共查询到20条相似文献,搜索用时 15 毫秒
1.
EJ Bilsky RN Bernstein GW Pasternak VJ Hruby D Patel F Porreca J Lai 《Canadian Metallurgical Quarterly》1994,55(2):PL37-PL43
Evidence in vivo has suggested the existence of subtypes of the delta opioid receptor (DOR), which have been termed delta 1 and delta 2. These proposed DOR subtypes are thought to be activated by [D-Pen2, D-Pen5]enkephalin (DPDPE, delta 1) and [D-Ala2, Glu4]deltorphin (delta 2). Recent work in which an antisense oligodeoxynucleotide (oligo) to a cloned DOR was administered by the intrathecal (i.th.) route has demonstrated a reduction in the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin, but not of [D-Ala2, NMPhe4, Gly-ol]enkephalin (DAMGO, mu agonist) in mice. The present investigation has extended these observations by administering the same DOR antisense oligo sequence by the intracerebroventricular (i.c.v.) route and evaluating the antinociceptive actions of i.c.v. agonists selective for delta, mu and kappa receptors. I.th. treatment with DOR antisense oligo, but not mismatch oligo, significantly inhibited the antinociceptive actions of both i.th. DPDPE and [D-Ala2, Glu4]deltorphin but not of i.th. DAMGO or U69,593 (kappa agonist), confirming previous data. In contrast, i.c.v. DOR antisense oligo, but not mismatch oligo, selectively inhibited the antinociceptive response to i.c.v. [D-Ala2, Glu4]deltorphin without altering the antinociceptive actions of i.c.v. DPDPE, DAMGO or U69,593. The data suggest that the cloned DOR corresponds to that pharmacologically classified as delta 2 and further, suggest that this delta receptor subtype may play a major role in eliciting spinal delta-mediated antinociception. 相似文献
2.
In vivo administration of an antisense oligonucleotide targeted toward the D2 dopamine (DA) receptor mRNA (D2 AS) markedly inhibited D2 receptor-mediated behaviors but produced only a relatively small reduction in the levels of D2 DA receptors in mouse striatum. This apparent dissociation between DA receptor-mediated behaviors and the levels of D2 DA receptors was addressed by inhibiting the total number of D2 DA receptors by intraperitoneal administration of the selective, irreversibly acting D2 DA receptor antagonist fluphenazine-N-mustard (FNM) and then determining the effects of D2 AS, administered intracerebroventricularly, on the rate of synthesis of D2 DA receptors and on the recovery of D2 receptor-mediated behaviors. FNM inactivated approximately 90% of D2 DA receptors within 4 hr of treatment, after which the receptors returned to normal levels by approximately 8 days. D2 AS treatment significantly inhibited the rate of recovery of D2 DA receptors in striatum of FNM-treated mice. FNM treatment also produced a number of behavioral alterations, including catalepsy, and the inhibition of stereotypic behavior induced by the D2/D3 DA receptor agonist quinpirole. Both of these behaviors returned to normal within 8 days after FNM treatment. D2 AS treatment delayed the restoration of these FNM-induced behaviors. Thus, it reduced the rate of disappearance of the cataleptic behavior induced by FNM and significantly delayed the restoration of the stereotypic behavior induced by quinpirole. The changes induced by D2 AS on D2 receptor-mediated behaviors were reversed on cessation of D2 AS treatment. A random oligomer given in the same amount and for the same length of time as that of the D2 AS had no significant effects on either D2 DA receptor synthesis or DA receptor-mediated behaviors. These studies demonstrate that in vivo administration of D2 AS decreased the rate of recovery of D2 DA receptors and inhibited the recovery of D2 DA receptor-mediated behaviors after irreversible receptor inactivation and suggest that D2 AS treatment inhibits the synthesis of a functional pool of D2 DA receptors. 相似文献
3.
