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Item A Study of Some Aspects of the Role of Mast Cells in Experimental Autoimmune Uveitis(1994-06-01) Lee, Carol Hamberlin; Edward Orr; Robert Gracy; Laura S. LangLee, Carol Hamberlin, A Study of Some Aspects of the Role of Mast Cells in Experimental Autoimmune Uveitis. Doctor of Philosophy (Biomedical Sciences), June 1994, 141 pp., 6 tables, 29 illustrations, bibliography, 115 titles. Choroidal mast cells have been implicated in experimental autoimmune uveitis (EAU), an ocular inflammatory disease induced by S-antigen (Sag). Activation of ocular mast cells in Lewis rats was evaluated by determining changes in numbers of mast cells, levels of histamine, and wet weights of ocular tissues. A decrease in choroidal mast cells was confirmed statistically, and limbal mast cells were found to be activated earlier than choroidal mast cells. The ocular histamine distribution was altered during EAU, decreasing in the anterior eye, and increasing in the posterior eye. Retinal histamine levels increased when EAU symptoms occurred, but decreased while the disease was still intense. Levels of histamine methyltransferase, which degrades histamine, increased significiantly in retinal tissue when histamine levels fell. Signficant weight increases indicated edema, which can result from mast cell mediator action. Leflunomide, an immunomodulating drug that is known to affect mast cells in vitro, prevented induction of EAU. Leflunomide also suppressed changes in the mast cell-related parameters, histamine levels and wet weights. Mechanisms for activation of ocular mast cells in EAU were investigated. Results suggest that mast cell activation does not occur through mast cell surface IgE-antigen crosslinking. The adjuvant used, complete Freund’s adjuvant, is not conducive to IgE production. Histamine releasing factors, HRFs, are produced by various immune system cellular components. Preliminary efforts did not demonstrate HRF activity. Mast cell numbers, histamine levels, and wet weights were also evaluated in a milder form of EAU induced by M-peptide (Mpep), a peptide fragment of Sag. Mpep/EAU produces few disease symptoms in the anterior eye, but destroys the same retinal area as Sag/EAU—photoreceptor cells and their outer segments. Inflammation is less intense, restricted primarily to the target area. Mast cell numbers did not change, but histamine levels and wet weights changed significantly, suggesting that mast cells are also involved in Mpep/EAU. Overall, the results of this study add to evidence that mast cells are involved in pathogenesis of EAU. The results also point to topics of further investigation into the role of mast cells in EAU and in normal function in ocular tissues.Item Cross-Tolerance Between the Discriminative Stimulus Properties of Ethanol, Diazepam and Pentobarbital(1995-12-01) Lytle, Douglas A.; Michael Forster; Glenn Dillon; Thomas YorioLytle, Douglas A., Cross-Tolerance Between the Discriminative Stimulus Properties of Ethanol, Diazepam and Pentobarbital. Doctor of Philosophy (Biomedical Sciences), December, 1995, 132 pp., 8 tables, 19 figures, bibliography, 176 titles. Ethanol, benzodiazepine agonists and barbiturates all facilitate GABA-mediated CT flux. The present experiments tested the hypothesis that, because these agents share this common action, tolerance to discriminative stimulus properties of one of these drugs would result in cross-tolerance to the others. Rats were trained to detect either ethanol (EtOH; 1.0 g/kg), the benzodiazepine diazepam, (DZP; 5.6 mg/kg), or the barbiturate pentobarbital (PB; 10.0 mg/kg) from vehicle using a two-lever choice procedure where food was available under a fixed-ration ten schedule of reinforcement. Subsequently, dose-effect curves for EtOH (0.1-1.78 g/kg), DZP (0.56-17.8 mg/kg), or PB (1.0-17.8 mg/kg) were tested before and after chronic administration of EtOH 96.0 g/kg/12hrs for seven days), DZP (20.0 mg/kg/8hrs for seven days), or PB (32.0 mg/kg/8hrs for seven days). The chronic administration of EtOH conferred tolerance to itself in all cases and cross-tolerance to DZP and PB in subjects trained to detect EtOH, but did not confer cross-tolerance to these agents in their respective discriminations. The chronic administration of DZP conferred tolerance to itself substituting for DZP. Although tolerance developed to DZP substituting for PB after treating animals with chronic DZP, this regimen