Missense Oligodeoxynucleotide Research Articles

The TRPA1 Channel Mediates Mechanical Allodynia and Thermal Hyperalgesia in a Rat Bone Cancer Pain Model.

Background: Bone cancer pain (BCP) significantly affects patient quality of life, results in great bodily and emotional pain, and creates difficulties in follow-up treatment and normal life. Transient receptor potential ankyrin 1 (TRPA1) is an essential transduction ion channel related to neuropathic and inflammatory pain. However, the role of TRPA1 in BCP remains poorly understood. This study aimed to explore the relationship between TRPA1 and BCP.Methods: A BCP model was induced by Walker256 cells to the left tibia. The sham group was induced by normal saline to the left tibia. Thereafter, pain behaviors and TRPA1 expression between the BCP group and the sham group were observed on the 14th day of modeling. The TRPA1 antagonist A967079 (10 mg/kg) was injected via tail vein. TRPA1 antisense oligodeoxynucleotide (AS-ODN, 5 nmol/10 μl) and missense oligodeoxynucleotide (MS-ODN, 5 nmol/10 μl) were intrathecally delivered via a mini-osmotic pump for 5 consecutive days to assess the effect of TRPA1 on BCP. Behavioral tests were assessed preoperatively and postoperatively. Real-time quantitative PCR and western blot analyses were used to measure TRPA1 levels among the different groups.Results: The BCP model was successfully established via X-ray and pathological sections at 14 days. Compared to the sham group, the BCP group was more sensitive to mechanical stimuli, cool stimuli and hot stimuli. Intravenously injected A967079 can relieve paw mechanical withdrawal threshold and paw withdrawal thermal latency in rats with BCP. Moreover, AS-ODN can relieve paw mechanical withdrawal threshold and paw withdrawal thermal latency in rats with BCP. Additionally, relative mRNA and protein expression of TRPA1 in the BCP group were much higher than those in the sham group (14.55 ± 1.97 vs. 1 ± 0.04, P < 0.01). Compared to the BCP group, the relative mRNA and protein expression of TRPA1 in the BCP+AS-ODN group was reduced (14.55 ± 1.97 vs. 2.59 ± 0.34, P < 0.01).Conclusions: The TRPA1 channel mediates mechanical allodynia and thermal hyperalgesia in a rat BCP model.

Effect of Cdc42 on myocardial ischemia-reperfusion of rats.

To investigate the effects and their possible mechanisms of cell division cycle 42 (Cdc42) to neonatal rat myocardial cells subjected to the ischemia-repefusion. Neonatal rat cardiomyocytes were cultured and then subjected to the ischemia-reperfusion. Experimental groups 1. Control group; 2. Ischemia-repefusion group (I/R group); 3. Oligofectamine group (Oli group); 4. Oligofectamine and antisense oligodeoxynucleotide (AS-ODN) group (As group); 5. Oligofectamine and missense oligodeoxynucleotide (MS-ODN) group (Ms group); 6. SP600125 and Oligofectamine and AS-ODN group (SP600125/As group); 7. SP600125 and Oligofectamine and MS-ODN group (SP600125/Ms group). The cardiacmyocyte apoptosis rate was detected by AnnexinV/PI with flow cytometry. Cdc42, JNK, p-JNK, Bax and Bcl-2 were detected by western blot. In comparison with control group, Cdc42, the cardiacmyocyte apoptosis rate and phosphorylation of JNK were increased and the ratio of Bcl-2/Bax was reduced in the I/R group; Cdc42, the cardiacmyocyte apoptosis rate and phosphorylation of JNK in As group was lower than the I/R group, Oli group and the Ms group, and the ratio of Bcl-2/Bax was the highest in the four groups; Cdc42, cardiacmyocyte apoptosis rate, phosphorylation of JNK and the ratio of Bcl-2/Bax showed no differences in the I/R group, Oli group and the Ms group. Compared with As group, phosphorylation of JNK was lower in the SP600125/As group, phosphorylation of JNK in SP600125/Ms group was lower than the Ms group, and it showed no differences between the SP600125 &amp; As group and the SP600125 &amp; Ms group. Cdc42 in myocardial I/R can promote cardiacmyocyte apoptosis rate. AS-ODN of Cdc42 can decrease the cardiacmyocyte apoptosis rate in I/R. Cdc42 may played a role in myocardial I/R via JNK , Bcl-2 and Bax signal pathway.

