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.

Recent Submissions

  • The Role of T Cells in High Fat Diet Induced Hypertension and Vascular Dysfunction in Female Rats

    Taylor, Lia; Department of Pharmacology and Toxicology (2019-01-08)
    Hypertension is a leading risk factor for the development of cardiovascular disease (CVD) worldwide, affecting ~86 million adults in the United States alone. In general, the prevalence of hypertension is lower in premenopausal women than in age-matched men. Despite having lower risk than men, however, women represent more than half of all hypertension cases, and CVD is the leading cause of death in women worldwide. Women are also more likely than men to be obese and two-thirds of hypertension cases in the U.S. positively correlate with excessive weight gain due to the consumption of diets high in saturated fat. Dahl salt-sensitive rats (DSS) have traditionally been used to study salt-sensitive hypertension. However, previously, it has been shown that male DSS also exhibit a high fat diet (HFD)-induced increase in blood pressure (BP) at normal salt intake, which is attenuated by treatment with the lymphocyte inhibitor mycophenolate mofetil (MMF), suggesting a role for B and T cells in HF-induced hypertension in male animals. However, there is limited data on the role of HFD and T cell activation on BP in females. We hypothesized that HFD will promote hypertension and vascular dysfunction in female DSS and that these cardiovascular changes will be mediated by T cells. We demonstrate that female DSS are susceptible to HFD-induced hypertension and vascular T cell infiltration and activation similar to that of males. Since local inflammation in perivascular adipose tissue (PVAT) is linked to HFD-induced increases in BP and vascular dysfunction in males, we further investigated the impact of HFD on vascular function and the buffering capacity of PVAT in female DSS. HFD alone did not impair vascular function compared to normal fat diet (NFD) in female DSS. Interestingly, the buffering capacity of PVAT was also maintained in female DSS in response to a HFD despite increases in BP and vascular inflammation, likely due to overproduction of nitric oxide (NO). To directly assess the role of T cells on HFD-induced alterations of BP and vascular function in female DSS, we utilized female CD247-/- rats on the DSS background. HFD-induced increases in BP were completely abolished in CD247-/- . Further, the buffering capacity of PVAT was preserved in CD247-/- in response to a HFD. We conclude that T cells underlie the susceptibility of female DSS to HFD-induced hypertension. Further, vascular function and PVAT buffering capacity are maintained in female DSS despite fat-induced increases in BP and vascular inflammation, suggesting an adaptive response in the early stages of diet-induced obesity in which nitric oxide (NO) may play a key role.
  • Effect of chronic oral treatment with risperidone or quetiapine on cognitive performance and neurotrophin-related signaling molecules in rats

    Poddar, Indrani; Department of Pharmacology and Toxicology (8/7/2018)
    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
  • Epigenetic Regulation of Adipogenic Differentiation And Lipid Metabolism: Role of Enhancer of Zeste Homolog 2 (EZH2)

    YIEW, KAN HUI; Department of Pharmacology and Toxicology (2017)
    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.
  • A Data-Mining Strategy That Identifies Drugs and Genes Associated With Anti-Cancer Drug Sensitivity

    Schleifer, Robert John; Department of Pharmacology and Toxicology (2017)
    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.
  • Circadian Clock in Angiotensin II Induced Hypertension and Vascular Disease

    Pati, Paramita; Department of Pharmacology and Toxicology (2015)
    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.
  • Involvement of arginase upregulation in diabetes- and angiotensin II-induced vascular dysfunction

    Bhatta, Anil; Department of Pharmacology and Toxicology (2015)
    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.
  • A Novel Function of ADP-Ribosylation Factor 1 in Prostate Cancer Cell Proliferation through Activating the Mitogen-Activated Protein Kinase Pathway

    Davis, Jason E.; Department of Pharmacology and Toxicology (8/23/2016)
    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
  • Calpain-2 Activates Akt via the TGF~ 1-mTORC2 Pathway in Pulmonary Artery Smooth Muscle Cells

    Abeyrathna, Prasanna; Deparment of Pharmacology and Toxicology (8/23/2016)
    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.
  • Attenuating the Interaction Between Delta Protein Kinase C and the "d" Subunit of FIFo ATp Synthase Protects Against Cardiac Ischemia/Repferusion injury