on DZP did not confer tolerance to itself substituting for EtOH. This regimen of DZP failed to confer significant cross-tolerance to either EtOH or PB under any conditions. The chronic administration of PB conferred tolerance to itself substituting for PB. Although tolerance developed to PB substituting for DZP after treating animals with chronic PB, this regimen of PB did not confer tolerance to itself substituting for EtOH. This regimen of PB failed to confer significant cross-tolerance to either EtOH or DZP under any conditions. In summary, EtOH was found to confer cross-tolerance to DZP and PB only in animals trained to detect EtOH. The chronic administrations of DZP and PB failed to confer tolerance to themselves substituting for EtOH. These results are parsimonious with the heterogeneous nature of the GABA receptor. Finally, tolerance to either DZP or PB does not result in cross-tolerance to the discriminative stimulus properties of the other drug. These results suggest that the mechanisms mediating tolerance to BZs and barbiturates are not linked.Item Endothelin-1-Induced Signaling Involved in Extracellular Matrix Remodeling(2006-12-01) He, Shaoqing; Thomas Yorio; Neeraj Agarwal; Peter KoulenET-1-Induced Signaling in ECM Remodeling in Astrocytes. Shaoqing He, Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107. ET-1 levels are elevated under pathophysiological conditions, including glaucoma, however, ET-1’s ocular functions are not fully documented. Therefore, ET-1-induced signaling and ECM remodeling in astrocytes and at the optic nerve head were determined in this study. Three signaling pathways, including ERK1/2, PKC, and P13 kinase, were involved in ET-1-medicated cell proliferation of U373MG astrocytoma cells. Blocking one of these pathways completely abolished cell proliferation. It appeared that ERK1/2 activation was involved, but was independent of PKC and P13 kinase activation by ET-1. It was also determined that the ETB receptor was the dominant receptor involved in ERK1/2 phosphorylation and cell proliferation. In addition, ERK1/2 phosphorylation was not transactivated by the EGF receptor by ET-1. The studies also indicated that there was no activation of c/nPKC, although PKC was involved in cell proliferation. In U373MG astrocytoma cells, MAPK-ERK, PKC and P13K pathways appear to exert their roles in parallel without a direct, apparent “cross-talk”. Based on the signaling pathways obtained from U373MG astrocytoma cells, the regulation of MMPs/TIMPs and fibronectin in ET-1-activated human optic nerve head astroctyes (hONAs) was also determined. ET-1 not only induced rapid phosphorylation of ERK1/2 and PKC βI/ βII/δ but also increased the activity of MMP-2 and the expression of TIMP=1 and 2. The activity of MMP-2 was enhanced in the presence of inhibitors of MAPK or PKC in hONAs, whereas the expression of TIMP-1 and 2 was abolished. ET-1 increased the soluble fibronectin (FN) expression as well as FN matrix formation, however, the expression and deposition of FN were MAPK- and PKC-independent, whereas expression and activity of MMps and TIMPs were MAPK- and PKC-dependent. Therefore, ET-1 shifted the balance of MMPs/TIMPs and substrates that altered the ECM composition and subsequently let to ECM remodeling in activated hONA cells. ET-1’s effects on ECM remodeling at the optic nerve head were also examined following intravitreal administration of ET-1 in rats. The increased expression of MMP-9 and collagen VI was detected in both ETB deficient rats and wildtype Wistar rats post ET-1 intravitreal injection for 2 and 14 days, whereas the deposition of FN and collagen IV was unchanged. There was no significant difference in staining of MMP-9 and collagen VI between ETB deficient rats and wildtype Wistar rats. In this study, ECM remodeling was demonstrated in rats injected with ET-1 into the vitreous. Such changes in the ECM seen in the current study provide additional insight into the mechanisms that might explain the glaucomatous changes observed in ET-1-injection or perfusion models. In summary, ET-1 not only activated several signaling pathways in cell proliferation of astrocytes, but also modulated the expression of ECM molecules in vitro and in vivo, indicating that ET-1 plays a regulatory role in ECM remodeling. These effects coupled with observations that ET-1 levels are elevated in glaucoma patients, suggests that ET-1 may be involved in glaucomatous optic neuropathy.Item