J Kamei Y Iwamoto M Misawa H Nagase Y Kasuya 《Canadian Metallurgical Quarterly》1994,55(6):PL121-PL126
We assessed the effect of diabetes on antinociception produced by intracerebroventricular injection of delta-opioid receptor agonists [D-Pen2,5]enkephalin (DPDPE) and [D-Ala2]deltorphin II. The antinociceptive effect of DPDPE (10 nmol), administered i.c.v., was significantly greater in diabetic mice than in non-diabetic mice. The antinociceptive effect of i.c.v. DPDPE was significantly reduced in both diabetic and non-diabetic mice following pretreatment with 7-benzylidenenaltrexone (BNTX), a selective delta 1-opioid receptor antagonist, but not with naltriben (NTB), a selective delta 2-opioid receptor antagonist. There were no significant differences in the antinociceptive effect of [D-Ala2]deltorphin II (3 nmol, i.c.v.) in diabetic and non-diabetic mice. Furthermore, the antinociceptive effect of i.c.v. [D-Ala2]deltorphin II was significantly reduced in both diabetic and non-diabetic mice following pretreatment with NTB, but not with BNTX. In conclusion, mice with diabetes are selectively hyper-responsive to supraspinal delta 1-opioid receptor-mediated antinociception, but are normally responsive to activation of delta 2-opioid receptors. 相似文献
4.
The regulation of 5-HT2A receptor expression by an antisense oligodeoxynucleotide, complementary to the coding region of rat 5-HT2A receptor mRNA, was examined in a cortically derived cell line and in rat brain. Treatment of A1A1 variant cells, which express the 5-HT2A receptor coupled to the stimulation of phosphatidylinositol (PI) hydrolysis, with antisense oligodeoxynucleotide decreased the maximal stimulation of PI hydrolysis by the partial agonist quipazine and the number of 5-HT2A receptor sites as measured by the binding of 2-[125I]-iodolysergic acid diethylamide. Treatment of cells with random, sense, or mismatch oligodeoxynucleotide did not alter the stimulation of PI hydrolysis by quipazine or 5-HT2A receptor number. Intracerebroventricular infusion of antisense, but not mismatch, oligodeoxynucleotide for 8 days resulted in a significant increase in cortical 5-HT2A receptor density and an increase in headshake behavior induced by the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane. The density of cortical 5-HT2A receptors was not altered by administration of antisense oligodeoxynucleotide for 1, 2, or 4 days. We hypothesize that in brain this antisense oligodeoxynucleotide relieved some form of translational suppression, resulting in an increase in 5-HT2A receptor expression. 相似文献
5.
CD-1 mice were treated intravenously with streptozotocin, 200 mg/kg, and tested 2 weeks later or treated with 60 mg/kg and tested 3 days later. Both treatments changed the tail flick response of heroin and 6-monoacetylmorphine (6 MAM) given intracerebroventricularly from a mu- to delta-opioid receptor-mediated action as determined by differential effects of opioid receptor antagonists. The response to morphine remained mu. Heroin and 6 MAM responses involved delta1 (inhibited by 7-benzylidenenaltrexone) and delta2 (inhibited by naltriben) receptors, respectively. These delta-agonist actions did not synergize with the mu-agonist action of morphine in the diabetic mice. The expected synergism between the delta agonist, [D-Pen2-D-Pen5]enkephalin (DPDPE), and morphine was not obtained in diabetic mice. Thus, diabetes disrupted the purported mu/delta-coupled response. In nondiabetic CD-1 mice, heroin and 6 MAM produced a different mu-receptor response (not inhibited by naloxonazine) from that of morphine (inhibited by naloxonazine). Also, these mu actions, unlike that of morphine, did not synergize with DPDPE. The unique receptor actions and changes produced by streptozotocin suggest that extrinsic in addition to genetic factors influence the opioid receptor selectivity of heroin and 6 MAM. 相似文献
6.
