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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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.
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
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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.
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Regulators of G Protein Signaling (RGS Proteins) Regulate Presynaptic Inhibition at Rat Hippocampal Synapses
(2000-11) Chen, Huanmian; Department of Pharmacology and Toxicology
Presynaptic inhibition o f elicited neurotransmitter release mediated by G protein-coupled receptors (GPCRs) can develop and decay in a few seconds. This time course is too rapid to be accounted for by the intrinsic GTPase activity o f Ga subunits alone. Here we test the hypothesis that endogenous regulators o f G protein signaling (RGS proteins), which are GTPase activating proteins (GAPs) for Ga, are required for rapid, brief presynaptic inhibition. Endogenous G protein a subunits were uncoupled from GPCRs by treating hippocampal microisland cultures with pertussis toxin (PTX). Adenovirusmediated expression o f mutant PTX-insensitive (PTX-i) Gau.3 or Ga0 subunits rescued adenosine-induced presynaptic inhibition in neurons. Expression o f double mutant Gan or Ga0 subunits that were both PTX-insensitive and unable to bind RGS proteins (PTX/RGS-i) also rescued presynaptic inhibition. Presynaptic inhibition mediated by PTX/RGS-i subunits decayed much more slowly after agonist removal than that mediated by PTX-i subunits or native G proteins. The onset o f presynaptic inhibition mediated by PTX/RGS-i Ga0 was also slower than that mediated by PTX-i Ga0. In contrast, the onset o f presynaptic inhibition mediated by PTX/RGS-i Gan was similar to that mediated by PTX-i Gan. These results suggest that endogenous RGS proteins are present in presynaptic terminals and essential for fast recovery o f presynaptic inhibition. The effect o f endogenous RGS proteins on the onset o f presynaptic inhibition appears dependent on the particular Ga subunits involved. We also performed experiments to test whether the functions o f RGS proteins are sensitive to upregulation. Over-expression o f RGS8 in neurons without pretreatment o f PTX not only accelerated the time course o f the onset but also increased the steady state level o f presynaptic inhibition. Overexpression o f RGS4 also enhanced the steady state. These results suggest that RGS8 and probably RGS4 as well can be transported to presynaptic terminals and upregulate the activation o f Gy0 protein signaling. Interestingly, overexpression o f these RGS proteins failed to accelerate the recovery o f presynaptic inhibition, although it is well established that both RGS8 and RGS4 are strong GAPs for GcCj/0. This result suggests GAP activity for Gai/0 in presynaptic terminals is physiologically “ saturated” by endogenous RGS proteins.
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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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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.
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The Role of Lateral Diffusion in G-Protein-Coupled Receptor Signaling
(2006-05) Lober, Robert M.; Department of Pharmacology and Toxicology
In the standard model of G-protein-coupled receptor (GPCR) signaling, receptors and G-proteins are free to diffuse laterally within the plane of the plasma membrane, and these molecules encounter each other by random collision. It is possible that formation of the receptor-G-protein (R-G) complex precedes receptor activation, although the dynamics of this process have been challenging to observe in live cells. We have approached this problem by measuring the membrane diffusion and binding reactions of receptors and Gproteins. We examined the functional consequences of immobilizing receptors, G-proteins, and inwardly rectifying potassium (GIRK) channels at the cell surface by biotinylation and avidin crosslinking, and monitored intermolecular binding events reflected by receptor-imposed constraints on G-protein diffusion, as measured by fluorescence recovery after photobleaching. In whole-cell voltage-clamp recordings, we found that both mobile and immobile heterologous p-opioid receptors (MOR) activated endogenous GIRK channels in cerebellar granule neurons with kinetics and agonist sensitivity resembling native synaptic responses. In HEK 293 cells, immobile GIRK channels were activated by multiple populations of immobilized receptors and Gproteins. Immobilization of MOR constrained the apparent mobility of freely diffusing fluorescent Gaj/o-family proteins, indicating measurable binding reactions between the two protein types, but had no effect on the diffusion of unrelated membrane proteins or Gaq subunits. Transient binding reactions were highly specific, as determined by competition with unlabeled binding partners. RG binding was disrupted by receptor agonist or GTPase-deficient G-protein mutants. Neither receptor antagonists nor pertussis toxin blocked basal R-G binding. Furthermore, the Ga subunit amino terminus (amino acids 1-31) was sufficient for mediating R-G binding. Our results provide evidence for the free diffusion of receptors and Gproteins, as well as a pre-signaling binding-dissociation equilibrium between them that is altered upon activation. The frequency of collisions between receptors and G-proteins does not limit the rate of signaling in neurons, but by diffusion receptors can “swap” G-proteins that are not stably associated with GIRK channels. A three-stage sequential fit model of R-G coupling is suggested.
