Biomedical Sciences Presentations (UMKC)
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Items in this collection are the scholarly output of the Department of Biomedical Sciences faculty, staff, and students, either alone or as co-authors, and which may or may not have been published in an alternate format.
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Item Inhibition of nitric oxide in LPS-stimulated macrophages of young and senescent mice by delta-tocotrienol and quercetin.(2011-12-20) Qureshi, Asaf A.; Tan, Xiaoyu; Reis, Julia C.; Badr, Mostafa Z., 1950-; Papasian, Christopher J.; Morrison, David C., 1941-; Qureshi, NiloferAbstract Background Changes in immune function believed to contribute to a variety of age-related diseases have been associated with increased production of nitric oxide (NO). We have recently reported that proteasome inhibitors (dexamethasone, mevinolin, quercetin, δ-tocotrienol, and riboflavin) can inhibit lipopolysaccharide (LPS)-induced NO production in vitro by RAW 264.7 cells and by thioglycolate-elicited peritoneal macrophages derived from four strains of mice (C57BL/6, BALB/c, LMP7/MECL-1-/- and PPAR-α-/- knockout mice). The present study was carried out in order to further explore the potential effects of diet supplementation with naturally-occurring inhibitors (δ-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-α, and other pro-inflammatory cytokines involved in the ageing process. Young (4-week-old) and senescent mice (42-week old) were fed control diet with or without quercetin (100 ppm), δ-tocotrienol (100 ppm), or dexamethasone (10 ppm; included as positive control for suppression of inflammation) for 4 weeks. At the end of feeding period, thioglycolate-elicited peritoneal macrophages were collected, stimulated with LPS, LPS plus interferon-β (IFN-β), or LPS plus interferon-γ (IFN-γ), and inflammatory responses assessed as measured by production of NO and TNF-α, mRNA reduction for TNF-α, and iNOS genes, and microarray analysis. Results Thioglycolate-elicited peritoneal macrophages prepared after four weeks of feeding, and then challenged with LPS (10 ng or 100 ng) resulted in increases of 55% and 73%, respectively in the production of NO of 46-week-old compared to 8-week-old mice fed control diet alone (respective control groups), without affecting the secretion of TNF-α among these two groups. However, macrophages obtained after feeding with quercetin, δ-tocotrienol, and dexamethasone significantly inhibited (30% to 60%; P < 0.02) the LPS-stimulated NO production, compared to respective control groups. There was a 2-fold increase in the production of NO, when LPS-stimulated macrophages of quercetin, δ-tocotrienol, or dexamethasone were also treated with IFN-β or IFN-γ compared to respective control groups. We also demonstrated that NO levels and iNOS mRNA expression levels were significantly higher in LPS-stimulated macrophages from senescent (0.69 vs 0.41; P < 0.05), compared to young mice. In contrast, age did not appear to impact levels of TNF-α protein or mRNA expression levels (0.38 vs 0.35) in LPS-stimulated macrophages. The histological analyses of livers of control groups showed lesions of peliosis and microvesicular steatosis, and treated groups showed Councilman body, and small or large lymphoplasmacytic clusters. Conclusions The present results demonstrated that quercetin and δ-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray DNA analyses, followed by pathway analyses indicated that quercetin or δ-tocotrienol inhibit several LPS-induced expression of several ageing and pro-inflammatory genes (IL-1β, IL-1α, IL-6, TNF-α, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40). The NF-κB pathway regulates the production of NO and inhibits the pro-inflammatory cytokines involved in normal and ageing process. These ex vivo results confirmed the earlier in vitro findings. The present findings of inhibition of NO production by quercetin and δ-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process.Item Suppression of nitric oxide induction and pro-inflammatory cytokines by novel proteasome inhibitors in various experimental models(2011-10-12) Qureshi, Asaf A.; Tan, Xiaoyu; Reis, Julia C.; Badr, Mostafa Z., 1950-; Papasian, Christopher J.; Morrison, David C., 1941-; Qureshi, NiloferAbstract Background Inflammation has been implicated in a variety of diseases associated