Gao C et al / Acta Pharmacol Sin 2003 Dec; 24 (12): 1253-1258
GAO Can3, CHEN Li-Wei, TAO Yi-Min, CHEN Jie, XU Xue-Jun , CHI Zhi-Qiang2
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031; 3Research Center for Biochemistry and Molecular Biology, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China
1 Project supported by the National Basic Research Program (G1999054002).
2 Correspondence to Prof CHI Zhi-Qiang. Phn 86-21-6471-8045, ext 513. Fax 86-21-5080-7088. E-mail firstname.lastname@example.org
Received 2003-03-28 Accepted 2003-07-14
KEY WORDS ohmefentanyl stereoisomers; cAMP-response element binding protein; mu opioid receptor; cyclic AMP; hippocampal neurons; phosphorylation
AIM: To define the effects and signal pathways of ohmefentanyl stereoisomers [(-)-cis-(3R,4S,2'R) OMF (F9202), (+)-cis-(3R,4S,2'S) OMF (F9204), and (-)-cis-(3S,4S,2'R) OMF (F9203)] on the phosphorylation of cAMP-response element binding protein (CREB) in cultured rat hippocampal neurons. METHODS: The effects of the three OMF stereoisomers and morphine (Mor) on cAMP accumulation and CREB phosphorylation were monitored by radioimmunoassay and Western blot analysis, respectively. results: The three OMF stereoisomers and Mor could all partially inhibit forskolin-stimulated (25 µmol/L, 15 min) cAMP accumulation in a dose-dependent manner and this effect could be reversed by naloxone. F9202, F9204, and Mor could significantly increase CREB phosphorylation from 2.88 to 3.59 folds over control levels after 30min exposure. This effect was reversed by naloxone, but F9203 failed to increase CREB phosphorylation. KN-62 and staurosporine significantly blocked the opioids- induced CREB phosphorylation, while H-89 and PD 98059 had no effect on the actions. CONCLUSION: Mor, F9202, and F9204, which could induce psychological dependence affected via the µ-opioid receptor, stimulated intracellular signal pathways involving Ca2+/calmodulin-dependent protein kinases (CCDPK) and protein kinase C (PKC) pathways, which in turn initiated CREB phosphorylation. F9203, which could not induce dependence, had no effect on CREB phosphorylation in hippocampal neurons. The increased CREB phosphorylation in hippocampal neurons may play a role in opioids dependence.
Ohmefentanyl(N-[1-(2-hydroxy-2-phenylethyl)-3- methyl-4-peperidyl]-N-phenyloropanamide, OMF) is a novel and potent analgesic agent with high affinity to µ opioid receptor[1,2]. Among the 8 stereoisomers of OMF (F9201-F9208), (-)-cis-(3R,4S,2'R) OMF (F9202), and (+)-cis-(3R,4S,2'S) OMF (F9204) had similarly potent analgesic action and high selectivity to µ opioid receptor, but showed a large difference in physical dependence. They could induce conditioned place preference (CPP) with minor difference. While (-)-cis-(3S,4S,2'R) OMF (F9203) shows the lowest binding affinity to µ-opioid receptor and very little analgesic activityin 8 stereoisomers. It could not induce CPP also.
The mechanisms that underlie the development of opioids dependence remain unclear. Recent findings indicated that learning and memory and drug addiction shared certain intracellular signaling cascades and depended on activation of transcription factor cAMP-response element binding protein (CREB). They were accompanied by alterations in neural plasticity. CREB acts as a gene transcription factor and modulates the expression of several cAMP-inducible genes. CREB can be phosphorylated on serine133 , which is required to induce the transcriptional activity of the protein, by several protein kinases, such as protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinases (CCDPK), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK). CREB is thought to play a key role in opioids dependence[8,9].
Our previous work showed that both F9202 and F9204 could increase CREB phosphorylation in hippocampus in CPP paradigm, while F9203 had no effect on CPP and CREB phosphoryoation. Hippocampus plays an important role in learning and memory. But the direct effect of opioids on the regulation of CREB phosphorylation has not yet been studied in cultured hippocampal neurons.
To better understand the mechanisms underlying the actions of opioids on neurons, the effects of F9202, F9204, F9203, and Mor on forskolin-stimulated cAMP accumulation and CREB phosphorylation in cultured rat hippocampal neurons were investigated. Several protein kinase inhibitors were also used to clarify the signal pathways of CREB phosphorylation induced by opioids.