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RGS protein modulation of neuronal Gaq-mediated signaling
(2006-05) Clark, Michael A.; Department of Pharmacology and Toxicology
The decay of excitatory postsynaptic potentials (EPSPs) in neurons is much shorter than the predicted lifetime of activated G aq and thus GTPase activating proteins (GAPs) are thought to accelerate inactivation. PLCp and regulators of G-protein signaling (RGS) proteins act as GAPs toward G aq in vitro. Thus, it is unknown which of these GAPs determine the fast decay rate observed in neuronal EPSP termination. We therefore test the hypothesis that endogenous RGS proteins regulate the kinetics of Gaqmediated signaling in cultured rat cerebellar granule neurons (CGNs). Electrophysiological recordings of G aq-regulated standing outward potassium currents (Ik(SO>) were performed using mutant RGS-insensitive (RGSi) G aq chimeras. RGS insensitivity was determined by these mutants’ inability to recruit RGS2-EGFP from the nucleus of HEK293 cells to the plasma membrane despite additional mutations that render constitutive activity. Recovery from Ik(so) inhibition mediated by RGSi mutants was 5-fold slower than their wild type counterparts, confirming the necessity of native RGS proteins for rapid termination of G aq mediated signals. In addition to regulating decay of EPSPs, evidence suggests RGS proteins control activation kinetics of G-protein-mediated signals by undetermined mechanisms. We therefore hypothesized that native RGS proteins regulate onset kinetics by acting as physical scaffolds to increase the availability of activated receptors to inactive G aq, bypassing cellular diffusional limitations. Fluorescence recovery after photobleaching (FRAP) was used to determine the diffusional mobility of G aq signaling components in the presence or absence of extracellular crosslinking reagents. Even though M3R recruited RGS2-EGFP to the plasma membrane in HEK293 cells, interaction between these two proteins was extremely transient, as RGS2-EGFP diffusion was unaltered upon immobilization of ECFP-M3R. In addition, G aq-EGFP constrained diffusion when interacting with ECFP-M3R was not further slowed by RGS2 expression. Finally, RGS proteins may act as kinetic scaffolds whereby RGS-accelerated GAP activity leads to multiple rounds of activation/inactivation per receptor-Gaq binding event. A mutation causing increased intrinsic GTPase activity of G aq significantly restored onset rates of G aq activation ( I k (s o > inhibition) in the background of RGS-insensitivity. These results indicate a minimal physical scaffolding function of RGS2 and provide evidence for native RGS protein-mediated kinetic scaffolding contributing to fast G aq activation kinetics observed in CGNs.
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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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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.
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Regulation of Endothelial Nitric Oxide Synthase by Subcellular Localization and
(2007-04) Zhang, Qian; Department of Pharmacology and Toxicology
Endothelial nitric oxide synthase (eNOS) is regulated by post-translational modifications that target eNOS to the plasma membrane (PM) and the perinuclear/Golgi region. It has been shown in COS-7 cells that targeting of eNOS to the Golgi or PM regulates the mechanism and degree of eNOS activation. However, little is known about the functional significance of eNOS targeting in endothelial cells (ECs). Our first goal was to isolate these two pools of enzyme in ECs and determine their functional significance in response to agonist stimulation and manipulation of membrane cholesterol levels. Using an RNAi strategy, we generated stable populations of EC that had greater than 90% inhibition of eNOS expression and lacked the ability to produce NO. Reconstitution of these eNOS “knockdown” EC with Golgi and PM targeted eNOS restored the ability of EC to produce NO. This approach can be broadly applied to endothelial cells from a number of different species and from different vascular beds and should have broad utility. Using these cells we found that the PM is the optimal location within the cell to produce NO, but it is also the most vulnerable to changes in cholesterol and oxidized LDL. Calcium-dependent agonists were the more efficient stimulus for the PM-restricted eNOS in EC. In contrast, Golgi eNOS was less responsive to both calcium and Akt-dependent agonists. The functional significance of the increased NO produced by the PM eNOS is reflected in the greater ability to elicit endothelium-dependent relaxation, greater suppression of vWF secretion, a key regulator of platelet aggregation, and inhibition of endothelial cell proliferation. Mechanistically, PM eNOS induces more nitrosylation of proteins such as NSF, but this is related to the amount of NO being produced, rather than its intracellular location. Increased superoxide formation in endothelial cells (ECs) has been identified as a causative factor in endothelial dysfunction by reducing nitric oxide (NO) bioavailability, uncoupling eNOS. A major source of intracellular superoxide is the NADPH oxidase (Nox) family of enzymes. In experiments to address the effect of superoxide on local eNOS activity, we found that Nox5 increased eNOS activity paradoxically in both cotransfected COS-7 cells and transduced bovine aortic ECs determined by chemiluminescence to measure the NO metabolite. Nox5 also activated eNOS in human aortic ECs as detected by a cGMP reporter assay that measured the release of biologically functional NO from cells in the presence of superoxide dismutase (SOD). To establish the functional significance of this observation in blood vessels, the endothelium of mouse aorta was tranduced with Nox5 or control adenoviruses. Nox5 potently inhibited Achinduced relaxation, potentiated contractile responses to phenylephrine. In precontracted blood vessels, acute exposure to SOD induced significant vascular relaxation in vessels exposed to Nox5 versus control and unmasked the ability of Nox5 to activate eNOS in blood vessel endothelium. These results are in contrast to a number of described mechanisms for eNOS inhibition and provide valuable clues that in complex diseases such as diabetes and hypertension that ROS production is not the sole cause of endothelial cell dysfunction.