Intermittent Hypoxia Training to Foster Brain Recovery after Ischemic Stroke in rats(2018-05) Ruelas, Steven S.; Mallet, Robert T.; Jung, Marianna E.; Schreihofer, Ann M.; Das, Hriday K.Purpose: Ischemic stroke is the leading cause of disability and #5 cause of death in the US. Annually, nearly 800,000 Americans suffer an ischemic stroke, and 130,000 die. The only FDA approved treatment for stroke is recombinant tissue plasminogen activator, but this thrombolytic agent neither protects the affected tissue, nor mitigates the motor or cognitive impairments resulting from stroke. Intermittent hypoxia training (IHT) has been shown to increase cerebral blood flow, reduce oxidative stress, mobilize cerebroprotective signaling cascades and minimize behavioral deficits in a rat model of Alzheimer's Disease. Moreover, a 20 d IHT program attenuated behavioral deficits and protected neurons in ethanol-withdrawn (EW) rats, even when EW began 35 d after IHT. Therefore, we hypothesize that IHT, initiated in rats after stroke, preserves motor and cognitive function, relative to non-IHT rats. Methods: Ischemic stroke will be produced in rats by 90 min occlusion and abrupt reperfusion of the middle cerebral artery (MCA). Motor function and coordination will be evaluated by the rotarod test before and at 1 week intervals after MCA occlusion (MCAO). Rats must balance on a rotating cylinder that accelerates at a constant speed. High fall latency represents intact motor function. The Morris Water Maze (MWM) assesses spatial learning and memory. Rats are placed in an open, circular pool and must find a sunken platform within 90 s. 24 h after stroke, rats undergoing IHT will breathe moderately hypoxic gas (10% O2) for 5-8 cycles, each lasting 5-10 min, with intervening 4 min room air breathing, for 20 consecutive days. These rats will be compared to an MCAO group continuously exposed to 21% O2. At 21 d post-stroke, the brain will be harvested for analyses of infarct and neuroprotective proteins. Results: In pre-stroke testing, the time taken to solve the MWM fell progressively over 10 days, indicating spatial learning and memory, and fall latency on the rotarod lengthened over 5 days, reflecting improved coordination and possibly a training effect. These studies have established the pre-stroke baselines for assessment of IHT's impact on post-stroke recovery. Conclusions: We expect that IHT given after stroke will minimize motor and cognitive impairment by activating neuroprotective signaling cascades culminating in expression of anti-oxidant and anti-inflammatory proteins.Item Investigation of Proteasome Chymotryptic Activities and Effects on their Inhibition in Rat and Human Natural Killer Cells(2003-04-01) Lu, Min; Goldfarb, Ronald H.; Borejdo, Julian; Easom, RichardLu, Min, Investigation of Proteasome Chymotryptic Activites and Effects of Their Inhibition in Rat and Human Natural Killer Cells. Doctor of Philosophy (Biochemistry and Molecular Biology), April, 2003, 185 pp., 3 tables, 32 illustrations, bibliography, 158 titles. The proteasome is a multicatalytic proteinase complex that is involved in the major extralysosomal pathway responsible for intracellular protein degradation in mammalian cells. This dissertation focuses on investigating proteasome chymotryptic activities and the effects of selective inhibitors of these activities on the function of natural killer (NK) cells. In this dissertation, 20S proteasomes derived from rat RNK16 cells were purified and some of their biochemical and biophysical properties were investigated extensively. The results indicated that RNK16 cell-derived proteasome differ from the proteasome of other origins in many aspects including substrate selectivity, inhibitor specificity, and kinetic regulation, although they may share some common biochemical properties with others. To investigate the effects of proteasomal inhibition on the function of NK cells, several proteasome inhibitors were used including MG115, MG132, clasto-lactacystin-β-lactone, EGCG and LLnL. MG115 and MG 132 were shown to induce apoptosis of RNK16 cells, as evidenced by DNA fragmentation, caspase-3 activation and the appearance of sub-G1 cell populations. Activation of multiple caspases and increased expression of cell surface Fas (CD95) protein were also observed following the treatment of RNK16 cells by these two inhibitors. This dissertation also tested the hypothesis that different cell types could respond