Abstract
Studies in part 1 of this dissertation were intended to develop selective inhibitors of (aiBAR and ajo-AR, respectively) subtype expression using antisense oligomer approaches. Using the nucleotide sequence of the (Xib-AR and ccid-AR subtypes, antisense oligomers were designed and tested for their effectiveness at inhibiting cti-AR subtype expression in vitro. For these studies two cell culture models were used: 1) DDTi-MF2 cells expressing aiB-AR’s were used to test the potency and maximum effects of aie-AR antisense oligomers and 2) transfected HEK293 cells expressing aio-AR’s were used to test a^-AR antisense oligomers. Separate oligomers containing mismatches and/or scrambled sequences relative to the antisense constructs served as controls and were always included in parallel experiments to test for potential nonspecific effects by the antisense oligomers. Cells incubated with media alone served as additional controls. The results of the in vitro experiments in DDTi-MF2 cells and HEK293 cells are summarized below.|DDTi-MF2 cells were incubated with an 18-mer phosphorothioate antisense oligomer designed to target the translation start-site of the aie-AR mRNA. Following incubation, I measured am-AR density, aie-AR mRNA concentration and aie-AR-mediated accumulation of total [JH]-inositol phosphates and compared the results to those of control oligomer and mediaalone treated cells. [JFI]-Prazosin saturation binding experiments indicated that the antisense oligomer reduced cxib-AR density in a time- and concentration-dependent manner in cell membranes relative to control oligomer and media-alone treated cells. Competitive RT-PCR experiments showed that antisense oligomer incubation decreased a^-AR mRNA concentration relative to media-alone and control oligomer treatments. Incubation of DDT[-MF2 cells with the antisense oligomer also inhibited a.iB-AR-stimulated accumulation of total [3H]-inositol phosphates vs. that of media-alone and control oligomer cells, whereas responses to histamine stimulation were unaffected. I found no differences in a^-AR density, am-AR mRNA concentration or aie-AR-mediated accumulation of total [JH]-inositol phosphates between cells incubated with Control oligomer and media-alone. Finally, the uptake of the antisense oligomer into DDTi-MF2 cells was shown by fluorescent microscopy studies using a fluorescently labeled form of the oligomer.|Additional studies in DDTi-MF2 cells were performed to investigate whether oligomer backbone modification (phosphorothioate vs. morpholino) altered the potency of the antisense oligomer sequence characterized above. I measured ccib-AR density in [3H]-prazosin saturation binding experiments using membranes from cells treated with varied concentrations of either phosphorothioate or morpholino antisense oligomers, or their respective control oligomers or media-alone. These experiments indicated that under their optimal treatment protocols, the morpholino oligomer was 10-fold more potent than the phosphorothioate oligomer in reducing aie-AR density, but both produced an identical maximum response. Studies of ais-ARmediated accumulation of total [JH]-inositol phosphates also showed that under their optimal treatment protocols, the morpholino antisense oligomer was a 10-fold more potent inhibitor of ccib-AR function than its phosphorothioate counterpart. Again, both oligomers produced identical maximal responses. In this study, there were no differences in cxib-AR density and/or aiB-AR-mediated accumulation of total [3H]-inositol phosphates between cells incubated with control oligomers and media-alone.|Five sites of the rat aio-AR mRNA were selected with the Genetics Computer Group (GCG, Inc. Madison, WI) computer program as potential targets for oligomer hybridization. 18-mer phosphorothioate oligomers were synthesized to complement each of these sites and tested for their ability to reduce am-AR density in HEK293 cells. Using [JH]-prazosin binding experiments, I found that only the oligomer directed immediately at the translation start-site significantly reduced aio-AR density compared to media-alone treatment. Additional binding studies with this oligomer and a random-sequence control oligomer revealed that the time-course of inhibition in am-AR density was consistent with an antisense mechanism of action. Comparative RT-PCR experiments showed that the antisense oligomer reduced the steady-state aio-AR mRNA concentration without affecting the integrity of p-actin mRNA. Incubation of HEK293 cells with the ccid-AR antisense oligomer also inhibited norepinephrine-stimulated accumulation of total [JH]-inositol phosphates relative to control oligomer treated cells. Experiments were also performed in vivo to determine the effectiveness of the phosphorothioate and morpholino am-AR antisense oligomers