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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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Soluble Epoxide Hydrolase Inhibition Attenuates Vascular Remodeling and Protects Against Cerebral Ischemia
(2008-05) Simpkins, Alexis Netis; Department of Pharmacology and Toxicology
Hypertension is linked to the incidence of cardiovascular events such as ischemic stroke due to several mechanisms, including vascular remodeling. The progression of vascular remodeling leads to increased arterial stiffness, plaque formation and rupture, and thrombosis, culminating in blockage of the arterial supply to the brain. A strategy to confer protection from hypertension linked cardiovascular events is inhibition of pathological remodeling. Notably, epoxyeicosatrienoic acids (EETs) modulate vascular smooth muscle cell (VSMC) proliferation and migration and vascular tone. However, their conversion by soluble epoxide hydrolase (SEH) enzyme to less active diols attenuates their protective properties. As a result, an alternative strategy to confer protection from the sequela of hypertension is SEH inhibition. Here we show that SEH inhibition and deletion of the gene responsible for the production of the SEH enzyme protects against pathological vascular remodeling in a model in which the endothelium is preserved via left carotid ligation. Interestingly, this protection was not observed in a model of arterial injury in which the femoral artery is denuded. This demonstrates that SEH antagonism has potential for protecting against pathological remodeling by an endothelium dependent mechanism. In line with this finding, we demonstrated that SEH inhibition restored the plasticity of carotid arteries in hypertensive rats with impaired responses to increases or decreases in flow. Importantly, we demonstrated that these protective properties of SEH inhibition were translatable to vascular protection from cerebral ischemia in an animal model of essential hypertension. Chronic SEH inhibition protected against cerebral ischemia in hypertensive rats by inhibiting vascular remodeling of the middle cerebral artery and increasing microvessel density. Interestingly, we also show that SEH inhibition is able to protect against cerebral ischemia without changing the structure of the vasculature of normotensive animals. In fact, we found that a potential for protection could be afforded by changes in the expression profile of genes involved in apoptosis, neurogenesis, and reactive oxygen species antagonism in models both of hypertension and normotension. The sum of these findings indicates that SEH inhibition has broad pharmacological potential for protecting against the occurrence and severity of ischemic stroke by mechanisms that are attributed to blocking the sequela of hypertension and neuroprotection.
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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Protein-Protein Interaction between G protein-coupled receptors and heterotrimeric G proteins
(2011-01) Qin, Kou; Department of Pharmacology and Toxicology
G protein-coupled receptors (GPCRs) interact directly with heterotrimeric G proteins to transduce physiological signals. Early studies of this interaction concluded that GPCRs (R) and G proteins (G) collide with each other randomly after receptor activation and that R-G complexes are transient (collision model). More recent studies have suggested that inactive R and G are preassembled as stable R-G complexes in cells (preassembly models). Using fluorescence recovery after photobleaching (FRAP) we examined the stability of complexes formed between cyan fluorescent protein-labeled a2Aadrenoreceptors (C-a2ARs) and G proteins in HEK293 cells. Labeled G proteins diffused in the plasma membrane with equal mobility in the absence and presence of immobile C- a2ARs. In contrast, a stable R-G interaction was detected when G proteins were deprived of nucleotides and C- a2ARs were active. Over-expression of regulator of G protein signaling 4 (RGS4) accelerated the onset of effector activation but did not alter the interaction between C- a2ARs and G proteins. At most a small fraction of C- a2ARs and G proteins exist as R-G complexes at any moment. However, applying similar technique and protocols, we demonstrated that immobilized M3R specifically decreases the mobility of Gaq heterotrimers on the plasma membranes of intact HEK293 cells, suggesting the existence of R-G preassembly. The C-terminus of M3R was determined to be both required and sufficient for preassembly. The M3R C-terminus contains a polybasic region (565KKKRRK570) located distal to the 8th a-helix domain. Substitution of this polybasic region with 6 electroneutral alanines (M3R6A) prevented preassembly. Permeabilization of cells with low ionic strength buffer resulted in enhanced R-G interaction, implicating electrostatic forces as a factor in the preassembly. We examined the functional properties of the mutant M3R6A, which showed decreases in acetylcholine potency compared with M3R. M3R6A produced active Gq at half the rate of M3R. Other Gq-coupled receptors, such as M1 and M5 muscarinic and a1a,a1b, aid adrenergic receptors, contain similar C-terminal polybasic regions. We found that both M5R and alb adrenoceptor (albAR) preassembled with Gq proteins. Our findings suggest that a polybasic regionmediated electrostatic mechanism could be a common mechanism of preassembly between Gq-coupled receptors and Gq proteins.