    Walker, Matthew; Deparment of Pharmacology and Toxicology (6/3/2016)
    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.
  • Protein-Protein Interaction between G protein-coupled receptors and heterotrimeric G proteins

    Qin, Kou; Department of Pharmacology and Toxicology (2011-01)
    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.
  • Modulation of Ion Channels by Cannabinoid Receptors in Rat Sympathetic Neurons

    Pan, Xianghua; Department of Pharmacology and Toxicology (1996-09)
    The rat brain cannabinoid receptor (CB1) is a member of the G protein coupled receptor family. The present study directly tested the functional coupling of CB1 receptor with neuronal ion channels. The rat CB1 receptor was heterologously expressed by microinjection o f cRNA into the enzymatically dissociated adult rat superior cervical ganglion (SCG) neurons. The cannabinoid receptor agonists WIN 55,212-2 and CP 55940 produced a voltagedependent inhibition of whole cell Ca2+ currents. The maximal inhibitions o f the Ca2+ current by WIN 55,212-2 and CP 55,940 were 73% and 38%, respectively. The E C 50 of WIN 55,212-2 was 47 nM and the EC50 of CP 55940 was 7 nM. The inhibition was mediated by pertussis toxin (PTX) sensitive G protein and the N-type Ca2+ channels are the targets. The L-type Ca2+ channels, M type K+ current and the A current were not modulated by WIN 55,212-2. An inwardly rectifying current was activated by WIN 55,212-2. The selective CB1 receptor antagonist SR 141716A and LY 320135 abolished the inhibition of the Ca2+ currents by WIN 55,212-2. However, when given alone, SR 141716A and LY 320135 increased the Ca2+ current in SCG neurons expressing the CB1 receptor. SR 141716A increased Ca2+ current with an EC50 of 32 nM and a maximal current increase of 41% at 1 p.M. This increase of Ca2+ current by SR 141716A was a reversal o f a tonic inhibition of Ca2+ current in neurons expressing CB 1 receptors. A K192A mutant of the human CB1 receptor was tested to determine whether the tonic activation o f the cannabinoid receptor is due to endogenous arachidonyl ethanolam ide (anandam ide). The K192A m utant receptor was expressed by microinjection of receptor cDNA into nucleus of the SCG neurons. WIN 55,212-2 inhibited the Ca2+ current in these SCG neurons, but SR 141716A did not increase the Ca2+ current. However, SR 141716A inhibited the effect of WIN 55,212-2. Ca2+ currents from male rat major pelvic ganglion neurons were examined for modulation by native cannabinoid receptors. WIN 55,212-2 inhibited and SR 141716A increased the \ '>ltage-dependent Ca2+ currents in a subpopulation of the rat major pelvic ganglion neurons. 1 (J.M WIN 55,212-2 inhibited current by 26.1±1.8% and 1 pM SR 141716A increased current by 27.4±6.9%. These findings indicate that both heterologously expressed CB1 cannabinoid receptors and the native neuronal cannabinoid receptors can inhibit voltage-dependent Ca2+ channels. There is a tonic activation of both the heterologously expressed rat and human CB1 receptor and the native rat cannabinoid receptor. Two possible mechanisms may be responsible for the tonic receptor activation: an endogenous agonist may exist or the cannabinoid receptor can adopt an active conformational state such that SR 141716A may act as an inverse agonist.
  • The CB1 Cannabinoid Receptor: Receptor States, Activity and G Protein Sequestration