with ageing, including cancer, cardiovascular, and neurologic diseases. We have recently established that the proteasome is a pivotal regulator of inflammation, which modulates the induction of inflammatory mediators such as TNF-α, IL-1, IL-6, and nitric oxide (NO) in response to a variety of stimuli. The present study was undertaken to identify non-toxic proteasome inhibitors with the expectation that these compounds could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing ageing related diseases. We evaluated the capacity of various proteasome inhibitors to suppress TNF-α, NO and gene suppression of TNF-α and iNOS mRNA, by LPS-stimulated macrophages from several sources. Further, we evaluated the mechanisms by which these agents suppress secretion of TNF-α, and NO production. Over the course of these studies, we measured the effects of various proteasome inhibitors on the RAW 264.7 cells, and peritoneal macrophages from four different strains of mice (C57BL/6, BALB/c, proteasome double subunits knockout LMP7/MECL-1-/-, and peroxisome proliferator-activated receptor-α-/- (PPAR-α-/-) knockout mice. We also directly measured the effect of these proteasome inhibitors on proteolytic activity of 20S rabbit muscle proteasomes. Results There was significant reduction of chymotrypsin-like activity of the 20S rabbit muscle proteasomes with dexamethasone (31%), mevinolin (19%), δ-tocotrienol (28%), riboflavin (34%), and quercetin (45%; P < 0.05). Moreover, quercetin, riboflavin, and δ-tocotrienol also inhibited chymotrypsin-like, trypsin-like and post-glutamase activities in RAW 264.7 whole cells. These compounds also inhibited LPS-stimulated NO production and TNF-α secretion, blocked the degradation of P-IκB protein, and decreased activation of NF-κB, in RAW 264.7 cells. All proteasome inhibitors tested also significantly inhibited NO production (30% to 60% reduction) by LPS-induced thioglycolate-elicited peritoneal macrophages derived from all four strains of mice. All five compounds also suppressed LPS-induced TNF-α secretion by macrophages from C57BL/6 and BALB/c mice. TNF-α secretion, however, was not suppressed by any of the three proteasome inhibitors tested (δ-tocotrienol, riboflavin, and quercetin) with LPS-induced macrophages from LMP7/MECL-1-/- and PPAR-α-/- knockout mice. Results of gene expression studies for TNF-α and iNOS were generally consistent with results obtained for TNF-α protein and NO production observed with four strains of mice. Conclusions Results of the current study demonstrate that δ-tocotrienol, riboflavin, and quercetin inhibit NO production by LPS-stimulated macrophages of all four strains of mice, and TNF-α secretion only by LPS-stimulated macrophages of C57BL/6 and BALB/c mice. The mechanism for this inhibition appears to be decreased proteolytic degradation of P-IκB protein by the inhibited proteasome, resulting in decreased translocation of activated NF-κB to the nucleus, and depressed transcription of gene expression of TNF-α and iNOS. Further, these naturally-occurring proteasome inhibitors tested appear to be relatively potent inhibitors of multiple proteasome subunits in inflammatory proteasomes. Consequently, these agents could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing a variety of ageing related diseases.Item Regulation of Phosphorylation of Dopamine D3 Receptors in Mouse Striatal Neurons in vivo(2010-03) Rivera, D.; Mao, Li-Min; Fibuch, Eugene; Wang, John Q.; University of Missouri (System); Missouri Life Sciences Summit (2010: University of Missouri--Kansas City)Introduction: Dopamine D3 receptors (D3Rs) are G-protein-coupled receptors. These D3Rs inhibit adenylyl cyclase and the downstream formation of cAMP. Due to their preferential expression in the mesolimbic areas, especially in the nucleus accumbens (NAc), they are known to play a major role in the mesolimbic function. Recently, we found that D3Rs are phosphorylated at serine 229 (Ser229) by Ca2+/calmodulin-dependent protein kinase II (CaMKII). This phosphorylation is subject to the modulation, and the phosphorylation level controls receptor function. In this study, an effort was made to develop a phospho- and site-specific antibody. Using this antibody, we hypothesized that the modulation of D3R phosphorylation at Ser229 by dopamine D1 receptors would occur in the mouse NAc in vivo. Methods: To detect phosphorylation of D3Rs