To determine the uptake and distribution of oligodeoxynucleotides in brain, a 20-mer phosphorothioated oligodeoxynucleotide complementary to a portion of the D2 dopamine receptor mRNA was fluorescently labeled with fluorescein isothiocyanate (FITC) and injected into the lateral cerebral ventricles of mice. At various survival times after the injection, the brains were removed, fixed, sectioned, and viewed under a fluorescent microscope. The results showed that the oligodeoxynucleotide was rapidly taken up into the brain. Initially the label was relatively diffusely spread throughout the interstitial spaces of the brain, then became redistributed to the cellular compartments. The signal extended from those forebrain nuclei located immediately in contact with the ventricles, such as the corpus striatum, septum, and hippocampus, to areas further removed from the ventricles, such as the cerebral cortex, nucleus accumbens, and substantia nigra. When the FITC-labeled D2 antisense oligodeoxynucleotide was given once daily for 4 d, the signal intensity seen 24 h after the last injection appeared to be of greater intensity overall compared to that seen after a single injection. At early time-points the oligodeoxynucleotide signals appeared to be punctuated and were found in cell bodies as well as in proximal dendritic processes. However, not all cells were equally labeled, suggesting an uneven uptake and accumulation of the D2 antisense into the various cell types. At later time-points the fluorescent signal appeared granular; at these times the injected material was largely degraded. These studies show that a D2 dopamine receptor antisense oligodeoxynucleotide is rapidly taken up from cerebral ventricles into brain, becomes widely distributed throughout the brain tissue to areas far removed from direct contact with the ventricles, and appears to accumulate to a different extent in the various brain areas and cell types. 相似文献
7.
PURPOSE: To evaluate the effects of drug therapy on the clinical course of acute acquired Toxoplasma retinochoroiditis and on the number of Toxoplasma cysts present in the brain and ocular tissues in the hamster animal model. METHODS: The Syrian golden hamster animal model of Toxoplasma retinochoroiditis was used. In acute disease, systemically administered atovaquone was compared with conventional therapies (pyrimethamine combined with sulfadiazine; clindamycin; and spiramycin). The clinical course of the ocular disease was determined with retinal examination and photography of the fundus. The number of Toxoplasma cysts remaining after treatment was evaluated in aliquots of brain homogenate and in retinal tissue. The effect of atovaquone on cerebral Toxoplasma cyst count was also studied in chronic disease. RESULTS: None of the drugs administered altered the course of the acute disease, judged by clinical examination. Atovaquone alone significantly reduced the number of cerebral Toxoplasma cysts after acute disease. Atovaquone also significantly reduced the cerebral Toxoplasma cyst count in chronic disease. CONCLUSIONS: Tissue cysts are believed to be responsible for reactivation of Toxoplasma retinochoroiditis. Atovaquone has the potential to reduce the risk of recurrent disease. 相似文献
8.
To analyze the selectivity of delta receptor subtypes to regulate different classes of G proteins, the expression of the alpha-subunits of Gi2, Gi3, Go1, Go2, Gq and G11 transducer proteins was reduced by administration of oligodeoxynucleotides (ODNs) complementary to sequences in their respective mRNAs. Mice receiving antisense ODNs to Gi2 alpha, Gi3 alpha, Go2 alpha and G11 alpha subunits showed an impaired antinociceptive response to all the delta agonists evaluated. An ODN to Go1 alpha specifically blocked the antinociceptive effect of the agonist of delta-1 receptors, [D-Pen2,5]enkephalin (DPDPE), without altering the activity of [D-Ala2]deltorphin II or [D-Ser2]-Leu-enkephalin-Thr (DSLET). In mice treated with an ODN to Gq alpha, the effects of the agonists of delta-2-opioid receptors were reduced, but not those of DPDPE. Thus, Go1 proteins are selectively linked to delta-1-mediated analgesia, and Gq proteins are related to delta-2-evoked antinociception. After impairing the synthesis of Go1 alpha subunits, DPDPE exhibited an antagonistic activity on the antinociception produced by [D-Ala2]deltorphin II. After treatment with ODNs complementary to sequences in Gq alpha or PLC-beta 1 mRNAs, the analgesic capacity of [D-Ala2]deltorphin II was diminished. However, the delta-2-agonist did not alter the antinociceptive activity of DPDPE. An ODN complementary to nucleotides 7 to 26 of the murine delta receptor reduced the analgesic potency of [D-Ala2]deltorphin II, but not that observed for DPDPE. In these mice, [D-Ala2]deltorphin II did not antagonize the effect of DPDPE. These results suggest the existence of different molecular forms of the delta opioid receptor, and the involvement of inositol-signaling pathways in the supraspinal antinociceptive effects of delta agonists. 相似文献
9.