    Nie, Jingjiang; Department of Pharmacology and Toxicology (2001)
    The human CB1 cannabinoid receptor is a member o f the G protein coupled receptor family. The CB1 cannabinoid receptor couples to pertussis toxin sensitive Gi/o proteins and inhibits neuronal voltage-gated Ca2+ channels. The hCBl receptor has two unusual properties: 1) it is constitutively active in the absence o f agonist and 2) it can prevent other G protein coupled receptors from signaling by sequestering a common pool of Gi/o proteins. The mechanism o f constitutive activity and G protein sequestration by the hCB 1 receptor is unknown. In this study, two carboxyl terminal truncation mutants (hCB 1-417 and hCBl-400) were used to test the hypothesis that the proximal carboxyl terminal couples to G proteins while the distal carboxyl terminal modulates G protein sequestration and constitutive activity. Additionally, mutation o f a single amino acid in the second transmembrane domain (rCBl-D164N) was used to test the hypothesis that this amino acid plays a critical role in the structural basis o f G protein coupling and constitutive activity. Receptor cDNA constructs were injected into the nucleus of superior cervical ganglion neurons. After an overnight incubation to allow for receptor expression, neurons were voltage clamped and Ca2+ current were recorded. Inhibition of the Ca2r current by the cannabinoid agonist WIN 55,212-2 was used as an index o f CB1 cannabinoid receptor G protein coupling and activation. Ca2' channels are inhibited by G{3y subunits released from activated Gi/o proteins. In contrast to the wild type CB1 cannabinoid receptor, the mutant receptor in which the entire carboxyl terminal (amino acids 401-472) was deleted (hCB 1-400) failed to inhibit the Ca2+ current. Deletion o f only the distal carboxyl terminal (amino acids 418-472; hCB 1-417) restored Ca2+ current inhibition. These results demonstrate the critical role o f the proximal domain (amino acids 401-417) o f the carboxyl terminal of the hCB 1 receptor in coupling to G proteins. Truncation o f the distal carboxyl terminal domain, however, changed the magnitude o f Ca2+ current inhibition. The hCBl-417 receptor produced significantly less inhibition of the Ca2+ current in the presence o f WIN 55,212-2 compared to the wild type receptor (22.6±3.0% vs 43.7±6.5%, respectively). Thus, the distal carboxyl terminal domain is important in modulating the magnitude of Ca2* current inhibition. In addition to the change in the magnitude o f Ca2+ current inhibition, deletion o f the distal carboxyl terminal significantly slowed the kinetics o f Ca2l~ current inhibition by WIN 55,212-2 (time to peak o f effect: 146.0+8.7 second). The distal carboxyl terminal tail of the CB1 cannabinoid receptor also played a role in constitutive activity and G protein sequestration. The hCB 1-417 receptor displayed enhanced constitutive activity. In neurons injected with 50 ng/pl hCBl-417 cDNA the inverse agonist SR141716A increased the Ca2+ current 101.1±21.3%. The inverse agonist acts to reverse the constitutive activity o f the receptor. The effect o f SRI 41716 A on the hCBl-417 receptor was significantly greater than the 42.9±7.6% Ca2+ current increase in neurons expressing the wild type hCB 1 receptor. G protein sequestration was also enhanced in neurons expressing the truncated hCBl-417 receptor. Wild type hCBl cannabinoid receptors when expressed by injecting 100 ng/p.1 cDNA completely abolish signaling by other G protein coupled receptors including a 2 -adrenergic receptors. Normally activation o f a 2 -adrenergic receptors inhibits the Ca2+ current 44.5±5.7%. In the presence o f hCBl receptors activation of the a 2- adrenergic receptors by UK14304 inhibited the Ca2+ current only 1.5±4.2%. Signaling by a 2-adrenergic receptors can be partially restored by injecting a lower concentration o f hCBl cDNA. In neurons injected with 50 ng/pl hCBl cD N A the ct2 -adrenergic agonist UK14304 inhibited the Ca2+ current 20.0±3.7%. In neurons injected with 50 ng/pl hCBl- 417 cDNA UK14304 inhibited the Ca2+ current 7.0±1.2%. Thus, signaling by the a 2- adrenergic receptor was abolished by the carboxyl terminal truncated hCBl-417 receptor. These results indicate that deletion of the distal carboxyl terminal enhances the ability o f the receptor to sequester G proteins. The aspartic acid residue in the second transmembrane domain of G protein coupled receptors is highly conserved. Mutation o f this aspartic acid (rCBl-D164N) had profound effects on the constitutive activity o f the C B 1 receptor as well as on the ability of the receptor to sequester G proteins. Both the constitutive activity and the ability to sequester G proteins were abolished by the rCBl-D164N receptor. The inverse agonist increased the Ca2+ current only 11.6+6.9% in neurons expressing the mutant rC Bl- D164N receptors. The mutant rCBl-D164N receptors failed to block signaling by the a 2- adrengic receptor. UK14304 inhibited the Ca2+ current 35.8+6.8% in neurons expressing the rCBl-D164N receptors and was not different from control neurons. Additionally, the D164N mutation in the second transmembrane domain decreased the time to peak o f the WIN 55,212-2 inhibition o f the Ca2+ current to 24+4 seconds. These results demonstrate that 1) the proximal carboxyl terminal domain o f the hCB 1 receptor is critical for G protein coupling, 2) the distal carboxyl terminal domain regulates constitutive activity, G protein coupling kinetics and G protein sequestration and 3) the aspartic acid in the second transmembrane domain plays a critical role in G protein sequestration, G protein coupling kinetics and constitutive activity o f the hCBl receptor. Taken together, the mutant receptors shift the CB1 receptor into different receptor states. The D164N-CB1 receptor exists primarily in an inactive state uncoupled from G proteins, the wild type CB1 receptor exists in both active and inactive G protein coupled states and the carboxyl terminal truncated CB 1-417 receptor exists primarily in an active G protein coupled state.
  • The Role of Lateral Diffusion in G-Protein-Coupled Receptor Signaling