at Ser229 in striatal neurons in vivo, we developed a phospho- and site-specific antibody. A short peptide containing phospho-Ser229 (KRILTRQNpSQCISI) was synthesized and used as an immunogen for producing a polyclonal antibody from rabbits. Upon demonstration of the selectivity of this antibody, we used it in Western blot to monitor changes in Ser229 phosphorylation in striatal neurons in response to dopamine D1 receptor stimulation by a D1 selective agonist SKF81297. Following IACUC approval, adult male mice (CL57/BL6) were randomly divided into 2 groups (n = 3 per group). The animals received an intraperitoneal injection of saline or SKF81297 (1 mg/kg). Animals were sacrificed by cervical dislocation 15 min after drug injection. The NAc was removed and homogenized. Homogenates were centrifuged (1000 g, 10 min), and the supernatant was used for Western blot. Densities of immunoblots were measured using optical scanning and the data were analyzed using Student's t-test (p < 0.05). Results: A series of control experiments validated the selectivity of the antibody against phosphorylated D3Rs at Ser229 (pD3R-Ser229). Using this antibody, we found that the level of pD3R-Ser229 in the NAc was significantly increased in mice treated with a systemic injection of the D1 receptor agonist SKF81297 (1 mg/kg, 15 min) compared to mice treated with saline. Conclusion: Using a phospho- and site-specific antibody against phospho-D3Rs at Ser229, we found that stimulation of dopamine D1 receptors increases phosphorylation of D3Rs in the mouse NAc in vivo. Discussion: Developing a phospho- and site-specific antibody is necessary for detecting changes in D3R phosphorylation in vivo. Using this newly acquired antibody, we found that Ser229 phosphorylation of D3Rs is subject to the modulation by dopamine D1 receptors. This seems to indicate a previously-unrecognized D1-dependent negative feedback control of D3R function given that Ser229 phosphorylation inhibits D3Rs1. In a subpopulation of striatal neurons that co-express D3Rs and D1 receptors, dopamine is able to concurrently enhance the D1-mediated phosphorylation of D3Rs to induce a heterologous desensitization of D3Rs after their activation. This regulation is deemed important for maintaining normal homeostasis of D3R function and could be altered to contribute to various neurological disorders.Item Amphetamine Alters Group I mGluR Expression in the Rat Striatum and Medial Prefrontal Cortex(2010-03) Shaffer, C. B.; Mao, Li-Min; Fibuch, Eugene; Wang, John Q.; University of Missouri (System); Missouri Life Sciences Summit (2010: University of Missouri--Kansas City)Introduction: Group I metabotropic glutamate receptors (mGluR1/mGluR5 subtypes) and their key scaffolding protein Homer1b/c are densely expressed in the striatum. These receptors are believed to play important roles in the regulation of psychostimulant action. The psychostimulant amphetamine increases extracellular glutamate levels, which in turn activates postsynaptic mGluR1/5 in striatal neurons. It is, however, unclear whether amphetamine has any impact on striatal mGluR1/5 expression. In this study, we hypothesized that alterations in mGluR1/5 and Homer 1b/c expression in the rat striatum and medial prefrontal cortex (mPFC) would occur in response to an acute injection of amphetamine in vivo. Methods: Following IACUC approval, adult male Wistar rats received an intraperitoneal injection of saline (n = 4) or amphetamine (5 mg/kg, n = 5). Motor responses to amphetamine were monitored continuously following drug administration. For detecting gene expression, rats were anesthetized and sacrificed 1 h after saline or amphetamine injection. Brains were removed, and the striatum, including the dorsal (caudate putamen) and ventral (nucleus accumbens) striatum, and mPFC were dissected. Synaptic proteins were extracted for Western blot analysis of changes in mGluR1, mGluR5, and Homer1b/c protein levels with specific antibodies. The density of immunoblots was measured using optical scanning. Data were statistically analyzed using Student's t-test (p<0.05). Results: A single injection of amphetamine induced a typical increase in motor activity, confirming that a behaviorally active dose of the drug was used. At this dose, amphetamine markedly reduced mGluR5 protein levels in the striatum, while increasing mGluR5 protein levels in the mPFC. Unlike