Effects of intrathecally postsynaptic density protein-95 antisense oligodeoxynucleotide on pain behaviors in mice model of neuropathic pain

Objective To investigate the effects of intrathecally postsynaptic density protein-95 (PSD-95) antisense oligodeoxynucleotide on neuropathic pain behaviors of sciatic nerve ligation mouse. Methods Forty-eight male C57BL/6 mice in which intrathecal catheter was successfully implanted were randomly divided into 4 groups (n=12): Sham group, normal saline group (group NS), PSD-95 antisense oligodeoxynucleotide group (group A) and PSD-95 missense oligodeoxynucleotide group (group M). In groups NS, A and M, normal saline 5 μl, antisense oligodeoxynucleotide 5 μg/5 μl and missense oligodeoxynucleotide 5 μg/5 μl were injected intrathecally once a day for 14 d, starting from the 1st day after chronic constriction injury(CCI), respectively. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWL) were measured on day 1 before CCI and on day 1, 3, 5, 7, 14, 17, 21 after CCI. Results During day 1-day 14, mice in group A maintained the pain thresholds consistent with Sham group [14 d, PWMT(5.69±1.34) g, P>0.05, PWL (9.65±1.44) s, P>0.05]. The withdrawal thresholds in the ipsilateral hind paws of the group A were significantly lower than sham group on day 17 after CCI[PWMT(4.24±1.83) g, P<0.05, PWL(7.18±0.41) s, P<0.05]. However, compared with group NS [PWMT (1.77±0.38) g, P<0.05, PWL (4.33±1.21) s, P<0.05]and group M[PWMT (1.6±0.37) g, P<0.05, PWL(4.38± 0.95) s, P<0.05], the withdrawal thresholds of group A was significantly elevated on day 17 after CCI. Conclusions Intrathecally treated with PSD-95 antisense oligodeoxynucleotide in the development of neuropathic pain induced by CCI completely improved pain behaviors during the course of injection, and the effects of pain relief lasted at least 7 d after no injection. Key words: Neuropathic pain; postsynaptic density protein-95; Antisense oligonucleotide; N-methyl-D-aspartate

Effect of NF-κB p65 antisense oligodeoxynucleotide on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2.

To study the inhibition of nuclear factor kappa-B p65 (NF-κB p65) antisense oligodeoxynucleotide (ASODN) on transdifferentiation of normal human lens epithelial cells induced by transforming growth factor-β2 (TGF-β2) in vitro. NF-κB p65 ASODN and NF-κB p65 missense oligodeoxynucleotide (MSODN) were designed and synthesized. Human lens epithelial cell line (HLE B-3) cells were prepared for study and divided into 7 groups. Control group was HLE B-3 cells cultured in vitro in dulbecco's modified eagle medium (DMEM). T1, T2, and T3 group were HLE B-3 cells cultured in vitro in DMEM with 10 ng/mL TGF-β2 for 6h, 12h, 24h respectively. A+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65 ASODN for 24h. M+T group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after transfected by NF-κB p65 MSODN for 24h. The negative control group was HLE B-3 cells cultured with 10 ng/mL TGF-β2 for 24h after cultured with transfer agent (HiPerFect) for 24h. Cell morphology was observed at different time points using an inverted microscope. The expression of NF-κB p65 mRNA was detected with reverse transcription-polymerase chain reaction (RT-PCR), and the expression of α-smooth muscle actin (α-SMA) protein was assayed with ELISA. With the TGF-β2 stimulation prolongation, the expression of NF-κB p65 mRNA and α-SMA protein increased in T1, T2, T3 groups compared with the control group, and the difference was statistically significant (P<0.05). NF-κB p65 ASODN lowered the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2. NF-κB p65 MSODN and HiPerFect did not lower the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2. The difference between control group and A+T group was not statistically significant (P>0.05), but the difference among A+T group and other groups was statistically significant (P<0.05). NF-κB p65 ASODN could lower the expression of NF-κB p65 mRNA and α-SMA protein induced by TGF-β2, and antagonized TGF-β2-induced transdifferentiation of HLE B-3 in vitro. NF-κB p65 ASODN could be used as a new biological therapeutic target of posterior capsular opacification.

Effect of glutathione S-transferaseπin COC1/Cisplatin cells on cisplatin resistance

Objective To study the expression of drug resistance related gene in COC1/Cisplatin (DDP)cell line,and the reversion of cisplatin resistance by antisense oligodeoxynucleotide(ASODN)of the overexpressed gene. Methods The expression of glutathione S-transferaseπ(GSTπ)in COC1/DDP cell line was examined by reverse transcription-polymerase chain reaction(RT-PCR), and compared with that in COC1 cell line.ASODN targeting GSTπwas designed and synthesized.GSTπ-ASODN and missense oligodeoxynucleotide with the concentration of 200 nmol/L were transferred into COC1/DDP cells by lipofectin.The inhibition rate of transferred cells to cisplatin was detected by methyl thiazol tetrazolium(MTT)colori-metric assay,and compared with that in untransferred COC1/DDP cells.And the altered expression of GSTπwas also detected by RT-PCR. Results (1)GSTπwas overexpressed in cisplatin resistant cell line of COC1/DDP as compared with COC1 cell line.(2) In comparison with COC1/DDP cells, GSTπexression in GSTπ-ASODN transfected cells was significantly decreased to 0. 058±0. 022 and resistance index(RI) was 3. 61 (P 0. 05),and the drug sensitivity of COC1/DDP cells either(P> 0. 05). Conclusion (1)The overexpression of GSTπmay play an important role in the induction of resistance of COC1/DDP cells to cisplatin; (2)GSTπ-ASODN can effectively reverse the resistance of COC1/DDP cells to cisplatin and increase their drug sensitivity in a sequence specific manner. Key words: Ovarian neoplasm; Cisplatin; Antisense oligonucleotide; Glutathione S-transferaseπgene; Drug resistance