    Lober, Robert M.; Department of Pharmacology and Toxicology (2006-05)
    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.
  • Characterization of the Physical and Functional Interaction between a Novel Protein CRIPla and the CBj Cannabinoid Receptor

    Liu, Yunguang; Department of Pharmacology and Toxicology (2006-10)
    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.
  • Cytoprotective Actions of Nicotine: The Increased Expression of a7 Nicotinic Receptors and NGF/TrkA Receptors

    Jonnala, Ramamohana R.; Department of Pharmacology and Toxicology (2001-07)
    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.
  • Effect of Homocysteine on Bovine Aortic Endothelial Cell Function

    Jin, Liming; Department of Pharmacology and Toxicology (2001-05)
    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.
  • Use-dependent Antagonism of Nicotinic Acetylcholine Receptors as a Novel Treatment for Drug Addiction

    Hall, Brandon J; Department of Pharmacology and Toxicology (2011-11)
    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.
  • Studies on the Regulatory Mechanisms of Induction of no Synthase in Rat Aortic Smooth Muscle Cells

    Go, Carolyn Y.; Department of Pharmacology and Toxicology (1996-08)
    The induction of NOS involves a growing number of stimuli which include a variety of substances, namely bacterial endotoxin (LPS), cytokines (e.g. IL-1, TNFa, DFNy), cAMPelevating agents, UV light, ozone, trauma, microbes, etc. (Nathan and Xie, 1994). In this study, we report the induction of NOS by protein synthesis inhibitors in rat aortic smooth muscle cells (RASM). We hypothesized that protein synthesis inhibitors increase iNOS mRNA stability and cause the depletion of protein repressor IkBcc, resulting in the activation of the transcription factor NF-kB and subsequent iNOS gene expression. We tested our hypothesis by determining the effects of two protein synthesis inhibitors, namely cycloheximide (CH) and anisomycin (ANI), on iNOS function and gene expression in R ASM. We observed the absence of NMMA-sensitive and L-arginine-dependent NOS activity. However, in the presence of either lOpM CH or 250ng/ml ANI for 24 hours, there is significant iNOS activity, as determined by cGMP accumulation in the presence of IBMX, a phosphodiesterase inhibitor. The increase in cGMP levels is sensitive to NOS inhibition by LNAME and this inhibition is reversed by the addition of L-arginine, but not D-arginine. The response to an exogenous NO donor, SNP, is not altered after prolonged exposure to either protein synthesis inhibitor. These results suggest that the cGMP production is due to iNOS activation by CH or ANI. In the presence of a known iNOS agonist e.g. LPS, IL-1 P or TNF-a, iNOS activity is superinduced by CH. LPS-induced NOS activity which peaks a. ter 6 hours is inhibited by CH, however subsequent LPS-induced NOS (>6hrs) is superinduced after prolonged exposure to CH. In the presence of increasing concentration of CH, peak iNOS activity was obtained at 10pM CH, whereas reduced activity was obtained at higher concentrations (>50pM). A similar biphasic curve was observed in the additional presence of LPS (lOOng/ml), however, the levels were 100-fold higher than with CHstimulation alone. To determine whether protein synthesis is altered by CH, total methionine incorporation was measured. A dose-dependent attenuation of protein synthesis was observed, concommitant to a biphasic increase in iNOS activity in response to increasing CH concentration. The additional presence of LPS did not affect CH-inhibition of protein synthesis. It was also observed that maximum inhibition of protein synthesis is reversible within 2 hours of removal of CH. To determine whether cell proliferation was affected by