MATERIALS AND METHODS
Drugs and reagents OMF stereoisomers were synthesized in Shanghai Institute of Materia Medica (China). Morphine·HCl (Mor) was purchased from Qinghai Pharmaceutical LTD (China). DMEM media, neurobasal medium, B27 supplement, and trypsin were purchased from GibcoBRL (USA). Antibodies for phospho-CREB (P-CREB, Ser133) and CREB were purchased from Cell Signaling Technology, Inc (USA). Naloxone (Nal), forskolin (For), poly-l-lysine, 1-methyl-3-isobutylxanthine (IBMX) were purchased from Sigma (USA). Horseradish peroxidase-conjugated anti-rabbit IgG was purchased from Rockland (USA). [N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide ·2HCl (H-89) and [1-[N,O-bis-(5-Isoquinolinesulfonyl)N-methyl-L-tyrosyl]-4-phenylpiperazine] (KN-62) were purchased from Alexis (Switzerland). 2'-Amino-3'-methoxyflavone (PD 98059) was purchased from Promega (USA). cAMP radioimmunoassay kit was purchased from Shanghai Second Medical University (China). Enhanced chemiluminescence reagents (ECL) were purchased from Amersham Pharmacia Biotech (England).
Hippocampal neurons culture Hippocampal neurons culture was prepared using methods with minor modification. Hippocampal neuron cultures were prepared from E18(embryonic day)-E19 Sprague-Dawley rats (Provided by Shanghai Institute of Materia Medica, Grade II) embryos dissociated with trypsin and plated on 6-well plates coated with poly-l-lysine in DMEM with glutamine 4 mmol/L, 10 % horse and 10 % fetal bovine serum. Neurobasal medium containing B27 supplement was changed on d 2 and neurons were fed twice weekly. During the incubation the neurons were kept in the 5 % CO2 incubator. Experiments were performed on 7-9 d (postisolation) old neurons.
cAMP assay Cells were washed with phosphate buffered saline (PBS), then cells were treated with different concentrations of OMF stereoisomers or Mor in the presence of forskolin 25 µmol/L and IBMX 500 µmol/L at 37 ºC for 15 min. The reactions were terminated with perchloric acid 1 mol/L and neutralized with KOH 1 mol/L. The cAMP level of each sample was measured using radioimmunoassay as previously described. The values calculated as 100×[cAMP (For+agonist)-cAMP (basal)]/[cAMP (For)-cAMP (basal)] where cAMP (For+agonist) is cAMP accumulation in the presence of forskolin and agonist, cAMP (basal) is cAMP in the absence of forskolin and agonist, and cAMP (For) is cAMP in the presence of forskolin alone. In the effect of naloxone experiments, cells were pretreated with Nal 1 µmol/L, then challenged with OMF 100 nmol/L stereoisomers or Mor 1 µmol/L, and the cAMP levels were measured as described above.
Cell stimulation Cells were treated with Mor (1 µmol/L), F9202 (100 nmol/L), F9204 (100 nmol/L), or F9203 (100 nmol/L) in DMEM medium. Nal (1 µmol/L) or several inhibitors of protein kinases were added 10 min ahead of drug stimulation for 30 min. After treatment, cells were washed three times with ice-cold PBS and were extracted with SDS sample buffer and boiled for 10 min.
Western blot analysis The lysates were separated by 10 % SDS-PAGE and transferred to nitrocel lulose membrane. The blots were blocked for 1 h in TBST buffer (in mmol/L) Tris-HCl 20, pH 7.4, NaCl 137, 0.1 % Tween 20 with 5 % nonfat milk at room temperature and then were incubated with monoclonal rabbit anti-P-CREB (1:1000) antibody at 4 ºC overnight. After being washed for three times with TBST, the blots were incubated with horseradish peroxidase conjugated anti-rabbit secondary antibody (1:5000) for 1 h at room temperature. The washings were repeated, and the blots were developed by ECL on an X-ray film and visualized by exposure to GS-800 Calibrated Densitometer (Bio-Rad, USA). The members were stripped of previous antibody and reprobed with anti-CREB antibody (1:1000).
Data analysis The data was presented as mean± SD. Statistical significant differences were determined with t-test.