differentially to proteasome inhibitors. The effects of several proteasome inhibitors were determined on the purified 20S proteasomal and 26S proteasomal chymotrypsin-like activity in whole cell extracts and intact YT and Jurkat cells, human NK and T cell lines respectively. Following such treatment, caspase-3 activation occurred much earlier in Jurkat cells than YT cells; cell cycle analysis indicated a sub-G1 apoptotic cell population in Jurkat cells and G2/M arrest in YT cells. In addition, accumulation of p27 and IκB-α was detected only in Jurkat cells, but not YT cells. Therefore, proteasome inhibitors appear to act differentially in cell cycle progression and apoptosis signaling pathways between human NK and T cells. These studies indicate that the generation of ideal proteasome inhibitors for the treatment of malignancies could be screened or designed to specifically induce cancer cells to undergo programmed cell death, while having little or no apoptosis-inducing abilities for natural killer cells and other cells of the immune response, thus enhancing the selectivity and specificity of the anti-cancer, apoptosis-inducing capabilities of proteasome inhibitors.Item Pharmacological Evaluation of the D2 and D3 Dopamine Receptor Selective Compounds on L-Dopa Dependent Abnormal Involuntary Movements in Rats(2008-05-01) Kumar, Rakesh; Robert Luedtke; Hriday K. Das; Nathalie SumienKumar Rakesh, Pharmacological Evaluation of the D2 and D3 Dopamine Receptor Selective Compounds on L-dopa Dependent Abnormal Involuntary Movements in Rats. Master of Science (Pharmacology & Neuroscience), May 2008, 106 pp, 21 illustrations, references, 31 titles. Parkinson’s Disease (PD) is a progressive, neurodegenerative disease of the dopamine neurons that innervate the striatum and is characterized by resting tremor, rigidity, bardykinesia and postural instability. L-dopa treatment is the most common and effective therapy for PD. However, both motor (wear-off phenomena, rigidity and dyskinesia) and non-motor (sweating, tachycardia, restless leg syndrome, anxiety, depression, confusion, reduced alertness, psychosis and/or dementia) side effects are associated with long term L-dopa therapy. Motor complications depend on the duration of L-dopa treatment and the abnormal involuntary movements are known as L-dopa-induced dyskinesia (LID). Several studies have suggested a possible role of the dopamine D3 receptor subtype in LID. Here I evaluated the effects of various D2 and D3 dopamine receptor selective compounds on LID in 6-hydroxydopamine-induced complete lesioned hemi-parkinsonian model of rat. D3 dopamine receptor selective compounds (agonists, partial agonists or antagonists) have antidyskinetic effects on LID. Co-administration of D3 dopamine receptor agonist and D3 dopamine receptor antagonist has additive effects in attenuating the LID instead of antagonizing the effects of each other in vivo. D3 dopamine receptor selective compounds attenuated apomorphine-induced dyskinesia.Item Regulation and Characterization of Cardiac Phosphoinositide-Specific Phospholipase C (PLC) Isoenzymes(1997-12-01) Wang, Juan; Eugene E. Quist; Thomas Yorio; Ming-Chi WuWang, Juan, Regulation and Characterization of Cardiac Phosphoinositide-Specific Phospholipase C Isoenzymes. Master of Biomedical Science, Dec., 1997, 79 pp., 20 illustration, bibliography, 62 titles. It is hypothesized that myocardial phosphoinositide-specific phospholipase C (PLC) isoenzymes are regulated by physiological intracellular Ca2+ and by cytosol-membrane translocation. The regulation and identification of PLC isoenzymes in rat and dog ventricular subcellular fractions were studied. PLC-β1, PLC-β3 and PLC-δ1 were identified in rat and dog cytosol and microsomal membranes by chromatographic separation, enzyme assays and western blotting. Truncated PLC-β isoforms with molecular weights of 69 kDa and 114 kDa were isolated from rat and dog cytosol, respectively. Species differences in the relative distribution of PLC isoenzymes were evident as PLC-δ dominant in rat whereas PLC-β isoenzymes were dominant in dog. A 91 kDa cytosolic protein which did not contain PLC activity alone markedly led to PLC activation when combined with microsomes. The activator protein was immunoprecipitated with an anti-PLC-δ identifying this activator as an inactive PLC-δ isoenzyme. These studies indicate that cytosolic PLC-δ may be activated by translocating to membranes. In addition, proteolysis