Role of phosphatidylinositol 3-kinase p110.BETA. in spinal dorsal horn neurons in the development of arthritic pain in rats: relationship with TRPV1 and ASICla

Objective To evaluate the role of phosphatidylinositol 3-kinase (PI3K) p110β in spinal dorsal horn neurons in the development of arthritic pain (AP) in rats and the relationship with transient receptor potential vanilloid 1 (TRPV1) and acid-sensing ion channel (ASIC)1 a.Methods Forty adult female Sprague-Dawley rats in which intrathecal catheters were successfully placed,aged 3 months,weighing 250-300 g,were randomly divided into 4 groups (n =10 each):control group (group C),group AP,AP + PI3K p110β missense oligo-deoxynucleotide group (group MS) and AP + PI3K p110β antisense oligo-deoxynucleotide group (group AS).AP was induced by injecting complete Freund's adjuvant into the ankle joint cavity of right hindpaw.Normal saline 20 μl,missense oligo-deoxynucleotide 15 μg (20μl) and antisense oligo-deoxynucleotide 15 μg (20 μl) were administered intrathecally once a day for 6 consecutive days starting from the time immediately after arthritis was induced in groups AP,MS and AS,respectively.Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured 1 day before operation (T0) and on days 4,7,10 after operation (T1-3).The rats were then sacrificed after the last measurement of pain threshold at T3.L4-6 segment of the spinal cord was removed for detection of expression of PI3K p110β (by Western blot),and TRPV1 and ASICla (by immunohistochemistry)in spinal dorsal horn neurons.Results Compared with group C,MWT and TWL were significantly decreased atT1-3,and the expression of PI3K p110β,TRPV1 and ASIC1a was up-regulated in the other 3 groups(P＜ 0.01).MWT and TWL were significantly higher at T1-3,and the expression of PI3K p110β,TRPV1 and ASIC1a was lower in group AS than in groups AP and MS (P ＜ 0.01).Conclusion PI3K p110β in spinal dorsal horn neurons is involved in the development of AP in rats,and the mechanism is related to up-regulation of TRPV1 and ASIC1a expression in spinal dorsal horn neurons. Key words: 1-Phosphatidylinositol 3-kinase; Arthritis; Pain; Capsaicin; Receptors, cell surface; Ion channels; Posterior horn cells

Effects of repeated intrathecal CREB antisense oligodeoxynucleotide on the expression of NR2A in spinal cord in a mouse model of neuropathic pain

Objective To investigate the effects of repeated intrathecal cyclic AMP response elementbinding protein (CREB) antisense oligodeoxynucleotide (ODN) on the expression of NR2A in spinal cord in mice with neuropathic pain produced by chronic constrictive injury of the sciatic nerve (CCI).Methods Forty C57BL/6 male mice in which intrathecal catheter was successfully implanted were randomly divided into 4 groups ( n =10 each):normal saline group (group NS),CREB sense ODN group (group S),CREB missense ODN group (group M),and CREB antisense ODN group (group A).In groups NS,S,M and A,normal saline 5μl,sense ODN 5 μg/5 μl,missense ODN 5 μg/5 μl and antisense ODN 5 μg/5 μl were injected intrathecally once a day for 6 days,starting from the 1st day after CCI,respectively.Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured on day 1 before CCI and on day 1,3,5 and 7 after CCI.Five mice from each group were sacrificed on day 7 and 14 after CCI and the lumbar segment of the spinal cord (L3-5 )was removed for determination of NR2A expression using Western blot and RT-PCR.Results Compared with the baseline value,no significant change was found in PWMT and PWTL on day 1-7 after CCI in group A ( P ＞0.05),while PWMT and PWTL were significantly decreased on day 1-7 after CCI in groups NS,S and M (P ＜0.05).Compared with groups NS,S and M,the expression of NR2A mRNA and protein was significantly downregulated on day 7 and 14 after CCI in group A ( P ＜ 0.05).The expression of NR2A mRNA and protein was significantly up-regulated on day 14 after CCI compared with that on day 7 after CCI in all the groups.Conclusion Intrathecal CREB antisense ODN during the development of neuropathic pain can attenuate neuropathic pain and inhibition of the expression of NR2A in mouse spinal cord may be involved in the mechanism. Key words: Oligonucleotides,antisense; Neuralgia; Receptors,N-methyl-D-aspartate