J Karle P Laudrup F Sams-Dodd JD Mikkelsen M Nielsen 《Canadian Metallurgical Quarterly》1997,340(2-3):153-160
Binding energy of DNA-Cro protein complexes is analyzed in terms of DNA elasticity, using a sequence-dependent anisotropic bendability (SDAB) model of DNA, developed recently [M.M. Gromiha, M.G. Munteanu, A. Gabrielian and S. Pongor, J. Biol. Phys. 22(1996) 227-243.]. The protein is considered to bind aspecifically to DNA that reduces the freedom of movement in the DNA molecule. In cognate DNA, the Cro protein moves on to form specific interactions and bends DNA. A comparison of the experimental data [Y. Takeda, A. Sarai and V.M. Rivera, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 439-443.] with the calculated DNA stiffness data shows that delta G of the complex formation increases with stiffness of the ligand when the interactions are nonspecific ones, while an opposite trend is observed for specific binding. Both of these trends are in agreement with our approach using the SDAB model. A decomposition of the energy terms suggests that binding energy in the nonspecific case is used maily to compensate the free energy changes due to entropy lost by DNA, while the energy of specific interactions provide enough energy both to bend the DNA molecule and to change the conformation of the Cro protein upon ligand binding. 相似文献
10.
A model for the 3D structure of the transmembrane domain of the delta opioid receptor was predicted from the sequence divergence analysis of 42 sequences of G-protein coupled peptide hormone receptors belonging to the opioid, somatostatin and angiotensin receptor families. No template was used in the prediction steps, which include multiple sequence alignment, calculation of a variability profile of the aligned sequences, use of the variability profile to identify the boundaries of transmembrane regions, prediction of their secondary structure, optimization of the packing shape in a helix bundle, prediction of side chain conformations and structural refinement. The general shape of the model is similar to that of the low resolution rhodopsin structure in that the TM3 and TM7 helices are most buried in the bundle and the TM1 and TM4 helices are most exposed to the lipid phase. An initial assessment of this model was made by determining to what extent a binding site identified using four structurally disparate high affinity delta opioid ligands was consistent with known mutational studies. With the assumption that the protonated amine nitrogen, a feature common to all delta opioid ligands, interacts with the highly conserved Asp127 in TM3, a pocket was found that satisfied the criteria of complementarity to the requirements for receptor recognition for these four diverse ligands, two delta selective antagonists (the fused ring naltrindole and the peptide Tyr-Tic-Phe-Phe-NH2) and the two agonists lofentanil and BW373U86 deduced from previous studies of the ligands alone. These ligands could be accommodated in a similar region of the receptor. The receptor binding site identified in the optimized complexes contained many residues in positions known to affect ligand binding in G-protein coupled receptors. These results also allowed identification of key residues as candidates for point mutations for further assessment and refinement of this model as well as preliminary indications of the requirements for recognition of this receptor. 相似文献
11.
B Weiss G Davidkova LW Zhou SP Zhang M Morabito 《Canadian Metallurgical Quarterly》1997,31(4):571-580
We reported that 3'-azidothymidine-3'-deoxythymidine (AZT) plus 5-fluorouracil or methotrexate produces additive cytotoxicity in HCT-8 cells: a reflection of increased AZT metabolism when de novo thymidylate (dTMP) synthesis was inhibited. We now report that AZT plus human recombinant interferon alpha-2a (rIFN-alpha 2a) produces synergistic growth inhibition in these cells. Evaluation of the effect of rIFN-alpha 2a on dTMP metabolism revealed that exposure to rIFN-alpha 2a (+/-AZT) did not affect dTMP synthase activity significantly but increased thymidine (dThd) kinase activity significantly. Consequently, AZT nucleotide production and incorporation into DNA were increased by coexposure to rIFN-alpha 2a. This alone, however, cannot explain the observed synergism. Therefore, the effect of these agents on DNA excision/repair processes was assessed. Isotope clearance studies demonstrated that rIFN-alpha 2a did not alter the rate of [3H]AZT excision from DNA. In contrast, filter-elution studies revealed that rIFN-alpha 2a (+/-AZT) produced more DNA damage and delayed repair compared with the effects produced by AZT alone. Since DNA polymerases alpha and beta are directly involved in gap-filling repair synthesis, experiments next assessed the effect of rIFN-alpha 2a and/or 3'- azido-3'-deoxythymidine-5'-triphosphate (AZTTP) on their activities. Polymerase alpha was inhibited slightly by AZTTP but not by rIFN-alpha 2a. Polymerase beta activity, however, was inhibited dramatically by rIFN-alpha 2a + AZTTP. Finally, western analysis revealed that a 24-hr exposure to 5000 IU/mL rIFN-alpha 2a (+/-20 microM AZT) significantly reduced wild-type p53 expression compared with AZT-exposed cells. We conclude that rIFN-alpha 2a enhances AZT-induced tumor cell growth inhibition by (i) increasing AZT metabolism, and (ii) inhibiting DNA repair and p53-mediated cell cycle control processes. 相似文献
12.