may be involved in long term activation of cytosolic PLC isoenzymes. Further studies will be required to resolve the physiological significance of these modes of cardiac PLC activation.Item Store-Operated Calcium Entry in Glomerular Mesangial Cells(2008-04-01) Sours-Brothers, Sherry; Rong Ma; Joan Carroll; Glenn DillonSours-Brothers, Sherry. Store-operated Calcium Entry in Glomerular Mesangial Cells. Doctor of Philosophy (Integrative Physiology, Molecular Cardiovascular Science Track), April, 2008, 181 pp; 1 table, 25 figures, bibliography, 241 titles. Mesangial cells (MCs) are found within the glomerulus, where they contribute to the regulation of glomerular filtration (GFR). Their contractile function is similar to that of vascular smooth muscle cells, regulated by a number of different Ca2+ release and entry mechanisms in response to vasoactive substances. Among these are store-operated channels (SOC), which have been identified in MC, but whose molecular components are unknown. Deficiency of store-operated Ca2+ entry (SOCE) has also been associated with loss of MC contractile function found during early diabetic renal hyperfiltration. For these reasons, it is imperative to clarify the mechanisms underlying SOCE in MCs. Members of the canonical transient receptor potential (TRCP) family of proteins have been identified as candidates for SOC function in a number of cell types. The distribution of TRPC subtypes, and their combination to form heterotetrameric channels is cell-type specific, possibly allowing for variation SOCE mechanisms in different cells. Recently, the endoplasmic reticulum (ER) resident protein stromal interaction molecule 1 (STIM1) has been identified as a regulator of SOCs, including TRPCs. With this in mind, the following studies were carried out to identify the distribution and function of TRPC proteins in MCs, including their role in the mediation of MC contractile function and potential regulation by STIm1. In the first study, TRPC1, -3, -6, and -7 were identified in cultured human MMCs as well as rat and human kidney sections. TRPC1 was found to associate with TRPC4 and TRPC6 by co-immunoprecipitation and colocalization by immunocytochemistry. Overexpression of TRPC1 by transient transfection increased, while knockdown of TRPC1 expression by RNAi decreased thapsigargin-mediated SOCE. These results indicate a role for TRPC1 in SOCE in MCs. In the second study, the contribution of TRPC1-mediated SOCE to Ang II-stimulated MC contractile function was examined. Ang II-mediated SOCE was attenuated by TRPC1-RNAi or by treatment with a TRPC1 antibody known to block channel activity. TRPC1-RNAi and antibody blockade also inhibited Ang II-stimulated single channel activity as measured by cell-attached patch clamp, while TRPC1-RNAi attenuated Ang II-mediated MC contraction. This effect was also examined in vivo in rats. Infusion of TRPC1 antibody blocked Ang II-induced decline in GFR. In the final study, the formation of SOC by TRPC heteromultimerization was assessed. Both TRPC1 and TRPC4 were found to contribute to TG-stimulated SOCE and single-channel activity in cultured MCs. The interaction between these two subtypes increased upon store-depletion with TG, while translocation of TRPC1 but not TRPC4 to the plasma membrane was induced by TG. STIM1 was also found to contribute to regulation of SOC, but co-immunoprecipitation demonstrated an interaction with TRPC1 but not TRPC4. These data suggest that SOC activity is mediated by interaction between TRPC1 and TRPC4, and translocation of TRPC1 to the plasma membrane may be responsible for increasing channel activity upon store depletion. STIM1 may play a regulatory role by activity channel complexes via TRPC4. Taken together these studies indicate an important role for TRPC function in MCs. Not only do these studies further understanding of SOC function in MCs specifically, they also contribute to the delineation of TRPC channel activity, complex formation, and regulation by STIM1. Futures studies are needed to further examine TRPC activation mechanisms and their potential role in other physiological and pathophysiological MC functions.Item The Influence of CNS stimulants, opioid antagonists and an NMDA antagonist on the reinforcing effect of cocaine using a progressive-ratio schedule(1996-07-01) Li, Donghang; Forster, Michael J.; Martin, Michael; Luedtke, Robert R.It has been hypothesized that there is a common dopaminergic pathway