S-Nitrosylation of Mixed Lineage Kinase 3 Contributes to Its Activation after Cerebral Ischemia

Previous studies in our laboratory have shown that mixed lineage kinase 3 (MLK3) can be activated following global ischemia. In addition, other laboratories have reported that the activation of MLK3 may be linked to the accumulation of free radicals. However, the mechanism of MLK3 activation remains incompletely understood. We report here that MLK3, overexpressed in HEK293 cells, is S-nitrosylated (forming SNO-MLK3) via a reaction with S-nitrosoglutathione, an exogenous nitric oxide (NO) donor, at one critical cysteine residue (Cys-688). We further show that the S-nitrosylation of MLK3 contributes to its dimerization and activation. We also investigated whether the activation of MLK3 is associated with S-nitrosylation following rat brain ischemia/reperfusion. Our results show that the administration of 7-nitroindazole, an inhibitor of neuronal NO synthase (nNOS), or nNOS antisense oligodeoxynucleotides diminished the S-nitrosylation of MLK3 and inhibited its activation induced by cerebral ischemia/reperfusion. In contrast, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (an inhibitor of inducible NO synthase) or nNOS missense oligodeoxynucleotides did not affect the S-nitrosylation of MLK3. In addition, treatment with sodium nitroprusside (an exogenous NO donor) and S-nitrosoglutathione or MK801, an antagonist of the N-methyl-D-aspartate receptor, also diminished the S-nitrosylation and activation of MLK3 induced by cerebral ischemia/reperfusion. The activation of MLK3 facilitated its downstream protein kinase kinase 4/7 (MKK4/7)-JNK signaling module and both nuclear and non-nuclear apoptosis pathways. These data suggest that the activation of MLK3 during the early stages of ischemia/reperfusion is modulated by S-nitrosylation and provides a potential new approach for stroke therapy whereby the post-translational modification machinery is targeted.

Involvement of kappa opioid receptors in formalin-induced inhibition of analgesic tolerance to morphine in mice

This study examined the role of kappa opioid receptors (KOR) in the mechanism underlying tolerance to the analgesic effects of morphine induced by chronic pain. The analgesic effect of morphine (10 mg kg(-1)), estimated by the tail-flick test in mice, gradually decreased during repeated daily morphine treatment. A significant decrease in the analgesic effect of morphine was seen on the fifth day of repeated morphine treatment compared with the first day. Chronic pain was induced by subcutaneous administration of 2% formalin into the dorsal part of the left hind paw, which significantly inhibited development of tolerance to morphine analgesia. The effect of formalin-induced pain on inhibition of morphine tolerance was reversed by the KOR antagonist nor-binaltorphimine. Furthermore, an antisense oligodeoxynucleotide, but not a missense oligodeoxynucleotide, against KOR completely suppressed the inhibitory effect of formalin-induced pain on morphine tolerance. Naltrindole, an antagonist of delta opioid receptor, did not affect chronic-pain-induced tolerance to morphine. Our findings show that the inhibitory effect of chronic pain on analgesic tolerance to morphine is mediated by KOR rather than delta opioid receptors.

Estrogen replacement therapy in diabetic ovariectomized female rats potentiates postischemic leukocyte adhesion in cerebral venules via a RAGE-related process

In this study, we tested the hypothesis that the documented transformation of 17beta-estradiol (E2) from a counterinflammatory hormone in nondiabetic (ND) rats to a proinflammatory agent in rats with diabetes mellitus (DM) is due to an enhanced contribution from the receptor for advanced glycation end products (RAGE). Rhodamine 6G-labeled leukocytes were observed through a closed cranial window in rats. In vivo pial venular leukocyte adherence and infiltration were measured over 10 h reperfusion after transient forebrain ischemia in DM (streptozotocin) versus ND intact, ovariectomized (OVX), and E2-replaced (for 7-10 days) OVX (OVE) females. The role of RAGE was examined in two ways: 1) RAGE knockdown via topical application of RAGE antisense versus missense oligodeoxynucleotide or 2) intracerebroventricular injection of the RAGE decoy inhibitor, soluble RAGE. Among diabetic rats, the lowest levels of cortical RAGE mRNA and immunoreactivity of the RAGE ligand, AGE, were seen in OVX females, with significantly higher levels exhibited in intact and OVE females. However, results from the analysis of cortical RAGE protein only partially tracked those findings. When comparing ND to DM rats, cortical AGE immunoreactivity was significantly lower in OVE and intact females but similar in OVX rats. In DM rats, the level of postischemic leukocyte adhesion and infiltration (highest to lowest) was OVE>intact>>untreated OVX. In NDs, adhesion was highest in the untreated OVX group. Leukocyte extravasation was observed at >6 h postischemia but only in diabetic OVE and intact females and in ND OVX (untreated) rats. Pretreatment with RAGE antisense-oligodeoxynucleotide or soluble RAGE attenuated postischemic leukocyte adhesion and prevented infiltration but only in the diabetic OVE and intact groups. These results indicate that the exacerbation of postischemic leukocyte adhesion by chronic E2 replacement therapy in diabetic OVX females involves a RAGE-related mechanism. Targeting RAGE may restore the neuroprotective effect of E2 replacement therapy in diabetic females.