Acute effects of the three OMF stereoisomers on forskolin-stimulated cellular cAMP levels in cultured hippocampal neurons All the three OMF stereoisomers and Mor were functionally coupled to the inhibitory G protein (Gi) and thus negatively regulated adenylyl cyclase in hippocampal neurons. After the cells were treated with various concentrations (1 nmol/L-10 µmol/L) of opioids for 15 min, they could all partially inhibit forskolin-stimulated cAMP accumulation in a dose-dependent manner (Fig 1A). The opioid receptor antagonist Nal (1 µmol/L) blocked the inhibitory effects of opioids on forskolin-stimulated cAMP accumulation (Fig 1B).
Fig 1. Acute inhibitory effects of OMF stereoisomers and Mor on forskolin-stimulated cAMP accumulation and the effects of naloxone (Nal) in cultured hippocampal neurons. A: the neurons were treated with different concentrations of OMF stereoisomers and Mor during a 15-min forskolin (25 µmol/L) stimulation. B: Nal (1 µmol/L) was added with OMF stereoisomers (100 nmol/L) and Mor (1 µmol/L) treatment during a 15 min forskolin (25 µmol/L) stimulation. n=3. Mean±SD. cP<0.01 vs corresponding control (Cont) group. fP<0.01 vs corresponding None-Nal preadministration group.
Different effects of the three OMF stereoisomers on CREB Ser133 phosphorylation in hippocampal neurons Mor (1 µmol/L), F9202 (100 nmol/L), and F9204 (100 nmol/L) stimulated CREB phosphorylation in hippocampal neurons. The effect of the three opioids had no significant difference. They potentiated 2.88 to 3.59 folds over control levels after 30 min exposure. In contrast, F9203 failed to increase CREB phosphorylation. Nal (1 µmol/L) reversed the effects of Mor, F9202, and F9204 on the CREB phospho-rylation. Immunoblot analysis with anti-CREB antiserum showed no changes in the total CREB protein levels. These results indicated that the transcription factor CREB in hippocampal neurons was a target for a signaling pathway of opioids, which could induce psychological dependence, that was initiated by activation of µ-opioid receptor (Fig 2).
Fig 2. Effect of naloxone on CREB phosphorylation in cultured hippocampal neurons induced by OMF stereoisomers and Mor. Naloxone (Nal, 1 µmol/L) was added 10 min ahead of OMF stereoisomers (100 nmol/L, 30 min) and Mor (1 µmol/L, 30 min) treatment. A: immunoactivities of P-CREB and CREB were measured by Western blot analysis. B: densitometry data. n=3. Mean±SD. cP<0.01 vs corresponding control (Cont) group. fP<0.01 vs corresponding none- Nal preadministration group.
Effects of several protein kinase inhibitors on the CREB phosphorylation induced by OMF stereoisomers and Mor in hippocampal neurons We next questioned which protein kinase initiated the CREB phosphorylation induced by F9202, F9204, and Mor in hippocampal neurons. H-89 (20 µmol/L), an inhibitor of PKA, and PD 98059 (50 µmol/L), an inhibitor of MAPK kinase, had no effect on the CREB phosphorylation. In contrast, KN-62 (10 µmol/L), an inhibitor of CCDPK caused significant inhibition of the CREB phosphoryla- tion. Staurosporine (STAU, 1 µmol/L), an inhibitor of PKC, caused an complete blockade of the CREB phosphorylation (Fig 3). When given alone, staurosporine and H-89 tended to decrease the phosphorylated CREB levels (data not shown). The total CREB did not change in any state (data not shown). These data indicated that stimulation of µ-opioid receptors in hippocampal neurons resulted in PKC and CCDPK activition, which in turn initiated the CREB phosphorylation.
Fig 3. Effect of several inhibitors of protein kinases on CREB phosphorylation in cultured hippocampal neurons induced by Mor and OMF stereoisomers. H-89 (20 µmol/L), KN-62 (10 µmol/L), PD98059 (50 µmol/L), or staurosporine (STAU, 1 µmol/L) was given ahead of Mor (1 µmol/L, 30 min), F9202 (100 nmol/L, 30 min), or F9204 (100 nmol/L, 30 min) treatment, respectively. A: immunoactivity of P-CREB was measured by Western blot analysis. B: densitometry data. n=3. Mean±SD. cP<0.01 vs corresponding agonist group.