mediating the reward properties of abused drugs, and that dopamine is involved in tolerance to the reinforcing effect of cocaine. The progressive-ratio (PR) schedule can be used to test both potentiation and reduction of the reinforcing effects of cocaine by other factors. Under the PR schedule, an increasing number of responses is required to obtain each subsequent cocaine injection, and failure to complete the required number of responses within 1 h of the previous cocaine injection terminates the session. The number of total reinforcers obtained during a session is defined as “the breaking point” and was used as the primary dependent measure. Fisher F344 male rats acquired the self-administration task under the PR schedule within forty sessions and showed a stable daily acquisition baseline. The breaking point and inter-reinforcer time (ISRT) were positively correlated within each ratio. A motor-incapacitating side effect of a pretreatment can be determined by a change in the relationship between the ISRT and the breaking point. d-Amphetamine pretreatment (0.32-3.2 mg/kg, i.p., 30 min) potentiates the reinforcing effect of cocaine as demonstrated by a higher breaking point of self-administration without changing the ISRT. Morphine pretreatment (0.32-3.2 mg/kg, i.p., 30 min failed to change the breaking point of cocaine self-administration but it did increase the ISRT. These results support an additive reinforcing effect for amphetamines and cocaine, but do not support an additive reinforcing effect of morphine and cocaine. The reinforcing effect of cocaine was reduced by pretreatment with ketamine (0.032-0.32 mg/kg, i.p., 20 min) as indicated by a reduction in the breaking point. In a concurrent experiment, animals were trained to self-administer cocaine under a fixed ratio 2 schedule (FR2). Ketamine pretreatment did not modify the ISRT in FR2 trained animals except at the highest dose (0.32 mg/kg, i.p., 20 min), where significant motor incoordination was observed. Both chronic treatment with cocaine (20 mg/kg/ 8hr x 7 days, iv) or amphetamine (3.2 mg/kg /12 hr x 7 days, i.p.) resulted in a reduction in breaking point at any given dose, providing direct evidence of tolerance and cross-tolerance to the reinforcing effects of cocaine. Chronic treatment with ketamine (0.32 mg/kg/8hr x 7 days, i.v.) failed to modify either the breaking point under a PR schedule of reinforcement or the ISRT under a FR2 schedule of reinforcement. Co-administration of ketamine (0.32 mg/kg/8hr x 7 days, i.v.) with chronic cocaine (20 mg/kg/8hr x 7days, i.v.) failed to prevent tolerance to the reinforcing effect of cocaine as indicated by either the breaking point under a PR schedule of reinforcement of the ISRT under an FR2 schedule of reinforcement. These data indicate that the breaking point in the PR schedule is more sensitive to changes in the dopamine reward system, whereas changes in rate of response are not consistently related to the changes in the dopamine reward system. These data support the use of PR schedule as a better method than FR schedule for determining reward properties of drugs of abuse with fewer complications due to the central nervous system inhibitory effects of some drugs of abuse.Item The Involvement of D1 and D2 Dopamine Receptors in Cocaine Self-Administration(1996-06-01) Peltier, Rachel; Michael Forster; Patricia A. Gwirtz; Thomas YorioPeltier, Rachel L., The Involvement of D1 and D2 Dopamine Receptors in Cocaine Self-Administration. Doctor of Philosophy (Biomedical Sciences), June 1996, 195 pp. introduction, 6 chapters, discussion, bibliography, 91 titles. D1 and D2 dopamine receptor subtypes have been implicated in producing the reinforcing properties of cocaine. Chronic exposure to cocaine produces tolerance to its reinforcing effects in rats trained to self-administer cocaine. The time between cocaine reinforcers (ISRT) is directly related to dose. A three-point dose-response curve (0.125, 0.25 and 0.5 mg/inj) for cocaine self-administration is obtained during a single test session, allowing determination of optimal tolerance effects of cocaine (20 mg/kg/8 hr/7 days; IP) as demonstrated by a shift of the curve to the right. To test if pharmacokinetic factors contribute to the development of tolerance to the reinforcing properties of cocaine (20 mg/kg/8hr/7days; IP), cocaine and benzoylecgonine (metabolite) were measured in the plasma and brains of rats given a challenge injection of cocaine (2.0 mg/kg; I.V.). Chronic cocaine did not reduce the concentration of cocaine must be due to pharmacodynamics changes. Acute pretreatment with either the direct dopamine agonists d-amphetamine (0.32-3.2 mg/kg) or methamphetamine (1.0 mg/kg) did not consistently change cocaine self-administration. Chronic high-dose treatment with d-amphetamine and methamphetamine produced cross-tolerance to the reinforcing effects of cocaine but apomorphine (0.32-3.2 mg/kg) did not. In contrast, acute pretreatment with dopamine antagonists; flupentixol (mixed D1 and D2, 0.032-1.0 mg/kg), SCH23390 (specific D1, 0.0032-0.32 mg/kg), or eticlopride (specific D2, 0.0032 -3.2 mg/kg); dose-dependently decreased the reinforcing effects of cocaine (ISRT). Chronic treatment with mixed of D1 antagonists (flupentixol, 3.2 mg/kg/12 hr/5 days; or SCH23390, 0.25 mg/kg/12 hr/7 days) produced sensitization to the reinforcing effects of cocaine, but the D2 antagonist eticlopride (0.25 mg/kg/12 hr/7 days) produced cross-tolerance to the reinforcing effects of cocaine. In summary, both the D1 and D2 receptor subtypes seem to be involved in the acute effects of cocaine; however, the development of tolerance to cocaine appears to involve only the D1 receptor subtype.Item The Role of a Membrane Androgen Receptron in the Brain(2007-02-01) Gatson, John Wayne; Simpkins, James; Koulen, Peter; Basu, AlakanandaGatson, Joshua Wayne, The Role of a Membrane Androgen Receptor in the Brain. Doctor of Philosophy (Biomedical Sciences), February 2007, pp187, 34 illustrations. In the brain, depending on the insult type, androgens have been shown to protect from or exacerbate the levels of cell death. This discrepancy is partly due to the array of receptors that androgens may activate during injury. For example, activation of intracellular androgen receptors (AR) leads to the activation of pro-survival pathways and protects from various toxins such beta-amyloid. In contrast, previous studies have demonstrated that testosterone causes an increase in lesion size following stroke. The damaging effects of androgens in the brain may be mediated by a membrane-associated AR (mAR), since activation of mAR in peripheral tissue results in a decrease in cell growth and an increase in apoptotic cell death during serum deprivation. Here, I hypothesize that activation of a mAR in cortical astrocytes, suppresses the ERK and Akt signaling pathways and increases cell death in the presence of a metabolic and oxidative stressor. In this study, we found that glia express both isoforms of the AR (AR-B and AR-A) and that dihydrotestosterone (DHT) elicits ERK and Akt phosphorylation in rat glioma (C6) cells. The effect of DHT on the activation of these signaling pathways is AR dependent, since flutamide blocked this effect. In contrast to the intracellular receptor, we concluded that DHT-BSA (membrane impermeant form of DHT) binds to DHT displaceable sites on the plasma membrane. Also, treatment with DHT-BSA in the C6 cells resulted in a significant decrease in phosphor-ERK and Akt levels, suggesting the existence of two different pathways through which DHT can influence the activity of these signaling pathways. With respect to cell survival, the C6 cells and primary cortical astrocytes were treated with the metabolic and oxidative insult, iodoacetic acid (IAA), in the presence or absence of DHT, DHT-BSA, or estradiol. Following treatment, DHT and estradiol protected the glia from IAA-induced toxicity, whereas DHT-BSA caused a significant increase in cell death in the presence of a sublethal concentration of IAA. These results indicate that activation of the intracellular pathway is protective and activation of a membrane pathway is damage-inducing during injury, further supporting our results from the ERK and Akt signaling studies. To further characterize this mAR in the brain, we decided to look for indices of apoptosis such as caspase activation and TUNEL staining. It was found that DHT-BSA treatment in the presence of IAA, resulted in an increase in caspase-3/7 activation and increased TUNEL staining. In addition, PKC-delta mediated DHT-BSA-induced cell death, since antagonism of PKC-delta with rottlerin afforded protection. In conclusion, we have partially characterized a novel mAR in astrocytes during injury. Here, the damaging effects of androgens, at least in astrocytes, may in fact be mediated by a mAR, which may be a therapeutic target stroke or reperfusion injury.