Unmixing Memory and Desire

Abstinent drug addicts often relapse following exposure to environmental cues associated with drug taking. The ability of some forms of previously acquired memory to influence future behavior may depend upon reconsolidation of retrieved memories (see Hernandez and Kelley), leading two groups to see if disrupting such reconsolidation also disrupts the association of addictive drugs with contextual cues. The acquisition of cocaine-conditioned place preference (COC-CPP) in rats depends on activation of the extracellular signaling-regulated kinase (ERK) signaling pathway; it is blocked by inhibiting mitogen-activated protein kinase kinase (MEK), which phosphorylates and thereby activates ERK. Miller and Marshall used immunocytochemistry and Western analysis to show increased phosphorylation of ERK and of the downstream transcription factors Elk-1 and CREB in the nucleus accumbens core (AcbC, a midbrain region associated with cue-elicited drug seeking) in rats that had acquired COC-CPP. Infusion of a MEK inhibitor into the AcbC shortly before testing for COC-CCP blocked COC-CCP-related behavior and an associated increase in ERK, CREB, and Elk-1 phosphorylation and Fos abundance in the AcbC of rats that had already undergone pairing of cocaine with a particular environment. When administered independently of behavioral testing, MEK inhibition did not affect COC-CCP tested 24 hours later. However, rats in which exhibition of COC-CPP was blocked by preadministration of a MEK inhibitor, or that received a MEK inhibitor right after testing (who exhibited COC-CCP during the test), failed to show COC-CCP when retested 24 hours or 14 days later. These rats also showed decreased activation of the AcbC ERK pathway. Thus, the authors concluded that disruption of AcbC ERK signaling during memory reconsolidation blocked the subsequent expression of COC-CCP. Expression of the transcription factor Zif268 in the basolateral amygdala increases following reexposure to stimuli associated with self-administration of cocaine. In the second study, by Lee et al. , rats learned to nosepoke for a cocaine infusion, which was accompanied by a light. When paired with a memory reactivation session, Zif268 antisense oligodeoxynucleotides (Zif268 ASO) infused into the basolateral amygdala eliminated the ability of light to promote acquisition of a new drug-seeking behavior, whereas missense oligodeoxynucleotides, administered with the reactivation session, or Zif268 ASO, administered without it, did not. C. A. Miller, J. F. Marshall, Molecular substrates for retrieval and reconsolidation of cocaine-associated contextual memory. Neuron 47 , 873-884 (2005). [PubMed] J. L. C. Lee, P. Di Ciano, K. L. Thomas, B. J. Everitt, Disrupting reconsolidation of drug memories reduces cocaine-seeking behavior. Neuron 47 , 795-801 (2005). [PubMed] P. J. Hernandez, A. E. Kelley, Cracking addiction the second time around: Reconsolidation of drug-related memories. Neuron 47 , 772-775 (2005). [PubMed]

NMDA-R1 antisense oligodeoxynucleotides modify formalin-induced nociception and spinal c-Fos expression in rat spinal cord

Noxious peripheral stimuli (thermal, mechanical, or chemical) produce long-term adaptations in the sensitivity of central nociceptive neurons to subsequent noxious stimuli. The mechanisms responsible for this central sensitization are multifactorial, but the activation of spinal N-methyl- d-aspartate (NMDA) receptors plays a pivotal role. Using antisense oligodeoxynucleotides, we tested the role of the NR1 subunit of the NMDA receptor in the nociception and expression of the immediate early gene c- fos following formalin-induced pain. Rats received NMDA-R1 antisense, sense, or missense oligodeoxynucleotides intrathecally three times over a 48-h interval. The day after the last injection of the oligodeoxynucleotide, the formalin test was performed. Pain-related behavior was quantified by counting the incidence of flinching of the injected paw for 60 min, and the animals were perfused and the spinal cord removed for c-Fos immunohistochemistry 60 min later. Immunopositive cells were counted in the laminae I/II 0 and V of the lumbar enlargement. Treatment with NR1 antisense oligodeoxynucleotide resulted in a marked decrease in flinching. Similarly, the antisense oligodeoxynucleotide virtually abolished formalin-induced expression of c-Fos-like immunoreactivity (Fos-IR) in the spinal cord dorsal horn ipsilateral to injection. In contrast, the corresponding sense or missense oligodeoxynucleotides had no effect on either formalin-evoked behavior or c-Fos immunoreactivity. We conclude that an NR1 antisense oligodeoxynucleotide inhibits both nociceptive behavior and c- fos expression following formalin injection in rats, demonstrating that NR1 plays an important role in the development of noxious stimulation induced c- fos expression in this model.