The effect of opioid receptor agonists and antagonists on the electrically evoked release of endogenous serotonin (5-hydroxytryptamine, 5-HT) was studied in superfused slices of the rat ventral lumbar spinal cord. Met-ENK (1 x 10(-8)M-1 x 10(-6)M) and DPDPE (1 x 10(-8)M-1 x 10(-6)M) reduced the evoked 5-Ht release in a concentration dependent fashion. DAMGO (1 x 10(-8)-1 x 10(-6)) and (-)-trans-(1S,2S)-U-50488 (1 x 10(-6)M) had no effect on the 5-HT release. The inhibitory effect of met-ENK was completely abolished by ICI-174,864, but neither by naloxonazine nor nor-binaltorphimine. Following i.c.v. treatment with 5,7-dihydroxytryptamine (5,7-DHT), the tissue concentration of 5-HT was reduced by 97%, whereas the concentration of noradrenaline was reduced by only 5%. The tissue concentration of met-ENK, as measured by radioimmunoassay, was not significantly altered. The results suggest that met-ENK is present in the rat ventral spinal cord mainly in non-serotonergic nerve terminals and exerts an inhibitory action on 5-HT release via delta opioid receptors. 相似文献
13.
D-Serine, a selective agonist for the strychnine-insensitive glycine allosteric site associated with the NMDA receptor-ion channel complex, was found to modulate differentially the antinociception produced by kappa and mu-opioid receptor agonists in the rat formalin test. D-Serine (100 micrograms, i.c.v.) attenuated the antinociception produced by the selective kappa-opioid agonist, enadoline (0.003-0.1 mg kg-1, s.c.) against the tonic, but not acute, phase of the formalin response. Conversely, D-serine potentiated the antinociception produced by morphine (0.3-10 mg kg-1, s.c.) against both the acute and tonic phases. These results demonstrate an important interaction between the opioid and NMDA/glycine systems in the control of nociceptive information possibly at different levels of the neuraxis. 相似文献
14.
Because the role of mu and delta opioid receptors in modulating gastric functions remains uncertain, we studied whether intracerebroventricular (i.c.v.) and subcutaneous (s.c.) injections of new opioid peptides with high selectivity for mu 1 (Lys7-dermorphin), mu 2 (Trp4-Asn7-dermorphin) and delta 2 (D-Ala2-deltorphin II) opioid receptors would modify gastric secretion (after 2 hr pylorus ligature) and transit (after a phenol red meal) in the rat. Neither i.c.v. nor s.c. injections of the delta 2 opioid agonist affected the gastric functions. In contrast, the mu opioid agonists decreased gastric acid secretion and emptying, i.c.v. injections inducing more potent inhibition than s.c. administration. The mu 1 selective opioid antagonist naloxonazine had no effect on the inhibition of the gastric secretory and motor response to these peptides but naloxone completely blocked their effects. Our findings suggest (1) that in rats, stimulation of central naloxonazine insensitive opioid receptors (mu 2 sites) inhibits gastric acid secretion and emptying; and (2) that delta opioid receptors take no part in mediating these functions. 相似文献
15.