following systemic administration in Sprague-Dawly rats. Animals treated with control oligomers and saline served as controls. Following administration, cxib-AR density in liver and kidney was measured by saturation and competition binding experiments, respectively, and the effect of antisense oligomer on spleen function was determined by contractility experiments. Saturation binding experiments with [3H]-prazosin showed that the morpholino antisense oligomer, and to a lesser extent the phosphorothioate antisense oligomer, reduced am-AR density in liver membranes of rats treated with either oligomer relative to control oligomer and saline treated animals. The morpholino oligomer was also twice as potent as the phosphorothioate oligomer. No effect on liver am-AR density was noted between control oligomer and saline-treated animals. Neither antisense oligomer was effective at inhibiting am-AR expression in kidney (as measured in competition-binding experiments with LY-757464-see Table 1 below) or am-AR function in spleen (as measured by ai-AR agonist mediated contraction).|In part 2 of this dissertation, experiments were designed to study the effects that traditional second messenger activation (increases in intracellular calcium and activation of protein kinase C) by ai-AR’s have on stimulation of mitogen activated protein (MAP) kinase pathways. Using CHO-K1 cells, we investigated the coupling of mouse ccia-AR’s to ERK1/2 and JNK pathway activation. characterization of the expressed a t-AR in CHO-K1 cells by competition binding experiments with [3H]-prazosin, 5-methyl-urapidil and BMY-7378 (see Table 1 below) revealed a receptor binding affinity typical of the cxia-AR. Experiments with the fluorescent calcium indicator fura-2 showed that the ai-AR agonist norepinephrine increased intracellular calcium levels in ciia-AR transfected CHO-K1 cells but not in untransfected CHOK1 cells. In transfected cells, norepinephrine stimulated the phosphorylation of ERK1/2 and JNK pathways in a time- and concentration-dependent fashion, as measured by western blotting. Blockade of MEK1/2 (required for ERK1/2 activation) prevented the phosphorylation of ERK1/2 by norepinephrine. Fura-2 experiments showed that the intracellular calcium chelator BAPTA blocked the norepinephrine-stimulated rise in intracellular calcium and western blotting demonstrated that the phosphorylation of ERK1/2 and JNK by norepinephrine was also abolished. Additional experiments showed that inhibition of protein kinase C abolished the norepinephrine-stimulated phosphorylation of both MAP kinases, whereas phorbol-ester-induced phosphorylation of ERK1/2 and JNK was blocked by protein kinase C inhibition.|Additional experiments in part 2 of this dissertation include studies of ERK1/2 activation by norepinephrine in freshly isolated liver slices. In these studies, I showed that norepinephrine (in the presence of a.2-AR and (3i-AR blockers) caused a time-dependent increase in the phosphorylation of the ERK1/2 MAP kinase. The stimulation of ERK1/2 MAP kinase phosphorylation by norepinephrine was partially blocked with 100 nM prazosin, whereas it was nearly abolished by L-757464 at concentrations shown to block only a^-AR’s in rat liver homogenates (Table 1).|Since MAP kinase activation has been shown to influence cellular growth and differentiation, a series of studies were designed to investigate the effect of cxia-AR stimulation on cellular morphology of transfected CHO-K1 cells. When stimulated with norepinephrine for 48 h, I found that a iA-AR transfected CHO-K1 cells changed in morphology from a stellate to a fibroblastic phenotype, characteristic of cell spreading. This morphology change was time- and concentration dependent, and did not occur in norepinephrine-stimulated untransfected CHO-K1 cells. Incubation with 100 nM prazosin prevented the norepinephrine-mediated morphology change and retarded growth of otiA-AR transfected CHO-K1 cells. In an attempt to investigate the mechanism of a iA-AR stimulated cell spreading, I incubated cells with the phorbol ester PMA (in order to down-regulate the activity of PKC) and/or PD98059 (an inhibitor of the ERK1/2 MAP kinase pathway). The effect of norepinephrine on cellular morphology was unaffected following prolonged PMA incubation, whereas incubation with PD98059 prevented the effect of norepinephrine on cellular morphology. In order to investigate the effect of a )A-AR stimulation on activation of FAK (a tyrosine kinase that has been reported to alter cell morphology upon stimulation by other GPCR’s), I immunoprecipitated FAK following norepinephrine stimulation of CHO-K1 cells. Preliminary experiments showed that norepinephrine stimulated the phosphorylation of FAK in transfected CHO-K1 cells at 5, 15 and 60 min of norepinephrine incubation relative to vehicle.