CAMP modulates cGMP-mediated cerebral arteriolar relaxation in vivo.

No studies have specifically addressed whether cAMP can influence nitric oxide (NO)/cGMP-induced cerebral vasodilation. In this study, we examined whether cAMP can enhance or reduce NO-induced cerebral vasodilation in vivo via interfering with cGMP efflux or through potentiating phosphodiesterase 5 (PDE5)-mediated cGMP breakdown, respectively, in cerebral vascular smooth muscle cells (CVSMCs). To that end, we evaluated, in male rats, the effects of knockdown [via antisense oligodeoxynucleotide (ODN) applications] of the cGMP efflux protein multidrug resistance protein 5 (MRP5) and PDE5 inhibition on pial arteriolar NO donor [S-nitroso-N-acetyl penicillamine (SNAP)]-induced dilations in the absence and presence of cAMP elevations via forskolin. Pial arteriolar diameter changes were measured using well-established protocols in anesthetized rats. In control (missense ODN treated) rats, forskolin elicited a leftward shift in the SNAP dose-response curves (approximately 50% reduction in SNAP EC50). However, in MRP5 knockdown rats, cAMP increases were associated with a substantial reduction in SNAP-induced vasodilations (reflected as a significant 35-50% lower maximal response). In the presence of the PDE5 inhibitor MY-5445, the repression of the NO donor response accompanying forskolin was prevented. These findings suggest that cAMP has opposing effects on NO-stimulated cGMP increases. On the one hand, cAMP limits CVSMC cGMP loss by restricting cGMP efflux. On the other, cAMP appears to enhance PDE5-mediated cGMP breakdown. However, because increased endogenous cAMP seems to potentiate NO/cGMP-induced arteriolar relaxation when MRP5 expression is normal, the effect of cAMP to reduce cGMP efflux appears to predominate over cAMP stimulation of cGMP hydrolysis.

Regulation of rat pial arteriolar smooth muscle relaxation in vivo through multidrug resistance protein 5-mediated cGMP efflux.

Multidrug resistance protein 5 (MRP5) has been linked to cGMP cellular export in peripheral vascular smooth muscle cells (VSMCs) and is widely expressed in brain vascular tissue. In the present study, we examined whether knockdown of MRP5 in pial arterioles [via antisense oligodeoxynucleotide (ODN) applications] affected nitric oxide (NO)/cGMP-induced dilations. The antisense or (as a control) missense ODN was applied to the cortical surface approximately 24 h before study via closed cranial windows. The efficacy of the antisense vs. missense ODN in eliciting selective reductions in MRP5 expression was confirmed by analysis of MRP5 mRNA in pial tissue. Unexpectedly, in initial studies, a significantly lower maximal pial arteriolar diameter increase in the presence of the NO donor S-nitrosoacetylpenicillamine (SNAP) was seen in the antisense vs. missense ODN-treated rats (35 vs. 48% diameter increase, respectively). It was suspected that this related to a reduced vascular smooth muscle cell sensitivity to cGMP due to prolonged exposure to increased intracellular cGMP levels elevated by overnight restriction of cGMP efflux. That postulate was supported by a finding of a diminished vasodilating response to the cGMP-dependent protein kinase-activating cGMP analog 8-p-chlorophenylthio-cGMP in antisense vs. missense ODN-treated rats. To prevent desensitization, additional rats were studied in the presence of chronic NOS inhibition via Nomega-nitro-L-arginine. In the NO synthase (NOS)-inhibited rats, the maximal SNAP response was much higher in the antisense (62% increase) vs. the missense ODN (40% increase) group. A similar result was obtained when monitoring responses to the soluble guanylyl cyclase-activating drugs YC-1 and BAY 41-2272. Moreover, in the presence of NOS inhibition, the normal SNAP-induced rise in periarachnoid cerebrospinal fluid cGMP levels, which reflects cGMP efflux, was absent in the antisense ODN-treated rats, a finding consistent with loss of MRP5 function. In conclusion, if one minimizes the confounding effects of basal cGMP production, a clearer picture emerges, one that indicates an important role for MRP5-mediated cGMP efflux in the regulation of NO-induced cerebral arteriolar relaxation.

