterial Hypertension
(2007-10) Zhu, Shu; Department of Pharmacology and Toxicology
Idiopathic pulmonary arterial hypertension (IPAH) is a severe and progressive vascular disease which ultimately leads to right-side heart failure and death. Pulmonary vasoconstriction is thought to be a key factor that leads to IPAH. The large-conductance, voltage- and calcium-activated potassium (BKCa) channel is a key element that controls vascular smooth muscle tone. Inhibition of BKCa channels in pulmonary arterial smooth muscle cells (PASMC) cause membrane depolarization and pulmonary vasoconstriction. We have shown that protein kinase C (PKC) inhibits BKCa channels in PASMC and constricts pulmonary arteries from the pulmonary hypertensive Fawn-Hooded rat (FHR). However, the underlying mechanism is unknown. Patch-clamp studies demonstrated that 100μM IBMX -a non-specific phosphodiesterase (PDE) inhibitor, 10μM milrinone -a specific PDE3 inhibitor, or 10μM zaprinast -a specific PDE5 inhibitor, reversed the inhibitory effect of 100nM thymeleatoxin (a PKC activator) on BKCa channels in FHR PASMC. Additionally, 100nM PMA (a PKC activator) increased PDE activity significantly, and also decreased cyclic nucleotide concentrations significantly. In inside-out patches, application of 2nM purified PKC catalytic subunits blocked the BKCa channel activity significantly. Furthermore, a cell-membrane permeable and PDE-resistant cyclic nucleotide analog, 100μM CPT-cAMP, reversed the inhibitory effect of 100nM thymeleatoxin on BKCa channels. Serine1076 (S1076) is a conserved PKC phosphorylation site on the C-terminus of the human BKCa channel α-subunit, and phospho-null (S1076A) and phospho-mimetic (S1076E) forms of this PKC phosphorylation site were created. Western blot revealed equal BKCa channel expression in wild-type BKCa-α/β1, BKCa-α(S1076A)/β1 and BKCa-α(S1076E)/β1-transfected HEK293 cells. Patch-clamp recordings demonstrated that wild-type BKCa-α/β1 was activated by PKC and PKG, while BKCa-(S1076A)/β1 responded to the stimulation of PKC to a lesser extent, but was unresponsive to PKG. In contrast, BKCa-α(S1076E)/β1 enhanced the stimulatory effect of PKC. Thus, PKC may inhibit BKCa channels in FHR PASMC primarily through PDE activity, and also by directly acting on BKCa channels. In addition, the PKC phosphorylation site S1076 on the human BKCa channel α-subunit is very important for the regulation of channel function. The findings in this study should increase our understanding of how PKC modulates BKCa channels in IPAH, and may contribute to the development of new approaches to treat IPAH.
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A Novel Function of ADP-Ribosylation Factor 1 in Prostate Cancer Cell Proliferation through Activating the Mitogen-Activated Protein Kinase Pathway
(8/23/2016) Davis, Jason E.; Department of Pharmacology and Toxicology
The enhanced activation of the mitogen-activated protein kinase (MAPK) Raf-MEK-ERK1/2 pathway directly correlates with the growth, androgen-independence, and poor prognosis of prostate cancer. However, the underlying molecular mechanisms remain poorly understood. Here, we have demonstrated that ADP-ribosylation factor 1 (ARF1), a Ras-like small GTPase, was highly expressed in human prostate cancer cells and tissues. In addition, ARF1 was markedly activated in prostate cancer cells. More interestingly, oncogenic G protein-coupled receptors (GPCRs) strongly activated ARF1 and the activation was mediated through Gβγ subunits. These data indicate that GPCRs and heterotrimeric G proteins are the upstream activators of ARF1 in prostate cancer cells. Next, we determined the role of ARF1 in the MAPK activation and proliferation in prostate cancer cells. Lentiviral-mediated overexpression of ARF1 remarkably enhanced, whereas shRNA-mediated depletion of ARF1 dramatically reduced ERK1/2 activation in prostate cancer cells. In addition, disruption of both the Golgi localization of ARF1 and the Golgi structure substantially attenuated ERK1/2 activation in prostate cancer cells. In parallel with their effects on the MAPK activation, ARF1 overexpression greatly enhanced and ARF1 knockdown inhibited the proliferation of prostate cancer cells. These data suggest that ARF1, by its ability to activate the MAPK pathway likely at the Golgi, controls prostate cancer cell proliferation. We then investigated the consequence of pharmacologically inhibiting ARF1 activation. Small molecule inhibitors including brefeldin A, golgicide A, and Exo2 that specifically target Golgi-localized ARF1 markedly reduced both ERK1/2 activation and proliferation in prostate cancer cells. These results further indicate an important role of ARF1 activation in regulating the MAPK pathway and prostate cancer cell proliferation. Altogether, our data suggest a possible GPCR-G-ARF1-MAPK signaling pathway, which may be responsible for the hyperactivation of the MAPK ERK1/2 in prostate cancer and contributes to prostate cancer progression. Our results also imply a novel approach for prostate cancer therapy by targeting ARF1 activation
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Post Traumatic Stress Disorder: Insights from Cat Hair and Catfish
(2012-06) Nalloor, Rebecca Ipe; Department of Pharmacology and Toxicology
Post traumatic stress disorder (PTSD) is an anxiety disorder that develops in some, but not all, individuals following a traumatic experience. Established PTSD is difficult to treat, therefore prevention and early intervention is important to reduce prevalence. Identifying individuals susceptible to developing PTSD before trauma exposure and investigating neurophysiological processes that contribute to the disease will help develop better treatment and preventive methods. Limitations to such investigations in humans make animal models a necessary tool. Like humans, only some rats develop PTSD-like behavior after trauma but pre-trauma identification of these rats was not possible until now. We were able to reliably predict before trauma exposure which rats are susceptible (Susceptible) or resistant (Resistant) to developing two PTSD-like symptoms: impaired fear extinction and lasting elevation in acoustic startle responses. We hypothesized that Susceptible rats will have pre-existing alterations in plasticity-related responses in the hippocampus, a brain region whose altered size and function is associated with PTSD diagnosis. We also hypothesized that Susceptible rats will differ from Resistant rats in the acquisition of a traumatic event and tested this using Arc/H1a catFISH, a cellular imaging technique that detects neurons expressing plasticity-related immediate early genes (IEGs) during behavior. We found that, in Resistant rats a large proportion of the same dorsal CA1 (dCA1) neurons expressed IEGs during two identical explorations of the experimental box. This suggests that dCA1 responds to identical events with high fidelity. In Susceptible rats, however, different neuronal ensembles expressed IEGs during identical explorations suggesting a lack of fidelity in hippocampal response to identical events. In addition fewer ventral CA3 neurons expressed IEGs during the second exploration in Susceptible as compared to Resistant rats. We also examined the basolateral nucleus of the amygdala, but found no difference in IEG expression. Contrary to hypothesis, differences between Susceptible and Resistant rats during a foot shock paired exploration (traumatic event) were not pronounced. These findings show that rats susceptible to developing PTSD-like symptoms can be behaviorally identified and have altered hippocampal plasticity-related responses prior to the trauma. This study provides a frame-work for the investigation and remediation of susceptibilities.

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