CH, total cell number was determined in the presence or absence of CH, either in serumcontaining or serum-free medium. There was no significant changes in cell number within 24 hours of NOS induction. To investigate the mechanisms of NOS induction by CH, we determined whether CH-induced iNOS activity or superinduction of LPS-induced iNOS activity were dependent on continuous transcription. In the presence of actinomycin D, a transcription blocker, the CH-induction and superinduction of iNOS activity were significantly attenuated, confirming the hypothesis that CH-mediated increased iNOS activity requires new transcription. To directly investigate the mechanism of CH-induced iNOS activity in RASM, we determined iNOS protein by western blot analysis and mRNA levels by northern blot and RTPCR analysis. By western blot analysis, we observed a 130-kDa size protein, representing iNOS, only after a 24-hour exposure to both CH and IPS, corresponding to superinduced iNOS activity, as determined by radioimmunoassay of cGMP. Utilizing RTPCR, after 12 hours exposure to lOpM CH, there is significant iNOS mRNA. At this time, LPS-induced mRNA is minimal, however, in the combined presence of LPS and CH, relative mRNA levels are significantly increased. Comparison of relative iNOS mRNA half-lives induced by CH, LPS or combined CH and LPS, showed that in the presence of LPS alone, iNOS mRNA significantly decayed within 1.5 hours, in contrast to iNOS mRNA in the presence of CH and combined LPS and CH which were not timedependently reduced by inhibition of transcription. These data strongly suggested the presence of a labile protein involved in the regulation of iNOS transcription. To identify this putative labile repressor protein, we investigated the time-dependent effects of CH on the major inhibitory protein subunit of NF-kB, i.e. IkB oc. CH reduced IkB oc protein levels, concommitant with a delayed increase in its phosphorylated isoform, observed as the slower migrating species during electrophoresis. In the additional presence of protease inhibitors (to block the breakdown of IkB), the CH-mediated effects on IkB oc were prevented. Moreover, induction of NOS activity by CH was dose-dependently attenuated by protease inhibitors. To determine whether the CH-mediated effects on IkB oc activate NF-kB, nuclear extracts from cells treated with CH, LPS or combined CH and LPS were analyzed by electrophoretic mobility shift assays (EMSA). There was a significant shift in radiolabelled N F-kB groups stimulated by CH or LPS. Activation of NF-kB in the presence of both stimuli was enhanced, while there was no significant shift detected in unstimulated groups. In contrast, a strong basal AP-1 activity was detected in the presence of radiolabelled AP-I. CH-stimulation increased basal AP-1 activity. This correlated with a time-dependent increase in JUN, but not FOS, protein levels. These data strongly support the hypothesis for a modulatory role of labile repressors on iNOS gene expression by CH. In conclusion, results from the present studies strongly support the hypothesis, that CH-mediated iNOS gone expression and superinduction of LPS-induced NOS is due to both iNOS mRNA stabilization and depletion of ItcBa, subsequently leading to NF-kB activation and iNOS gene expression in RASM.
  • Impaired Cognition in Spontaneously Hypertensive Rats: Role of Central Cholinergic Receptors

    Gattu, Mahanandeeshwar; Department of Pharmacology and Toxicology (1996-09)
    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.
  • RGS protein modulation of neuronal Gaq-mediated signaling

    Clark, Michael A.; Department of Pharmacology and Toxicology (2006-05)
    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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