mGluR5, mGluR1 protein expression in both the striatum and mPFC was not significantly altered in amphetamine-treated rats relative to saline-treated rats. Homer1b/c protein levels in the two regions also remained stable in response to amphetamine administration. Actin protein levels showed no difference between amphetamine- and saline-treated groups. Conclusion: These data identify mGluR5 as a sensitive target of amphetamine. Acute amphetamine exposure is able to alter striatal mGluR5 expression in a subtype- and region-specific manner. Discussion: Amphetamine increases glutamate release in the striatum1 which can activate mGluRs in striatal neurons to produce drug effects. Group I mGluRs have been demonstrated to undergo rapid desensitization following ligand stimulation of the receptor1. Thus, our finding of a loss of synaptic mGluR5 after amphetamine suggests a previously unrecognized mechanism for such desensitization. Of note, amphetamine has no effect on glutamate release in the mPFC1. Future studies are needed to define the role of amphetamine-stimulated mGluR5 expression in this region.Item Amphetamine Alters Acid-Sensing Ion Channel Expression in the Rat Striatum(2010-03) Suman, A.; Mao, Li-Min; Fibuch, Eugene; Wang, John Q.; University of Missouri (System); Missouri Life Sciences Summit (2010: University of Missouri--Kansas City)Introduction: The acid-sensing ion channel (ASIC) is specifically activated by a drop in the extracellular pH level. These channels are widely expressed in mammalian brains and actively modulate synaptic transmission and a variety of neuronal activities. In the striatum, two ASIC subtypes (ASIC1 and ASIC2) are densely expressed. Given the fact that the striatum is a central site for processing biological actions of drugs of abuse, expression of abundant ASICs in this structure implies a potential involvement of the channel in drug effects. In this study, we examined the expression of ASIC1 and ASIC2 in the rat striatum in response to chronic exposure to the psychostimulant amphetamine in vivo. Methods: Following IACUC approval, adult male Wistar rats (2 groups, n = 6 per group) received intraperitoneal injections of saline or amphetamine (once daily for 7 days, 1.25 mg/kg for day 1 and day 7, 4 mg/kg for days 2-6). At 14 days after the termination of drug injection, rats were sacrificed after anesthesia. Brains were removed and sliced into coronal sections (400 [mu]m). The dorsal (caudate putamen, CPu) and ventral (nucleus accumbens, NAc) striatum were dissected in artificial cerebrospinal fluid. A membrane-impermeable cross-linking reagent bis(sulfosuccinimidyl)suberate (BS3) was added. BS3 only cross-links ASICs on the surface of live cells to form high-molecular weight aggregates which can be readily separated from normal intracellular monomer ASIC proteins. Densities of immunoblots were measured using optical scanning and the data were analyzed (t-test (p < 0.05)). Results: BS3-treated striatal tissue showed a high-molecular weight band of ASIC1 and ASIC2 (surface channels) and a monomeric molecular weight band of ASIC1 and ASIC2 (intracellular channels). Quantification analysis revealed that 70-80% of ASIC1 and ASIC2 are expressed in the surface membrane of normal striatal neurons. Chronic amphetamine administration induced parallel increases in ASIC1 protein levels in both surface and intracellular pools in the CPu at a 14-day withdrawal period. Similar results were also observed in the NAc. In contrast to ASIC1, ASIC2 and [alpha]-actinin in their protein levels remained unchanged in the CPu and NAc of amphetamine-treated rats. Conclusion: These data identified the central ASIC as a sensitive target to repeated stimulant exposure. Discussion: Various synaptic proteins have been screened for their responses to repeated drug exposure. Plastic changes in the expression and function of all responsive proteins are thought to operate in concert to control drug effects. In this study, a new responsive gene is identified. Following repeated amphetamine administration, ASIC expression was regulated in striatal regions. This identifies the channel as an important element of molecular adaptations to drug exposure. Indeed, ASICs have been implicated in various mental disorders1. This study represents an initial effort toward elucidating the precise role of ASICs in processing the addictive action of drugs of abuse.