Effects of phosphorothioate anti-sense oligodeoxynucleotides on colorectal cancer cell growth and telomerase activity

To investigate the inhibitory effect of phosphorothioate anti-sense oligodeoxynucleotides (PASODN) on colorectal cancer LS-174T cells in vitro and the mechanism of inhibition of telomerase activity in these cells. PASODN were used to infect LS-174T cells and block human telomerase RNA (hTR) through anti-sense technology. The inhibitory effect of PASODN was evaluated by colony-forming inhibition assay and growth curve. Changes of telomerase activity in LS-174T cells were detected by polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), and the level of apoptosis was analyzed by flow cytometry (FCM) assay. PASODN showed a dose and time-dependent inhibition of cell proliferation. The optimal dosage of PASODN was 10 mumol/L. The colony-forming efficiency was 10.3% in PASODN group after 10 d, whereas that in phosphorothioate mis-sense oligodeoxynucleotides (PMSODN) group with the same concentration and in PBS group (blank control) was 49.1% and 50.7%, respectively. PCR-ELISA results indicated that telomerase activity in the PASODN group was obviously inhibited in comparison with in the control groups (P<0.01, t = 3.317 and 3.241, t0.01(20) = 2.845). Meanwhile, before the number of cells was decreased, the morphological changes were observed in the cells of PASODN group. The cells in PASODN group showed the apoptotic peak at 72 h after infection, whereas the control group did not show. Specific sequence oligonucleotides can inhibit telomerase activity and lead to cell apoptosis, suggesting a novel treatment strategy for malignant tumors induced by telomerase.

Knockdown of spinal cord postsynaptic density protein-95 prevents the development of morphine tolerance in rats

The activation of spinal cord N-methyl- d-aspartate (NMDA) receptors and subsequent intracellular cascades play a pivotal role in the development of opioid tolerance. Postsynaptic density protein-95 (PSD-95), a molecular scaffolding protein, assembles a specific set of signaling proteins around NMDA receptors at neuronal synapses. The current study investigated the possible involvement of PSD-95 in the development of opioid tolerance. Opioid tolerance was induced by intrathecal injection of morphine sulfate (20 μg/10 μl) twice a day for 4 consecutive days. Co-administration of morphine twice daily and PSD-95 antisense oligodeoxynucleotide (50 μg/10 μl) once daily for 4 days not only markedly reduced the PSD-95 expression and its binding to NMDA receptors in spinal cord but also significantly prevented the development of morphine tolerance. In contrast, co-administration of morphine twice daily and PSD-95 missense oligodeoxynucleotide (50 μg/10 μl) once daily for 4 days did not produce these effects. The PSD-95 antisense oligodeoxynucleotide at the doses we used did not affect baseline response to noxious thermal stimulation or locomotor function. The present study indicates that the deficiency of spinal cord PSD-95 attenuates the development of opioid tolerance. These results suggest that PSD-95 might be involved in the central mechanisms of opioid tolerance and provide a possible new target for prevention of development of opioid tolerance.

Effect of knock down of spinal cord PSD-93/chapsin-110 on persistent pain induced by complete Freund's adjuvant and peripheral nerve injury

PSD-93/chapsin-110 is a neuronal PDZ domain-containing protein that binds to and clusters the N-methyl- d-aspartate receptor (NMDAR) at synapses in the central nervous system. It also assembles a specific set of signaling proteins around the NMDAR and mediates downstream signaling by the NMDAR. Thus, PSD-93/chapsin-110 might be involved in many physiological and pathophysiological actions triggered via the activation of the NMDAR. In the current study, we report that abundant PSD-93/chapsin-110 protein was detected in rat spinal cord, particularly in the superficial dorsal horn. The rats injected intrathecally with PSD-93/chapsin-110 antisense oligodeoxynucleotide every 24 h for 4 days displayed not only a remarkable decrease in spinal cord PSD-93/chapsin-110 expression but also a significant reduction in the paw withdrawal responses to thermal and mechanical stimuli during complete Freund's adjuvant-induced inflammatory pain and peripheral nerve injury-induced neuropathic pain. In contrast, the rats injected intrathecally with PSD-93/chapsin-110 missense oligodeoxynucleotide did not exhibit these changes. We also found that pretreatment with PSD-93/chapsin-110 antisense oligodeoxynucleotide did not change the locomotor activity or the responses to acute noxious thermal and mechanical stimuli in intact rats. The present results indicate that the deficiency of spinal cord PSD-93/chapsin-110 protein significantly attenuates thermal and mechanical hyperalgesia in complete Freund's adjuvant- or peripheral nerve injury-induced chronic pain. This suggests that spinal cord PSD-93/chapsin-110 might be involved in the central mechanism of chronic pain. Our work might provide a new target for the therapy of chronic pain.

Experimental parkinsonism is associated with increased pallidal GAD gene expression and is reversed by site-directed antisense gene therapy.