For 5 consecutive days repeated intracerebroventricular (i.c.v.) administration of antisense oligodeoxynucleotides (ODNs) to G alpha subunit mRNAs was used to impair the function of mouse Gi1, Gi2, Gi3 and Gx/z regulatory proteins. Decreases of 20 to 60% on the G alpha-like immunoreactivity could be observed in neural structures of mouse brain, an effect that was not produced by a random-sequence ODN used as a control. The ODN to Gi1 alpha subunits lacked effect on opioid-evoked analgesia. In mice injected with the ODN to Gi2 alpha subunits the antinociceptive activity of all the opioids studied appeared greatly impaired. The ODN to Gi3 alpha subunits reduced the effects of the selective agonists of delta opioid receptors, [D-Pen2,5]-enkephalin and [D-Ala2]deltorphin II. Conversely, the analgesia evoked by opioids binding mu opioid receptors, [D-Ala2, N-MePhe4,Gly-ol5]enkephalin and morphine, appeared consistently and significantly attenuated in mice injected with the ODN to Gx/z alpha. The effect of the neuropeptide beta-endorphine-(1-31) agonist at mu and delta receptors was also reduced by ODNs to Gi3 alpha or Gx/z alpha subunits. l.c.v. injection of antibodies directed to these G alpha subunits antagonized opioid-induced analgesia with a pattern similar to that observed for the ODNs. Thus, the mu and delta opiod receptors regulate different classes of G transducer proteins to mediate the analgesic effect of agonists. The in vivo antisense strategy and the use of specific antibodies to G alpha subunits gave comparable results, indicating that in the neural tissue the mRNAs and the G alpha subunits can be accessed by the corresponding ODNs and IgGs. 相似文献
16.
The present study was designed to further investigate the nature of feeding induced by opioid stimulation of the nucleus accumbens through an examination of the effects of intra-accumbens (ACB) opioids on macronutrient selection. In 3-hr tests of free-feeding (satiated) rats, intra-ACB administration of the mu receptor agonist D-Ala2,N,Me-Phe4, Gly-ol5-enkephalin (DAMGO; 0, 0.025, 0.25 and 2.5 micrograms bilaterally) markedly enhanced the intake of fat or carbohydrate when the diets were presented individually (although the effect on fat intake was much greater in magnitude). Intra-ACB injections of DAMGO, however, produced potent preferential stimulatory effects on fat ingestion with no effect on carbohydrate ingestion when both fat and carbohydrate diets were present simultaneously. Moreover, this selective stimulation of fat intake was independent of base-line diet preference and could be blocked by systemic injection of naltrexone (5 mg/kg). We also examined the effect of 24-hr food deprivation on the pattern of macronutrient intake in rats with access to both carbohydrate and fat. In contrast to the DAMGO-induced selective enhancement of fat intake, food deprivation significantly increased the intake of both diets to the same extent; however, in this case, only the stimulated fat intake was blocked by systemic naltrexone. Intra-ACB administration of DAMGO in hungry rats produced an effect similar to that observed in free-feeding rats; preference was strongly shifted to fat intake. Similarly, the opioid antagonist naltrexone (20 micrograms) infused directly into ACB preferentially decreased fat intake in hungry rats. These findings suggest that endogenous opioids within the ventral striatum may participate in the mechanisms governing preferences for highly palatable foods, especially those rich in fat. 相似文献
17.
18.
The effects of angiotensin II are mediated by a family of seven transmembrane receptors. In the adult, the majority of the receptors are of the AT1 isoform, which is coupled to heterotrimeric G proteins (either Gqalpha or Gialpha). In contrast, the AT2 receptor is expressed at low levels in the adult but is the major form expressed in the fetal and neonatal animal. Previous results have failed to show G protein coupling of the AT2 receptor in the fetus. We now provide evidence that the AT2 receptor is G protein-coupled. An antibody that binds several Galpha subunits immunoselected angiotensin II receptor-Galpha complexes. In addition, Gialpha1-3 antibody, which recognizes Gialpha1, Gialpha2 and Gialpha3, also co-immunoselect the AT2 receptor. Anti-Gialpha2 and anti-Gialpha3 antibodies were both able to co-immunoselected AT2 receptor-Gialpha complexes, but consistent with the lack of Gialpha1 in the fetal extracts, anti-Gialpha1 antibodies did not nor did any other G protein-directed antisera. The finding that AT2 receptor couples to both Gialpha2 and Gialpha3 raises the possibility that selective interactions between AT2 receptor and different G proteins may result in specific cellular effects mediated by AT2 stimulation. 相似文献
19.