The present study indicated that the three of OMF stereoisomers and Mor could partially inhibit forskolin-stimulated cAMP accumulation. Opioids exert an inhibitory action on neurons because it is well known that they are usually coupled to Gi/Go classes of the G proteins. Activation of opioid receptors inhibits adenylyl cyclase, opens K+ channels, and closes voltage-dependent Ca2+ channels. These signaling pathways suppress neuronal excitability, however, opioid receptors also trigger various poorly defined signaling pathways that can stimulate cellular activity. Our results revealed that F9202, F9204, and Mor could increase CREB phosphorylation in cultured hippocampal neurons. The inductions of CREB phosphorylation following Mor, F9202, and F9204 treatment were blocked by Nal, which indicated that the effect under study was mediated by activation of µ-opioid receptor.
CREB has been shown to play a key role in opioid dependence. Opioids regulate CREB level, its phosphorylation and binding to its corresponding response element in the promoters of several genes implicated in drug addiction. At the cellular level CREB acts as convergence point for different cellular pathways. Considering the important role of CREB in learning and memory in hippocampal neurons, we studied the opioids effect on CREB phosphorylation in cultured rat hippocampal neurons in order to find plasticity-associated neuroadaptation of opioid dependence. Our previous studies indicated that in CPP paradigm, F9202, F9204, and Mor could induce CREB phosphorylation in hippocampus, while F9203 , which had very little analgesic activity could not induce CPP and CREB phosphorylation. This suggested that the increased CREB phosphorylation in hippocampus be relevant to psychological dependence. But the signal pathways of it were not well known.
CREB is a transcription factor expressed in many tissues. Several kinases are known to activate CREB by phosphorylation of Ser133 including PKA, CCDPK, ribosomal S6 kinase, and MAPK. Our results from hippocampal neurons indicated that CREB phosphorylation appeared to involve both CCDPK and PKC pathways, but did not relate to cAMP pathway. The acute effect of the three of OMF stereoisomers and Mor on cAMP accumulaton induced by forskolin was negative. Furthermore, although H-89, an inhibitor of PKA, slightly inhibited CREB phorphorylation, H-89 itself could also tend to decrease the phosphorylated CREB levels. The effect might contribute to that H-89 itself could decrease the normal CREB phosphorylation by inhibiting PKA pathway. Staurosporine, an inhibitor of PKC, significantly decreased the CREB phosphoryla-tion, which was even lower than that of control group. This might contain the effect of decreasing the normal CREB phosphorylation by inhibiting PKC pathway. While KN-62, an inhibitor of CaMK, caused significant inhibition of CREB phosphorylation without affecting basal phosphorylated CREB levels.
Opioid receptors have been shown to enhance Ca2+ influx/release via Gi/Go proteins. Recent studies revealed that in HEK-293 cells and SH-SY5Y cells, Ca2+ influxed into the cells resulting from µ-opioid receptor activation was required for nuclear calmodulin (CaM) translocation. Ca2+ influx resulted in nuclear CaM translocation and increased nuclear CaM content, which was associated with enhanced phosphorylation of the nuclear transcription factor CREB. These appeared to be mediated by CCDPK and also by a pathway including PKC. Furthermore, -specific opioids had also been shown to produce rapid transient increase in the [Ca2+]i level in NG108-15 cells. Mor exerted a stimulatory effect on CREB phosphorylation. This stimulation neither depended on newly synthesized proteins nor involved the cAMP pathway, but required Ca2+/CaM and activation of PKC. Our results indicated that CREB phosphorylation in hippocampal neurons induced by OMF stereoisomers and Mor was not relevant to cAMP pathway, but required activation of CCDPK and PKC.
Opioid receptors are known to activate MAPK through release and phosphatidylinositol 3-kinase activation in transfected Chinese hamoter ovary cells, and, furthermore, MAPK can phosphorylate CREB. How-ever, PD 98059, an inhibitor of MAPK kinase had no effect both on the CREB phosphorylation and the total protein level. Recent study on activity-dependent CREB phosphorylation revealed that CCDPK IV signaling was rapid and MAPK signaling was slow and sustained. The slowness of the MAPK pathway to CREB was due in part to the fact that P-MAPK formation was slow, perhaps because many intermediates lay upstream of P-MAPK in the activity-dependent signaling cascade. So, the role of MAPK pathway in the slow phase of opioids-induced CREB phosphorylation should be further investigated.
These results may lead to this conclusion that F9202, F9204, and Mor, which can induce psychological dependence, can increase CREB phosphorylation mainly through PKC and CCDPK pathways in cultured hippocampal neurons. The stimulation of CREB phosphorylation is likely contributed to the long-lasting consequences of exposure to opioids.