The levels of mRNA encoding the two isoforms of glutamic acid decarboxylase (GAD(65) and GAD(67)) were measured throughout the pallidal complex in normal and acutely (i.e., 1 month duration) and chronically (i.e., 5 years duration) parkinsonian 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) -treated monkeys as well as in monkeys exposed to MPTP but asymptomatic for parkinsonism. GAD(65) mRNA labeling was modestly increased in the mid/caudal internal globus pallidus (GPi) but not in the external globus pallidus (GPe) in parkinsonian monkeys, compared with normal and asymptomatic monkeys. GAD(67) mRNA expression was highly increased in the mid/caudal GPi, and modestly increased in the GPe in parkinsonian monkeys compared with normal and asymptomatic animals. Infusion of GAD(67) antisense oligodeoxynucleotides bilaterally into the GPi resulted in a transient reversal of akinesia and bradykinesia that was not produced by infusion of missense oligodeoxynucleotides. These data emphasize the role of GAD enzyme (particularly GAD(67)) and GABA in the GPi for the expression of parkinsonian motor signs and suggest that selective manipulation of GABAergic neurotransmission in the GPi may have therapeutic potential for treating parkinsonism.

Helicobacter pylori induces apoptosis of rat gastric parietal cells.

Gastric Helicobacter pylori infection may lead to multifocal atrophic corpus gastritis associated with loss of epithelial cells as well as glandular structures. The current work investigated H. pylori effects on cell death of isolated, nontransformed rat parietal cells (PC). Highly enriched rat PC (>97%) were isolated from gastric mucosa and cultured in serum-free medium over 24 h. The cells were cocultured over 8 h with cytotoxin-associated immunodominant protein (cagA)(+)/vacuolating toxin (vacA)(+) or with cagA(-)/vacA(-) H. pylori laboratory strains and also with H. pylori mutants deleted in several genes of the cag pathogenicity island. Staphylococcus aureus or Campylobacter jejuni were used as controls. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy. Interleukin (IL)-8 and cytokine-induced neutrophil chemoattractant (CINC)-1 secretion was measured by ELISA. Activation of nuclear factor-kappaB (NF-kappaB) was studied in nuclear extracts of PC by electrophoretic mobility shift assay. Apoptosis of PC was induced in a concentration- and time-dependent manner by cagA(+)/vacA(+) H. pylori strains but not by cagA(-)/vacA(-) negative strains or by the cagE knockout mutant. S. aureus and C. jejuni had no effect. PC showed no IL-8 or CINC-1 secretion on exposure to cagA(+)/vacA(+) H. pylori. cagA(+)/vacA(+) strains induced activation of NF-kappaB complexes in nuclear extracts of PC, which were composed of p65 and p50 subunits. No significant stimulation of NF-kappaB activation was detected by incubation of PC with the cagE knockout mutant. Preincubation of PC with antisense but not missense oligodeoxynucleotides against the p65 subunit significantly reduced DNA binding to the kappaB recognition sequence. The p65 oligonucleotides as well as the proteasome inhibitor N-CBZ-isoleucin-glutamin-(o-t-butyl-)-alanin-leucin and the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine completely prevented PC apoptosis induced by cagA(+)/vacA(+) strains. In summary, cagE presence appears to be essential for H. pylori-induced apoptosis of gastric parietal cells, and this effect is dependent on the activation of NF-kappaB and production of nitric oxide.

Dynorphin A(1-17)-induced feeding: pharmacological characterization using selective opioid antagonists and antisense probes in rats.

Ventricular administration of the opioid dynorphin A(1-17) induces feeding in rats. Because its pharmacological characterization has not been fully identified, the present study examined whether a dose-response range of general and selective opioid antagonists as well as antisense oligodeoxynucleotide (AS ODN) opioid probes altered daytime feeding over a 4-h time course elicited by dynorphin. Dynorphin-induced feeding was significantly reduced by a wide range of doses (5-80 nmol i.c.v.) of the selective kappa(1)-opioid antagonist nor-binaltorphamine. Correspondingly, AS ODN probes directed against either exons 1 and 2, but not 3 of the kappa-opioid receptor clone (KOR-1) reduced dynorphin-induced feeding, whereas a missense oligodeoxynucleotide control probe was ineffective. Furthermore, AS ODN probes directed against either exons 1 or 2, but not 3 of the kappa(3)-like opioid receptor clone (KOR-3/ORL-1) also attenuated dynorphin-induced feeding. Although the selective mu-antagonist beta-funaltrexamine (20-80 nmol) reduced dynorphin-induced feeding, an AS ODN probe directed only against exon 1 of the mu-opioid receptor clone was transiently effective. Neither general (naltrexone, 80 nmol) nor delta (naltrindole, 80 nmol)-selective opioid antagonists were particularly effective in reducing dynorphin-induced feeding, and an AS ODN probe targeting the individual exons of the delta-opioid receptor clone failed to significantly reduce dynorphin-induced feeding. These converging antagonist and AS ODN data firmly implicate the kappa(1)-opioid receptor and the KOR-1 and KOR-3/ORL-1 opioid receptor genes in the mediation of dynorphin-induced feeding.