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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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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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Use-dependent Antagonism of Nicotinic Acetylcholine Receptors as a Novel Treatment for Drug Addiction
(2011-11) Hall, Brandon J; Department of Pharmacology and Toxicology
The contributions of nicotinic acetylcholine receptors (nAChRs) to the onset and maintenance of drug addiction are well known, but these receptors are too often overlooked as potential targets for addiction treatment. The goal of this study was to demonstrate that use-dependent antagonism of nAChRs by the compound bis (2, 2, 6, 6-tetramethyl-4-piperidinyl) sebacate (BTMPS) offers a novel approach to treatment for drug addiction, and that positive outcomes of this treatment can be demonstrated across different classes of abusive drugs, nicotine or morphine in all three phases of an animal model of what is known as the drug abuse cycle: 1) binge-intoxication, 2) withdrawal-negative affect, and 3) preoccupation-anticipation. Different groups of rats were allowed to self-administer drugs of abuse (nicotine or morphine) on a 24 hr basis for a period of 14 days to establish binge-intoxication. Upon completion of self-administration, each rat was evaluated for withdrawal-negative affect. Subsequent to acute withdrawal the rats were placed in standard housing cages for a period of six weeks. At the end of the six week period, each rat was examined for unrewarded drug seeking responses, or preoccupation-anticipation, for another 14 day period preoccupation-anticipation. Injections of vehicle or BTMPS were administered to the animals during each behavioral phase of the study. Treatment with BTMPS significantly reduced the self-administration of both nicotine and morphine compared to vehicle treated animals. BTMPS treated animals also displayed reduced acute withdrawal symptoms when compared to their vehicle treated counterparts. When intervention occurred during self-administration or acute withdrawal, BTMPS treatment resulted in a significant reduction in drug-seeking responses after a protracted period of abstinence from drug. However, delaying treatment with the compound until the drug seeking phase of the study was ineffective against reducing drug seeking behavior. Administration of BTMPS alone did not appear to elicit adverse side effects in the animals, neither affecting their motivation to obtain food nor compromising the animals' performance during the behavioral procedures in the study. Thus, the resultsof this study support the hypothesis that use-dependent antagonism of nAChRs offers the potential for an alternative approach to treatment of substance abuse and drug addiction.
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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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A Variable Prenatal Stress Paradigm as a Valid Drug Discovery Platform for Cognitive Deficits Associated with Neuropsychiatric Disorders
(2012-10) Wilson, Christina Ann; Department of Pharmacology and Toxicology
Cognitive dysfunction is now recognized to be central to the functional disability of several neuropsychiatric disorders. However, treatment options for the management of cognitive symptoms are limited and the development of novel therapeutics has been made difficult by the lack of appropriate animal models. It has been suggested that variable prenatal stress (PNS) in rodents might be an etiologically appropriate model for some components of schizophrenia. Thus, the overall goal of this dissertation project was to conduct a comprehensive behavioral study of the model to assess face validity, and to make a preliminary assessment of its construct and predictive validity. Our results indicate that exposure to PNS results in elevated corticosterone levels following exposure to acute stress, increased aggressive behaviors, as well as increased locomotor activity and stereotypic behaviors. Further, PNS rats had altered innate fear responses to predator odor as well as impaired fear extinction. Additionally, PNS in rats was associated with impairments of sustained attention, inhibitory response control, and recognition memory all of which could be attenuated by the norepinephrine reuptake inhibitor, atomoxetine. Collectivity, these data support the premise that PNS in rodents is a valid model system for studying some behavioral components of neuropsychiatric disorders as well as their treatment.

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