A Ziegler GH Luedke D Fabbro KH Altmann RA Stahel U Zangemeister-Wittke 《Canadian Metallurgical Quarterly》1997,89(14):1027-1036
BACKGROUND: The emergence of resistance to chemotherapy remains a major problem in the treatment of patients with small-cell lung cancer. Elevated expression of Bcl-2, a protein that inhibits programmed cell death or apoptosis, has been associated with radiation and drug resistance and has been observed in the majority of small-cell lung cancer specimens and cell lines. PURPOSE: To test the hypothesis that Bcl-2 expression levels are critical for inhibiting apoptosis in small-cell lung cancer cells, we used an antisense strategy to reduce Bcl-2 expression in these cells in an attempt to restore the natural occurrence of apoptosis. METHODS: Thirteen antisense oligodeoxynucleotides (ODNs) targeting various regions of the bcl-2 messenger RNA and a control scrambled-sequence ODN were tested to identify the most effective sequence(s) for reducing Bcl-2 protein levels. Northern and western blot analyses were used to examine basal bcl-2 messenger RNA and protein levels, respectively, in four human small-cell lung cancer cell lines (SW2, NCI-H69, NCI-H82, and NCI-N417). SW2 cells were treated with the antisense ODNs in the presence of cationic lipids (to facilitate uptake), and cytotoxic effects were measured by use of a cell viability assay. Flow cytometric analysis of DNA fragmentation and cell morphology was also performed. The cytotoxic effect of the most potent antisense ODN was also tested on the three other cell lines. RESULTS: The viability of SW2 cells was effectively reduced by ODNs that targeted the translation initiation and termination sites of the bcl-2 messenger RNA, but ODN 2009 that targeted the coding region was the most cytotoxic. Treatment of SW2 cells with 0.15 microM ODN 2009 for 96 hours reduced their viability by 91% (95% confidence interval [CI] = 88%-94%) and caused a dose-dependent reduction in Bcl-2 levels that became detectable 24 hours after treatment and persisted up to 96 hours; analysis of cellular morphology demonstrated that viability was reduced through apoptosis. Moreover, ODN 2009 at 0.15 microM was cytotoxic to NCI-H69, NCI-H82, and NCI-N417 cells, resulting in decreases in cell viability of 82% (95% CI = 78%-86%), 100%, and 100%, respectively, after 96 hours of treatment. The cytotoxic effects were inversely correlated with the basal Bcl-2 levels in the cell lines (r = -9964). A control scrambled-sequence oligodeoxynucleotide had no statistically significant effect on the cell lines (P values ranging from .38 to .89). CONCLUSION: We have identified a novel antisense ODN sequence (ODN 2009) that effectively reduces the viability of small-cell lung cancer cells by reducing Bcl-2 levels and facilitating apoptosis. 相似文献
20.
Receptor binding studies and electrophysiological studies demonstrated the existence of at least two kappa opioid receptors, which have been designated kappa-1 and kappa-2. Several agonists and antagonists are selective for the kappa-1 receptor whereas no known ligands are selective for the kappa-2 receptor. In this study, the kappa opioid GR89,696 was tested in the guinea pig hippocampal slice preparation for kappa-1 versus kappa-2 activity. The perforant path-evoked population spike in the dentate was use to evaluate activity at the kappa-1 receptor, and the Schaffer collateral-evoked N-methyl-D-aspartate (NMDA) receptor-mediated synaptic current in CA3 pyramidal cells was used to measure kappa-2 receptor activation. GR89,696 had no effect on the perforant path-evoked dentate population spike; however, it did reverse the effects of the selective kappa-1 agonist U69,593 when co-perfused over the slices. In the CA3, GR89,696 inhibited the NMDA receptor-mediated synaptic current. The inhibition was antagonized by naloxone. The EC50 for GR89,696 on the NMDA current was 41.7 nM (95% CL, 7.0-248 nM). These findings indicate that GR89,696 is an agonist for kappa-2 opioid receptors and an antagonist at kappa-1 receptors in the guinea pig hippocampus. 相似文献